Effects of garlic oil, nitrate, saponin and their combinations supplemented to different substrates on in vitro fermentation, ruminal methanogenesis, and abundance and diversity of microbial populations.

PubMed

Patra, A K; Yu, Z

2015-07-01

To investigate the effect of garlic oil (G), nitrate (N), saponin (S) and their combinations supplemented to different forage to concentrate substrates on methanogenesis, fermentation, diversity and abundances of bacteria and Archaea in vitro. The study was conducted in an 8 × 2 factorial design with eight treatments and two substrates using mixed ruminal batch cultures obtained. Quillaja S (0·6 g l(-1) ), N (5 mmol l(-1) ) and G (0·27 g l(-1) ) were used separately or in binary and tertiary combinations. The two substrates contained grass hay and a dairy concentrate mixture at a 70 : 30 (high-forage substrate) ratio or a 30 : 70 (high-concentrate substrate) ratio. Ruminal fermentation and cellulolytic bacterial populations were affected by interaction between substrate and anti-methanogenic compounds. The inhibitor combinations decreased the methane production additively regardless of substrate. For the high-concentrate substrate, S decreased methane production to a greater extent, so did G and N individually for the high-forage substrate. Feed degradability and total volatile fatty acid (VFA) concentrations were not decreased by any of the treatments. Fibre degradability was actually improved by N+S for the high-forage substrate. VFA concentrations and profiles were affected differently by different anti-methanogenic inhibitors and their combinations. All treatments inhibited the growth of Archaea, but the effect on Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens varied. The results suggest that substrate influences the efficacy of these inhibitors when they are used separately, but in combinations, they can lower methanogenesis additively without much influence from the substrate. The presented research provided evidence that binary and tertiary combination of garlic oil, nitrate and saponin can lower the methane production additively without adversely impacting rumen fermentation and degradability, and forage to concentrate ratio does not change the above effects. These anti-methanogenic inhibitors in combination may have practical application to mitigate methane emission from ruminants. © 2015 The Society for Applied Microbiology.

Inhibitors of Proton Pumping

PubMed Central

Bisson, Mary A.

1986-01-01

Reported inhibitors of the Characean plasmalemma proton pump were tested for their ability to inhibit the passive H+ conductance which develops in Chara corallina Klein ex Willd. at high pH. Diethylstilbestrol inhibits the proton pump and the passive H+ conductance with about the same time course, at concentrations that have no effect on cytoplasmic streaming. N-Ethylmaleimide, a sulfhydryl reagent which is small and relatively nonpolar, also inhibits both pumping and passive conductance of H+. However, it also inhibits cytoplasmic streaming with about the same time course, and therefore could not be considered a specific ATPase inhibitor. p-Chloromercuribenzene sulfonate (PCMBS), a sulfhydryl reagent which is large and charged and hence less able to penetrate the membrane, does not inhibit pumping or conductance at low concentration. At high concentration, PCMBS sometimes inhibits pumping without affecting H+ conductance, but since streaming is also inhibited, the effect on the pump cannot be said to be specific. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water soluble carbodiimide, weakly inhibits both pump and conductance, apparently specifically. PMID:16664807

Recombinant factor concentrates may increase inhibitor development: a single centre cohort study.

PubMed

Strauss, T; Lubetsky, A; Ravid, B; Bashari, D; Luboshitz, J; Lalezari, S; Misgav, M; Martinowitz, U; Kenet, G

2011-07-01

Recent reports have raised concerns regarding potential risk factors for inhibitor development. In Israel, all haemophilia patients (n = 479) are followed by the National Hemophilia Center. Most children are neonatally exposed to factor concentrate (due to circumcision performed at the age of 8 days). The impact of early exposure and recombinant FVIII products (rFVIII) administration (approved in Israel since 1996) upon inhibitor occurrence in our cohort of haemophilia A (HA) patients was analysed. Two hundred ninety-two consecutive paediatric cases with a first symptomatic onset of HA were enrolled and followed over a median time of 7 years [min-max: 9 months to 17 years]. Study endpoint was inhibitor development against factor VIII. In addition, the treatment regimens applied, i.e. bolus administration or 'continuous infusion' and the family history of inhibitor development were investigated. During the follow-up period 31/292 children (10.6%) developed high titre inhibitors. Inhibitors occurred in 14/43 (32.5%) HA patients neonatally exposed to rFVIII, as compared to 22/249 previously treated with Plasma Derived (PD) products (8.8%). The odds ratio for inhibitor formation in rFVIII treated HA patients was 3.43 (95% CI: 1.36-8.65). Transient inhibitor evolved among 2/43 paediatric HA patients, only among those treated with rFVIII. The risk of inhibitor detection significantly increased among HA children treated by continuous infusion (P = 0.025). Our experience shows that the risk of inhibitor formation may be increased by early exposure to recombinant concentrates. The multiple variables affecting inhibitor incidence deserve further attention by larger prospective studies. © 2011 Blackwell Publishing Ltd.

Concentration and Separation of Active Proteins from Potato Industry Waste Based on Low-Temperature Evaporation and Ethanol Precipitation

PubMed Central

Ahokas, Mikko; Järvinen, Juho; Toivanen, Juho; Tanskanen, Juha P.

2017-01-01

Purpose. Potato fruit juice, a residue of starch industry, contains up to 2.5% [w/w] of proteins that are potentially valuable raw-materials of food, cosmetic, and pharma industries. The recovery of protein from the potato fruit juice is limited by the lack of industrially feasible concentration and separation technologies. The present research thus aimed at development of such process for the separation of active protease inhibitors from potato fruit juice. Methods. Low temperature mechanical vapor recompression evaporation was applied for concentration of potato fruit juice followed by ethanol precipitation for recovery of active proteins. The effects of precipitation temperature and precipitative agents were investigated employing response surface modeling methodology. Results. Concentration of potato fruit juice by evaporation was successful without loss of trypsin inhibition activity. Precipitation using 6.5 M ethanol at low temperature (0–+4°C) was found suitable for the recovery of active protease inhibitors from the concentrate. Piloting at starch industry yielded 50% of total proteins, with a high quantity of active protease inhibitors and a minor inclusion of other proteins. Conclusion. Concentration by low-temperature evaporation, followed by ethanol precipitation of protease inhibitors at optimized temperature, is an attractive option for valorization of potato fruit juice. PMID:28299232

AGEs Induce Apoptosis in Rat Osteoblast Cells by Activating the Caspase-3 Signaling Pathway Under a High-Glucose Environment In Vitro.

PubMed

Liu, Jiaqiang; Mao, Jing; Jiang, Yi; Xia, Lunguo; Mao, Lixia; Wu, Yong; Ma, Pan; Fang, Bing

2016-03-01

Advanced glycation end products (AGEs) accumulate under high-glucose conditions and affect the healing of bone damage through various pathways; however, the detail mechanisms underlying these changes are unknown. In this study, we investigated the effects of AGEs on the apoptosis of in vitro-cultured rat osteoblasts under high-glucose conditions and explored the underlying mechanisms of these effects. First, we cultured rat osteoblasts and determined the accumulation of AGEs in the culture medium under high-glucose conditions. Then, we cultured rat osteoblasts under a high glucose concentration (35 mM), a normal glucose concentration (5.5 mM), and a normal glucose concentration (5.5 mM) in the presence of AGEs. We examined the effects of high glucose and AGEs on the apoptosis of rat osteoblasts at different time points and further analyzed the activity and changes in the levels of procaspase-3, caspase-3, and the caspase-3 substrate poly ADP-ribose polymerase (PARP). Finally, we added sRAGE (soluble RAGE) (an AGE inhibitor) or DEVD (a caspase-3 inhibitor) to each culture group and examined apoptosis under each culture condition and the changes in the levels of procaspase-3, caspase-3, and its substrate PARP. The results showed that the high-glucose condition and the addition of AGEs increased the apoptosis of rat osteoblast cells and simultaneously increased the activity and quantity of caspase-3. These increases could be inhibited by the AGE inhibitor sRAGE or the caspase-3 inhibitor DEVD. The above results demonstrate that high-glucose conditions lead to the accumulation of AGEs and activation of the caspase-3 signaling pathway, resulting in the increased apoptosis of cultured rat osteoblast cells.

Were kinetics of Archean calcium carbonate precipitation related to oxygen concentration?

NASA Technical Reports Server (NTRS)

Sumner, D. Y.; Grotzinger, J. P.

1996-01-01

Archean carbonates commonly contain decimetre- to metre-thick beds consisting entirely of fibrous calcite and neomorphosed fibrous aragonite that precipitated in situ on the sea floor. The fact that such thick accumulations of precipitated carbonate are rare in younger marine carbonates suggests an important change in the modes of calcium carbonate precipitation through time. Kinetics of carbonate precipitation depend on the concentration of inhibitors to precipitation that reduce crystallization rates and crystal nuclei formation, leading to kinetic maintenance of supersaturated solutions. Inhibitors also affect carbonate textures by limiting micrite precipitation and promoting growth of older carbonate crystals on the sea floor. Fe2+, a strong calcite-precipitation inhibitor, is thought to have been present at relatively high concentrations in Archean seawater because oxygen concentrations were low. The rise in oxygen concentration at 2.2-1.9 Ga led to the removal of Fe2+ from seawater and resulted in a shift from Archean facies, which commonly include precipitated beds, to Proterozoic facies, which contain more micritic sediment and only rare precipitated beds.

Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries

PubMed Central

Watt, Eric D.; Hornung, Michael W.; Hedge, Joan M.; Judson, Richard S.; Crofton, Kevin M.; Houck, Keith A.; Simmons, Steven O.

2016-01-01

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using guaiacol as a substrate to confirm the activity profiles of putative TPO inhibitors. This effort represents the most extensive TPO inhibition screening campaign to date and illustrates a tiered screening approach that focuses resources, maximizes assay throughput, and reduces animal use. PMID:26884060

Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors.

PubMed

Esteva-Font, Cristina; Cil, Onur; Phuan, Puay-Wah; Su, Tao; Lee, Sujin; Anderson, Marc O; Verkman, A S

2014-09-01

Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ∼1 μM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by ∼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias. © FASEB.

Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology.

PubMed

Schmedes, Sarah; Marshall, Pamela; King, Jonathan L; Budowle, Bruce

2013-07-01

Various types of biological samples present challenges for extraction of DNA suitable for subsequent molecular analyses. Commonly used extraction methods, such as silica membrane columns and phenol-chloroform, while highly successful may still fail to provide a sufficiently pure DNA extract with some samples. Synchronous coefficient of drag alteration (SCODA), implemented in Boreal Genomics' Aurora Nucleic Acid Extraction System (Boreal Genomics, Vancouver, BC), is a new technology that offers the potential to remove inhibitors effectively while simultaneously concentrating DNA. In this initial study, SCODA was tested for its ability to remove various concentrations of forensically and medically relevant polymerase chain reaction (PCR) inhibitors naturally found in tissue, hair, blood, plant, and soil samples. SCODA was used to purify and concentrate DNA from intentionally contaminated DNA samples containing known concentrations of hematin, humic acid, melanin, and tannic acid. The internal positive control (IPC) provided in the Quantifiler™ Human DNA Quantification Kit (Life Technologies, Foster City, CA) and short tandem repeat (STR) profiling (AmpFℓSTR® Identifiler® Plus PCR Amplification Kit; Life Technologies, Foster City, CA) were used to measure inhibition effects and hence purification. SCODA methodology yielded overall higher efficiency of purification of highly contaminated samples compared with the QIAquick® PCR Purification Kit (Qiagen, Valencia, CA). SCODA-purified DNA yielded no cycle shift of the IPC for each sample and yielded greater allele percentage recovery and relative fluorescence unit values compared with the QIAquick® purification method. The Aurora provided an automated, minimal-step approach to successfully remove inhibitors and concentrate DNA from challenged samples.

Tiered High-Throughput Screening Approach to Identify ...

EPA Pesticide Factsheets

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

Comparison of Monoamine Oxidase Inhibitors in Decreasing Production of the Autotoxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde in PC12 Cells

PubMed Central

Jinsmaa, Yunden; Sullivan, Patti; Holmes, Courtney; Kopin, Irwin J.; Sharabi, Yehonatan

2016-01-01

According to the catecholaldehyde hypothesis, the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to the loss of nigrostriatal dopaminergic neurons in Parkinson’s disease. Monoamine oxidase-A (MAO-A) catalyzes the conversion of intraneuronal dopamine to DOPAL and may serve as a therapeutic target. The “cheese effect”—paroxysmal hypertension evoked by tyramine-containing foodstuffs—limits clinical use of irreversible MAO-A inhibitors. Combined MAO-A/B inhibition decreases DOPAL production in rat pheochromocytoma PC12 cells, but whether reversible MAO-A inhibitors or MAO-B inhibitors decrease endogenous DOPAL production is unknown. We compared the potencies of MAO inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and auto-oxidation. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All three drugs also increased dopamine and norepinephrine, decreased 3,4-dihydroxyphenylalanine, and increased cysteinyl-dopamine concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine spontaneous oxidation. In conclusion, clorgyline, rasagiline, and selegiline decrease production of endogenous DOPAL. At relatively high concentrations, the latter drugs probably lose their selectivity for MAO-B. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in Parkinson’s disease. PMID:26574516

Kinetics of suicide substrates. Practical procedures for determining parameters.

PubMed Central

Waley, S G

1985-01-01

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error. PMID:4004802

Density-dependent regulation of growth of BSC-1 cells in cell culture: growth inhibitors formed by the cells.

PubMed Central

Holley, R W; Armour, R; Baldwin, J H

1978-01-01

Inhibitors formed by a monkey epithelial cell line, BSC-1, play an important role in limiting growth at high cell densities. At least three inhibitors are formed: lactic acid, ammonia, and an unidentified inhibitor that may be an unstable protein. The unidentified inhibitor is destroyed by shaking the conditioned medium, by bubbling gas through the medium, or by heating or storing the medium in the absence of cells. The concentrations of lactic acid and ammonia that accumulate in conditioned medium inhibit growth when added to fresh medium. These results, together with earlier studies, indicate that density-dependent regulation of growth of BSC-1 cells results from the combined effects of (a) inhibitors formed by the cells, (b) decreased availability of receptor sites for serum growth factors as the cells become crowded, and (c) limiting concentrations of low molecular weight nutrients in the medium. In contrast, density-dependent regulation of growth in 3T3 mouse embryo fibroblasts results almost entirely from inactivation of serum factors. PMID:273914

Experimental and theoretical studies of Schiff bases as corrosion inhibitors.

PubMed

Jamil, Dalia M; Al-Okbi, Ahmed K; Al-Baghdadi, Shaimaa B; Al-Amiery, Ahmed A; Kadhim, Abdulhadi; Gaaz, Tayser Sumer; Kadhum, Abdul Amir H; Mohamad, Abu Bakar

2018-02-05

Relatively inexpensive, stable Schiff bases, namely 3-((4-hydroxybenzylidene)amino)-2-methylquinazolin-4(3H)-one (BZ3) and 3-((4-(dimethylamino)benzylidene)amino)-2-methylquinazolin-4(3H)-one (BZ4), were employed as highly efficient inhibitors of mild steel corrosion by corrosive acid. The inhibition efficiencies were estimated based on weight loss method. Moreover, scanning electron microscopy was used to investigate the inhibition mechanism. The synthesized Schiff bases were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy and micro-elemental analysis. The inhibition efficiency depends on three factors: the amount of nitrogen in the inhibitor, the inhibitor concentration and the inhibitor molecular weight. Inhibition efficiencies of 96 and 92% were achieved with BZ4 and BZ3, respectively, at the maximum tested concentration. Density functional theory calculations of BZ3 and BZ4 were performed to compare the effects of hydroxyl and N,N-dimethylamino substituents on the inhibition efficiency, providing insight for designing new molecular structures that exhibit enhanced inhibition efficiencies.

ELISA analysis of soybean trypsin inhibitors in processed foods.

PubMed

Brandon, D L; Bates, A H; Friedman, M

1991-01-01

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

PubMed

Li, Yun-Cheng; Mitsumasu, Kanako; Gou, Zi-Xi; Gou, Min; Tang, Yue-Qin; Li, Guo-Ying; Wu, Xiao-Lei; Akamatsu, Takashi; Taguchi, Hisataka; Kida, Kenji

2016-02-01

Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol.

Bonding-Compatible Corrosion Inhibitor for Rinsing Metals

NASA Technical Reports Server (NTRS)

Saunders, C. R.; Wurth, L. A.; Radar, A.

2005-01-01

A corrosion-inhibiting mixture of compounds has been developed for addition to the water used to rinse metal parts that have been cleaned with aqueous solutions in preparation for adhesive bonding of the metals to rubber and rubber-like materials. Prior to the development of this corrosion inhibitor, the parts (made, variously, of D6AC steel and 7075-T73 aluminum) were rinsed by deionized water, which caused corrosion in some places on the steel parts especially in such occluded places as sealing surfaces and threaded blind holes. An integral part of the particular cleaning process is the deposition of a thin layer of silicates and silane primers that increase the strength of the adhesive bond. The corrosion inhibitor is formulated, not only to inhibit corrosion of both D6AC steel and 7075- T73 aluminum, but also to either increase or at least not reduce the strength of the adhesive bond to be formed subsequently. The corrosion inhibitor is a mixture of sodium silicate and sodium tetraborate. The sodium silicate functions as both a corrosion inhibitor and a bond-strength promoter in association with the silane primers. The sodium tetraborate buffers the rinse solution at the optimum pH and functions as a secondary corrosion inhibitor for the steel. For a given application, the concentrations of sodium silicate and sodium tetraborate must be chosen in a compromise among the needs to inhibit corrosion of steel, inhibit corrosion of aluminum, and minimize cosmetic staining of both steel and aluminum. Concentrations of sodium silicate in excess of 150 parts of silicon per million parts of solution (ppm Si) have been determined to enhance inhibition of corrosion; unfortunately, because of the alkalinity of sodium silicate, even a small concentration can raise the pH of the rinse solution to such a level that aluminum becomes corroded despite the inhibiting effect. The pH of a solution that contains a high concentration of sodium silicate can be decreased by adding sodium tetraborate. On the other hand, the addition of sodium tetraborate increases the concentration of dissolved solids to such a high level that cosmetic staining becomes an issue.

The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines.

PubMed

Kolbinger, Fiona R; Koeneke, Emily; Ridinger, Johannes; Heimburg, Tino; Müller, Michael; Bayer, Theresa; Sippl, Wolfgang; Jung, Manfred; Gunkel, Nikolas; Miller, Aubry K; Westermann, Frank; Witt, Olaf; Oehme, Ina

2018-06-09

High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.).

PubMed

Asano, N; Yamashita, T; Yasuda, K; Ikeda, K; Kizu, H; Kameda, Y; Kato, A; Nash, R J; Lee, H S; Ryu, K S

2001-09-01

New polyhydroxylated alkaloids, (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxypyrrolidine-N-propionamide from the root bark of Morus alba L., and 4-O-alpha-D-galactopyranosyl-calystegine B(2) and 3 beta,6 beta-dihydroxynortropane from the fruits, were isolated by column chromatography using a variety of ion-exchange resins. Fifteen other polyhydroxylated alkaloids were also isolated. 1-Deoxynojirimycin, a potent alpha-glucosidase inhibitor, was concentrated 2.7-fold by silkworms feeding on mulberry leaves. Some alkaloids contained in mulberry leaves were potent inhibitors of mammalian digestive glycosidases but not inhibitors of silkworm midgut glycosidases, suggesting that the silkworm has enzymes specially adapted to enable it to feed on mulberry leaves. The possibility of preventing the onset of diabetes and obesity using natural dietary supplements containing 1-deoxynojirimycin and other alpha-glucosidase inhibitors in high concentration is of great potential interest.

Inhibitors of Leishmania mexicana CRK3 Cyclin-Dependent Kinase: Chemical Library Screen and Antileishmanial Activity

PubMed Central

Grant, Karen M.; Dunion, Morag H.; Yardley, Vanessa; Skaltsounis, Alexios-Leandros; Marko, Doris; Eisenbrand, Gerhard; Croft, Simon L.; Meijer, Laurent; Mottram, Jeremy C.

2004-01-01

The CRK3 cyclin-dependent kinase of Leishmania has been shown by genetic manipulation of the parasite to be essential for proliferation. We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target. A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors. Twenty-seven potent CRK3 inhibitors were discovered and screened against Leishmania donovani amastigotes in vitro. Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED50) of less than 10 μM. These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine. The paullones and staurosporine derivatives were toxic to macrophages. The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations. The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED50s for these inhibitors were 5.8 and 7.6 μM, respectively. In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control. Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in Leishmania and provides pharmacophores for further drug development. PMID:15273118

Effect of α1-acid glycoprotein on the intracellular accumulation of the HIV protease inhibitors saquinavir, ritonavir and indinavir in vitro

PubMed Central

Jones, K; Hoggard, P G; Khoo, S; Maher, B; Back, D J

2001-01-01

Aims Since α1-acid glycoprotein (AGP) levels may be raised during HIV infection, we have examined in vitro the effect of increasing the concentration of AGP on the intracellular accumulation of the HIV protease inhibitors saquinavir (SQV), ritonavir (RTV) and indinavir (IDV). Methods U937 cells (5 × 106 cells in 5 ml RPMI growth medium) were incubated at 37 °C for 18 h with [14C]-SQV (0.1 µCi), [3H]-RTV and [3H]-IDV (0.135 µCi) to a final concentration of 1 µm in the presence of 0, 0.5 and 2.0 mg ml−1 AGP. Following extraction in 60% methanol the intracellular drug concentration was determined by liquid scintillation counting. Results Binding to AGP (2.0 mg ml−1) reduced the mean intracellular concentration of SQV from 31.5 µm to 7.4 µm (P < 0.0001; 95% CI 19.4–28.8). RTV concentration was also reduced (8.8 µm to 1.6 µm; P < 0.0001; 95% CI 5.4–9.0) as was the concentration of IDV (3.0 µm to 1.5 µm; P < 0.0001; 95% CI 1.1–1.9). Conclusions Reduced intracellular protease inhibitor concentrations in the presence of increasing concentrations of AGP will certainly impact on the antiviral activity in vitro. However, since protease inhibitors are high clearance drugs, free drug concentration will likely remain unaffected in the presence of elevated AGP during chronic oral dosing although there will be an increase in total plasma drug concentration. PMID:11167671

DMH1, a Highly Selective Small Molecule BMP Inhibitor Promotes Neurogenesis of hiPSCs: Comparison of PAX6 and SOX1 Expression during Neural Induction

PubMed Central

2012-01-01

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-β1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors. PMID:22860217

Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay.

PubMed

Cai, Long-Fei; Zhu, Ying; Du, Guan-Sheng; Fang, Qun

2012-01-03

We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of β-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold. © 2011 American Chemical Society

Kinetic assay for high-throughput screening of in vitro transthyretin amyloid fibrillogenesis inhibitors.

PubMed

Dolado, Ignacio; Nieto, Joan; Saraiva, Maria João M; Arsequell, Gemma; Valencia, Gregori; Planas, Antoni

2005-01-01

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cary, Robert E.

2015-12-08

Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cary, Robert B.

Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

Browning inhibition mechanisms by cysteine, ascorbic acid and citric acid, and identifying PPO-catechol-cysteine reaction products.

PubMed

Ali, Hussein M; El-Gizawy, Ahmed M; El-Bassiouny, Rawia E I; Saleh, Mahmoud A

2015-06-01

The titled compounds were examined as PPO inhibitors and antibrowning agents; their various mechanisms were investigated and discussed. All compounds reduced significantly both the browning process and PPO activity. Browning index gave strong correlation with PPO activity (r(2) = 0.96, n = 19) indicating that the browning process is mainly enzymatic. Ascorbic acid could reduce the formed quinone instantly to the original substrate (catechol) at high concentration (>1.5 %) while at lower concentrations acted as competitive inhibitor (KI = 0.256 ± 0.067 mM). Cysteine, at higher concentrations (≥1.0 %), reacted with the resulted quinone to give a colorless products while at the low concentrations, cysteine worked as competitive inhibitor (KI = 1.113 ± 0.176 mM). Citric acid acted only as PPO non-competitive inhibitor with KI = 2.074 ± 0.363 mM. The products of PPO-catechole-cysteine reaction could be separation and identification by LC-ESI-MS. Results indicated that the product of the enzymatic oxidation of catechol, quinone, undergoes two successive nucleophilic attacks by cysteine thiol group. Cysteine was condensed with the resulted mono and dithiocatechols to form peptide side chains.

Predictive blood plasma biomarkers for EGFR inhibitor-induced skin rash.

PubMed

Hichert, Vivien; Scholl, Catharina; Steffens, Michael; Paul, Tanusree; Schumann, Christian; Rüdiger, Stefan; Boeck, Stefan; Heinemann, Volker; Kächele, Volker; Seufferlein, Thomas; Stingl, Julia

2017-05-23

Epidermal growth factor receptor overexpression in human cancer can be effectively targeted by drugs acting as specific inhibitors of the receptor, like erlotinib, gefitinib, cetuximab and panitumumab. A common adverse effect is a typical papulopustular acneiform rash, whose occurrence and severity are positively correlated with overall survival in several cancer types. We studied molecules involved in epidermal growth factor receptor signaling which are quantifiable in plasma, with the aim of identifying biomarkers for the severity of rash. With a predictive value for the rash these biomarkers may also have a prognostic value for survival and disease outcome.The concentrations of amphiregulin, hepatocyte growth factor (HGF) and calcidiol were determined by specific enzyme-linked immunosorbent assays in plasma samples from 211 patients.We observed a significant inverse correlation between the plasma concentration of HGF and overall survival in patients with an inhibitor-induced rash (p-value = 0.0075; mean overall survival low HGF: 299 days, high HGF: 240 days) but not in patients without rash. The concentration of HGF was also significantly inversely correlated with severity of rash (p-value = 0.00124).High levels of HGF lead to increased signaling via its receptor MET, which can activate numerous pathways which are normally also activated by epidermal growth factor receptor. Increased HGF/MET signaling might compensate the inhibitory effect of epidermal growth factor receptor inhibitors in skin as well as tumor cells, leading to less severe skin rash and decreased efficacy of the anti-tumor therapy, rendering the plasma concentration of HGF a candidate for predictive biomarkers.

Electrochemical research on corrosion behavior of A3 steel in compound sodium molybadate and organic inhibitor solution

NASA Astrophysics Data System (ADS)

Sun, C. X.; Chen, Y. M.; Xu, H. W.; Zhang, M.; Chen, M.; Xue, M.; Wu, J. Y.; Huang, C. S.

2015-07-01

The electrochemical corrosion behavior of A3 in compound sodium molybdate and organic inhibitor solution was tested by the electrochemical workstation method. The concentration of the compound inhibitor set to range 250 mg/L to 3000 mg/L. The polarization curve results of A3 in different concentration inhibitor solutions show that the inhibitor markedly represses the anodic processes. The EIS has two time constant. The extreme concentration is 1500 mg/L.

Sustained release of the CCR5 inhibitors CMPD167 and maraviroc from vaginal rings in rhesus macaques.

PubMed

Malcolm, R Karl; Veazey, Ronald S; Geer, Leslie; Lowry, Deborah; Fetherston, Susan M; Murphy, Diarmaid J; Boyd, Peter; Major, Ian; Shattock, Robin J; Klasse, Per Johan; Doyle, Lara A; Rasmussen, Kelsi K; Goldman, Laurie; Ketas, Thomas J; Moore, John P

2012-05-01

Antiretroviral entry inhibitors are now being considered as vaginally administered microbicide candidates for the prevention of the sexual transmission of human immunodeficiency virus. Previous studies testing the entry inhibitors maraviroc and CMPD167 in aqueous gel formulations showed efficacy in the macaque challenge model, although protection was highly dependent on the time period between initial gel application and subsequent challenge. In this paper, we describe the sustained release of maraviroc and CMPD167 from matrix-type silicone elastomer vaginal rings both in vitro and in vivo. Both inhibitors were released continuously during 28 days from rings in vitro at rates of 100 to 2,500 μg/day. In 28-day pharmacokinetic studies in rhesus macaques, the compounds were measured in the vaginal fluid and vaginal tissue; steady-state fluid concentrations were ~10(6)-fold greater than the 50% inhibitory concentrations (IC(50)s) for simian human immunodeficiency virus 162P3 inhibition in macaque lymphocytes in vitro. Plasma concentrations for both compounds were very low. The pretreatment of macaques with Depo-Provera (DP), which is commonly used in macaque challenge studies, was shown to significantly modify the biodistribution of the inhibitors but not the overall amount released. Vaginal fluid and tissue concentrations were significantly decreased while plasma levels increased with DP pretreatment. These observations have implications for designing macaque challenge experiments and also for ring performance during the human female menstrual cycle.

Sustained Release of the CCR5 Inhibitors CMPD167 and Maraviroc from Vaginal Rings in Rhesus Macaques

PubMed Central

Veazey, Ronald S.; Geer, Leslie; Lowry, Deborah; Fetherston, Susan M.; Murphy, Diarmaid J.; Boyd, Peter; Major, Ian; Shattock, Robin J.; Klasse, Per Johan; Doyle, Lara A.; Rasmussen, Kelsi K.; Goldman, Laurie; Ketas, Thomas J.; Moore, John P.

2012-01-01

Antiretroviral entry inhibitors are now being considered as vaginally administered microbicide candidates for the prevention of the sexual transmission of human immunodeficiency virus. Previous studies testing the entry inhibitors maraviroc and CMPD167 in aqueous gel formulations showed efficacy in the macaque challenge model, although protection was highly dependent on the time period between initial gel application and subsequent challenge. In this paper, we describe the sustained release of maraviroc and CMPD167 from matrix-type silicone elastomer vaginal rings both in vitro and in vivo. Both inhibitors were released continuously during 28 days from rings in vitro at rates of 100 to 2,500 μg/day. In 28-day pharmacokinetic studies in rhesus macaques, the compounds were measured in the vaginal fluid and vaginal tissue; steady-state fluid concentrations were ∼106-fold greater than the 50% inhibitory concentrations (IC50s) for simian human immunodeficiency virus 162P3 inhibition in macaque lymphocytes in vitro. Plasma concentrations for both compounds were very low. The pretreatment of macaques with Depo-Provera (DP), which is commonly used in macaque challenge studies, was shown to significantly modify the biodistribution of the inhibitors but not the overall amount released. Vaginal fluid and tissue concentrations were significantly decreased while plasma levels increased with DP pretreatment. These observations have implications for designing macaque challenge experiments and also for ring performance during the human female menstrual cycle. PMID:22330914

A host defense role for a natural antiviral substance in the nervous system.

PubMed

Baron, S; Chopra, A K; Coppenhaver, D H; Gelman, B B; Poast, J; Singh, I P

1998-05-15

The pathogenesis of virus infections of the nervous system (NS) is regulated by host defenses. The defensive role of a major constitutive antiviral substance was studied by determining its distribution in the human nervous system, its concentration and the ability of this viral inhibitor to protect mice against viral infection. The 4000 kDa inhibitor complex in the human nervous system was detected in brain gray and white matter, spinal cord, and sciatic nerve but not in human cerebrospinal fluid. The inhibitor was found in the extracellular medium incubated with minced murine brain. The inhibitory titer ranged from approximately 50 to 200 antiviral units per gram against polio 1, Semliki Forest, Banzi, mengo, Newcastle disease and herpes simplex 1 viruses. The inhibitor is composed of lipid and essential protein and carbohydrate moieties as determined by enzymatic inactivation. Protection of inhibitor-treated mice was demonstrated against both an alphavirus and a picornavirus. Thus a natural defensive role for the broadly antiviral inhibitor is suggested by its constitutively high concentration, wide distribution in nervous system tissues, presence in extracellular fluid and its ability to provide protection in infected mice.

Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA-Encoded Chemical Library.

PubMed

Cuozzo, John W; Centrella, Paolo A; Gikunju, Diana; Habeshian, Sevan; Hupp, Christopher D; Keefe, Anthony D; Sigel, Eric A; Soutter, Holly H; Thomson, Heather A; Zhang, Ying; Clark, Matthew A

2017-05-04

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design and synthesis of phosphoryl-substituted diphenylpyrimidines (Pho-DPPYs) as potent Bruton's tyrosine kinase (BTK) inhibitors: Targeted treatment of B lymphoblastic leukemia cell lines.

PubMed

Ge, Yang; Yang, Haijun; Wang, Changyuan; Meng, Qiang; Li, Lei; Sun, Huijun; Zhen, Yuhong; Liu, Kexin; Li, Yanxia; Ma, Xiaodong

2017-01-15

A family of phosphoryl-substituted diphenylpyrimidine derivatives (Pho-DPPYs) were synthesized and biologically evaluated as potent BTK inhibitors in this study. Compound 7b was found to markedly inhibit BTK activity at concentrations of 0.82nmol/L, as well as to suppress the proliferations of B-cell leukemia cell lines (Ramos and Raji) expressing high levels of BTK at concentrations of 3.17μM and 6.69μM. Moreover, flow cytometry analysis results further indicated that 7b promoted cell apoptosis to a substantial degree. In a word, compound 7b is a promising BTK inhibitor for the treatment of B-cell lymphoblastic leukemia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Prompt immune tolerance induction at inhibitor diagnosis regardless of titre may increase overall success in haemophilia A complicated by inhibitors: experience of two U.S. centres.

PubMed

Nakar, C; Manco-Johnson, M J; Lail, A; Donfield, S; Maahs, J; Chong, Y; Blades, T; Shapiro, A

2015-05-01

Current guidelines recommend delaying the start of immune tolerance induction (ITI) until the inhibitor titre is <10 Bethesda units (BU) to improve success. This study was conducted to evaluate ITI outcome relative to time to start ITI from inhibitor detection irrespective of inhibitor titre. Data were retrospectively collected from two U.S. haemophilia treatment centres (HTCs) on subjects with severe/moderate factor VIII (FVIII) deficiency with inhibitors who underwent ITI. Outcomes were defined pragmatically: success--negative inhibitor titre and ability to use FVIII concentrate for treatment/bleed prevention; partial success--inhibitor titre 1 to <5 BU with ability to use FVIII concentrate for treatment of bleeding; failure--ITI ongoing >3 years without achieving success/partial success, or ITI discontinuation. Fifty-eight subjects were included; 32 of 39 (82%) with high-responding inhibitor (HRI) achieved success, 7 failed. HRI subjects were subdivided based on ITI start time: 23/39 subjects started within 1 month of detection and 22/23 (96%) achieved success. Of these 23, 13 started ITI with an inhibitor titre ≥10 BU; all were successes. Eleven of 39 HRI subjects had an interval >6 months until ITI start; 7 (64%) achieved success. Time from inhibitor detection to ITI start may play a critical role in outcome. A titre ≥10 BU at ITI start did not influence outcome in subjects when ITI was initiated within 1 month of detection. Prompt ITI should be considered a viable therapeutic option in newly identified patients with inhibitors regardless of current inhibitor titre. © 2014 John Wiley & Sons Ltd.

First Penicillin-Binding Protein Occupancy Patterns of β-Lactams and β-Lactamase Inhibitors in Klebsiella pneumoniae.

PubMed

Sutaria, Dhruvitkumar S; Moya, Bartolome; Green, Kari B; Kim, Tae Hwan; Tao, Xun; Jiao, Yuanyuan; Louie, Arnold; Drusano, George L; Bulitta, Jürgen B

2018-06-01

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite β-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae , PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using β-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After β-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC 50 ]) were reported as the β-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by β-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC 50 , <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC 50 , <0.0075 mg/liter) and avibactam (IC 50 , 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC 50 , 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC 50 , 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC 50 , 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double β-lactam and β-lactam-β-lactamase inhibitor combinations. Copyright © 2018 American Society for Microbiology.

The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.

PubMed

Hansson, K M; Nielsen, S; Elg, M; Deinum, J

2014-10-01

Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. In the present work the specificity of CTI for factor (F) XIIa is questioned. In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3 ± 1.5 μm CTI (assay concentration 2.4 μm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki  = 8.1 ± 0.3 μm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF concentrations, no effect of CTI (plasma concentrations of 4.4 and 13.6 μm CTI, 25 resp. 100 mg L(-1) in blood) was found with ≥ 1 pm TF. At ≤ 0.1 pm TF in the FOR whole blood assay the coagulation time (CT) concentration dependently increased while the plasma CT became longer than the observation time. To avoid inhibition of FXIa and the thrombin feedback loop we recommend that for coagulation assays the concentration of CTI in blood should be below 20 mg L(-1) (1.6 μm) and in plasma below 3 μm. © 2014 International Society on Thrombosis and Haemostasis.

Mipomersen and lomitapide: Two new drugs for the treatment of homozygous familial hypercholesterolemia.

PubMed

Gouni-Berthold, Ioanna; Berthold, Heiner K

2015-05-01

Familial hypercholesterolemia (FH) is a disease associated with very high plasma concentrations of low-density lipoprotein cholesterol (LDL-C) and premature cardiovascular disease. It is difficult in these high risk patients, exposed lifelong to very high LDL-C, to reach target LDL-C concentrations, which require >50% LDL-C reduction, even when on maximally tolerated statin therapy and on apheresis if available. Therefore, there is an unmet need for new therapeutic options for these patients. In 2013 two new drugs were approved for the treatment of homozygous FH, namely the apolipoprotein B synthesis inhibitor mipomersen and the microsomal transfer protein inhibitor lomitapide. Objective of this narrative review is to discuss the available evidence on the safety and efficacy profile of these new drugs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

n-Octyl gallate as inhibitor of pyruvate carboxylation and lactate gluconeogenesis.

PubMed

Eler, Gabrielle Jacklin; Santos, Israel Souza; de Moraes, Amarilis Giaretta; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

2015-04-01

The alkyl gallates are found in several natural and industrial products. In the latter products, these compounds are added mainly for preventing oxidation. In the present work, the potencies of methyl gallate, n-propyl gallate, n-pentyl gallate, and n-octyl gallate as inhibitors of pyruvate carboxylation and lactate gluconeogenesis were evaluated. Experiments were done with isolated mitochondria and the isolated perfused rat liver. The potency of the gallic acid esters as inhibitors of pyruvate carboxylation in isolated mitochondria obeyed the following decreasing sequence: n-octyl gallate > n-pentyl gallate > n-propyl gallate > methyl gallate. A similar sequence of decreasing potency for lactate gluconeogenesis inhibition in the perfused liver was found in terms of the portal venous concentration. Both actions correlate with the lipophilicity of the compounds. The effects are harmful at high concentrations. At appropriate concentrations, however, octyl gallate should act therapeutically because its inhibitory action on gluconeogenesis will contribute further to its proposed antihyperglycemic effects. © 2014 Wiley Periodicals, Inc.

Cytochrome P450 3A4 in vivo ketoconazole competitive inhibition: determination of Ki and dangers associated with high clearance drugs in general.

PubMed

Boxenbaum, H

1999-01-01

Assuming complete hepatic substrate metabolism and system linearity, quantitative effects of in vivo competitive inhibition are investigated. Following oral administration of a substrate in the presence of a competitive inhibitor, determination of the inhibition constant (Ki) is possible when plasma concentration-time profiles of both substrate and inhibitor are available. When triazolam is the P450 3A4 substrate and ketoconazole the competitive inhibitor, Ki approximately 1.2 microg/mL in humans. The effects of competitive inhibition can be divided into two components: first-pass hepatic metabolism and systemic metabolism. For drugs with high hepatic extraction ratios, the impact of competitive inhibition on hepatic first-pass metabolism can be particularly dramatic. For example, human terfenadine hepatic extraction goes from 95% in the absence of a competitive inhibitor to 35% in the presence of one (ketoconazole, 200 mg po Q 12 h dosed to steady-state). First-pass extraction therefore goes from 5% in the absence of the inhibitor to 65% in its presence. The combined effect on first-pass and systemic metabolism produces an approximate 37 fold increase in terfenadine area under the plasma concentration-time curve. Assuming intact drug is active and/or toxic, development of metabolized drugs with extensive first-pass metabolism should be avoided if possible, since inhibition of metabolism may lead to profound increases in exposure.

Hemophilia and von Willebrand's disease: 2. Management. Association of Hemophilia Clinic Directors of Canada.

PubMed Central

1995-01-01

OBJECTIVE: To present current strategies for the treatment of hemophilia and von Willebrand's disease. OPTIONS: Prophylactic and corrective therapy with hemostatic and adjunctive agents: DDAVP (1-desamino-8-D-arginine vasopressin [desmopressin acetate]), recombinant coagulation products (human Factor VIII and human Factor VIIa) or virally inactivated plasma-derived products (high- or ultra-high-purity human Factor VIII or human Factor VIII concentrate containing von Willebrand factor activity, porcine Factor VIII, high-purity human Factor IX, human prothrombin-complex concentrate, human activated prothrombin-complex concentrate), adjunctive antifibrinolytic agents, topical thrombin and fibrin sealant. The induction of immune tolerance in patients in whom inhibitors develop should also be considered. OUTCOMES: Morbidity and quality of life associated with bleeding and treatment. EVIDENCE: Relevant clinical studies and reports published from 1974 to 1994 were examined. A search was conducted of our reprint files, MEDLINE, citations in the articles reviewed and references provided by colleagues. In the MEDLINE search the following terms were used singly or in combination: "hemophilia," "von Willebrand's disease," "Factor VIII," "Factor IX," "von Willebrand factor," "diagnosis," "management," "home care," "comprehensive care," "inhibitor," "AIDS," "hepatitis," "life expectancy," "complications," "practice guidelines," "consensus statement" and "controlled trial." The in-depth review included only articles written in English from North America and Europe that were relevant to human disease and pertinent to a predetermined outline. The availability of treatment products in Canada was also considered. VALUES: Minimizing morbidity and maximizing functional status and quality of life were given a high value. BENEFITS, HARMS AND COSTS: Proper prophylactic or early treatment with appropriate hemostatic agents minimizes morbidity and functional disability and improves quality of life. Economic gains are realized through the reduction of mortality and morbidity and their associated costs. The patient has a better opportunity to contribute to society through gainful employment and the fulfillment of social roles. Potential harms include HIV infection, hepatitis B, hepatitis C and the development of inhibitor antibodies to clotting-factor concentrates. The risk of viral transmission has been minimized through the development of procedures for the viral inactivation of plasma-derived clotting-factor concentrates and through the use of recombinant coagulation-factor concentrates and other non-plasma-derived hemostatic agents. RECOMMENDATIONS: DDAVP is the drug of choice for patients with mild hemophilia or type 1 or 2 (except 2B) von Willebrand's disease whose response to DDAVP in previous testing has been found to be adequate. Therapeutic blood components of choice include recombinant products and virally inactivated plasma-derived products. In Canada the recommended products are recombinant Factor VIII for hemophilia A, high-purity plasma-derived Factor IX for hemophilia B and plasma-derived Factor VIII concentrates containing adequate von Willebrand factor (e.g., Haemate P) for von Willebrand's disease. Dosages vary according to specific indications. Adjunctive antifibrinolytic agents, topical thrombin and fibrin sealant are useful for the treatment of oral or dental bleeds and localized bleeds in accessible sites. In patients with inhibitor antibodies, high-dose human or porcine Factor VIII is usually effective when the inhibitor titre is less than 5 Bethesda units/mL. In nonresponsive patients, or in those whose inhibitor titre is higher, "bypassing" agents (e.g., activated prothrombin-complex concentrate and recombinant Factor VIIa) are useful. Long-term management may include immune-tolerance induction.VALIDATION: These recommendations were reviewed and approved by the Association of Hemophilia Clinic Directors of Canada (AHCDC) and the Medical and Scientific Advisory Committee of the Canadian Hemophilia Society. No similar consensus statements or practice guidelines are available for comparison. SPONSORS: These recommendations were developed at the request of the Canadian Blood Agency, which funds the provision of all coagulation-factor concentrates for people with congenital bleeding disorders, and were developed and endorsed by the AHCDC and the Medical and Scientific Advisory Committee of the Canadian Hemophilia Society. PMID:7600466

Second-generation inhibitors demonstrate the involvement of p38 mitogen-activated protein kinase in post-transcriptional modulation of inflammatory mediator production in human and rodent airways.

PubMed

Birrell, Mark A; Wong, Sissie; McCluskie, Kerryn; Catley, Matthew C; Hardaker, Elizabeth L; Haj-Yahia, Saleem; Belvisi, Maria G

2006-03-01

The exact role of p38 mitogen-activated protein kinase (MAPK) in the expression of inflammatory cytokines is not clear; it may regulate transcriptionally, post-transcriptionally, translationally, or post-translationally. The involvement of one or more of these mechanisms has been suggested to depend on the particular cytokine, the cell type studied, and the specific stimulus used. Interpretation of some of the published data is further complicated by the use of inhibitors such as 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) used at single, high concentrations. The aim of this study was to determine the impact of two second-generation p38 MAPK inhibitors on the expression of a range of inflammatory cytokines at the gene and protein levels in human cultured cells. Similar assessment of the impact of these compounds on inflammatory cytokine expression in a preclinical in vivo model of airway inflammation was performed. The results in THP-1 cells and primary airway macrophages clearly show that protein expression is inhibited at much lower concentrations of inhibitor than are needed to impact on gene expression. In the rodent model, both compounds, at doses that cause maximal inhibition of cellular recruitment, inhibit tumor necrosis factor alpha (TNFalpha) protein production without impacting on nuclear factor kappaB pathway activation or TNFalpha gene expression. In summary, the data shown here demonstrate that, although at high compound concentrations there is some level of transcriptional regulation, the predominant role of p38 MAPK in cytokine production is at the translational level. These data question whether the effect of p38 inhibitors on gene transcription is related to their potential therapeutic role as anti-inflammatory compounds.

Isothiazolidinone (IZD) as a phosphoryl mimetic in inhibitors of the Yersinia pestis protein tyrosine phosphatase YopH

PubMed Central

Kim, Sung-Eun; Bahta, Medhanit; Lountos, George T.; Ulrich, Robert G.; Burke, Terrence R.; Waugh, David S.

2011-01-01

Isothiazolidinone (IZD) heterocycles can act as effective components of protein tyrosine phosphatase (PTP) inhibitors by simultaneously replicating the binding interactions of both a phosphoryl group and a highly conserved water molecule, as exemplified by the structures of several PTP1B–inhibitor complexes. In the first unambiguous demonstration of IZD interactions with a PTP other than PTP1B, it is shown by X-ray crystallography that the IZD motif binds within the catalytic site of the Yersinia pestis PTP YopH by similarly displacing a highly conserved water molecule. It is also shown that IZD-based bidentate ligands can inhibit YopH in a nonpromiscuous fashion at low micromolar concentrations. Hence, the IZD moiety may represent a useful starting point for the development of YopH inhibitors. PMID:21697602

An Additional Method for Analyzing the Reversible Inhibition of an 
Enzyme Using Acid Phosphatase as a Model.

PubMed

Baumhardt, Jordan M; Dorsey, Benjamin M; McLauchlan, Craig C; Jones, Marjorie A

2015-08-01

Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a novel method for analyzing reversible inhibition was carried out. Our alternative approach involves plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent constants can be derived from this plot: K max , K min , and K inflect . K max and K min represent the substrate to inhibitor concentration ratio for complete inhibition and minimal inhibition, respectively. K inflect represents the substrate to inhibitor concentration ratio at which the enzyme-substrate complex is equal to the inhibitory complex. These constants can be interpolated from the graph or calculated using the first and second derivative of the plot. We conclude that a steeper slope and a shift of the line to the right (increased x-axis values) would indicate a better inhibitor. Since initial velocity is not a linear function of the substrate/inhibitor ratio, this means that inhibition changes more quickly with the change in the [S]/ [I] ratio. When preincubating the enzyme with substrate before the addition of inhibitor, preincubating the enzyme with inhibitor before the addition of substrate or with concurrent addition of both substrate and inhibitor, modest changes in the slopes and y-intercepts were obtained. This plot appears useful for known competitive and non-competitive inhibitors and may have general applicability.

Interaction of difluoro-oxaloacetate with aspartate transaminase.

PubMed Central

Briley, P A; Eisenthal, R; Harrison, R; Smith, G D

1977-01-01

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme. PMID:849267

Emerging contaminants--pesticides, PPCPs, microbial degradation products and natural substances as inhibitors of multixenobiotic defense in aquatic organisms.

PubMed

Smital, Tvrtko; Luckenbach, Till; Sauerborn, Roberta; Hamdoun, Amro M; Vega, Rebecca L; Epel, David

2004-08-18

The environmental presence of chemosensitizers or inhibitors of the multixenobiotic resistance (MXR) defense system in aquatic organisms could cause increase in intracellular accumulation and toxic effects of other xenobiotics normally effluxed by MXR transport proteins (P-glycoprotein (P-gps), MRPs). MXR inhibition with concomitant detrimental effects has been shown in several studies with aquatic organisms exposed to both model MXR inhibitors and environmental pollutants. The presence of MXR inhibitors has been demonstrated in environmental samples from polluted locations at concentrations that could abolish P-gp transport activity. However, it is not clear whether the inhibition observed after exposure to environmental samples is a result of saturation of MXR transport proteins by numerous substrates present in polluted waters or results from the presence of powerful MXR inhibitors. And are potent environmental MXR inhibitors natural or man-made chemicals? As a consequence of these uncertainties, no official action has been taken to monitor and control the release and presence of MXR inhibitors into aquatic environments. In this paper we present our new results addressing these critical questions. Ecotoxicological significance of MXR inhibition was supported in in vivo studies that demonstrated an increase in the production of mutagenic metabolites by mussels and an increase in the number of sea urchin embryos with apoptotic cells after exposure to model MXR inhibitors. We also demonstrated that MXR inhibitors are present among both conventional and emerging man-made pollutants: some pesticides and synthetic musk fragrances show extremely high MXR inhibitory potential at environmentally relevant concentrations. In addition, we emphasized the biological transformation of crude oil hydrocarbons into MXR inhibitors by oil-degrading bacteria, and the risk potentially caused by powerful natural MXR inhibitors produced by invasive species.

Natural Gas Evolution in a Gas Hydrate Melt: Effect of Thermodynamic Hydrate Inhibitors.

PubMed

Sujith, K S; Ramachandran, C N

2017-01-12

Natural gas extraction from gas hydrate sediments by injection of hydrate inhibitors involves the decomposition of hydrates. The evolution of dissolved gas from the hydrate melt is an important step in the extraction process. Using classical molecular dynamics simulations, we study the evolution of dissolved methane from its hydrate melt in the presence of two thermodynamic hydrate inhibitors, NaCl and CH 3 OH. An increase in the concentration of hydrate inhibitors is found to promote the nucleation of methane nanobubbles in the hydrate melt. Whereas NaCl promotes bubble formation by enhancing the hydrophobic interaction between aqueous CH 4 molecules, CH 3 OH molecules assist bubble formation by stabilizing CH 4 bubble nuclei formed in the solution. The CH 3 OH molecules accumulate around the nuclei leading to a decrease in the surface tension at their interface with water. The nanobubbles formed are found to be highly dynamic with frequent exchange of CH 4 molecules between the bubble and the surrounding liquid. A quantitative analysis of the dynamic behavior of the bubble is performed by introducing a unit step function whose value depends on the location of CH 4 molecules with respect to the bubble. It is observed that an increase in the concentration of thermodynamic hydrate inhibitors reduces the exchange process, making the bubble less dynamic. It is also found that for a given concentration of the inhibitor, larger bubbles are less dynamic compared to smaller ones. The dependence of the dynamic nature of nanobubbles on bubble size and inhibitor concentration is correlated with the solubility of CH 4 and the Laplace pressure within the bubble. The effect of CO 2 on the formation of nanobubble in the CH 4 -CO 2 mixed gas hydrate melt in the presence of inhibitors is also examined. The simulations show that the presence of CO 2 molecules significantly reduces the induction time for methane nanobubble nucleation. The role of CO 2 in the early nucleation of bubble is explained based on the interaction between the bubble and the dissolved CO 2 molecules.

Polyphenol oxidase inhibitor from blue mussel (Mytilus edulis) extract.

PubMed

Schulbach, Kurt F; Johnson, Jodie V; Simonne, Amarat H; Kim, Jeong-Mok; Jeong, Yoonhwa; Yagiz, Yavuz; Marshall, Maurice R

2013-03-01

Enzymatic browning remains a problem for the fruit and vegetable industry, especially new emerging markets like pre-cuts. A crude inhibitor from blue mussel (Mytilus edulis) showed broad inhibition for apple (58%), mushroom (32%), and potato (44%) polyphenol oxidase (PPO) and was further characterized. Inhibition increased as the concentration of inhibitor increased in the reaction mixture eventually leveling off at a maximum inhibition of 92% for apple PPO. The inhibitor was capable of bleaching the brown color formed in the reaction mixture with apple PPO. Identification of the inhibitor by mass spectrometry and high-performance liquid chromatography revealed it to be hypotaurine (C2 H7 NO2 S). Hypotaurine and other sulfinic acid analogs (methane and benzene sulfinic acids) showed very good inhibition for apple PPO at various concentrations with the highest inhibition occurring at 500 μM for hypotaurine (89%), methane sulfinic acid (100%), and benzene sulfinic acid (100%). An inhibitor found in the expressed liquid from blue mussel shows very good inhibition on enzymatic browning. Since this enzyme is responsible for losses to the fruit and vegetable industry, natural inhibitors that prevent browning would be valuable. Finding alternative chemistries that inhibit browning and understanding their mode of action would be beneficial to the fruit and vegetable industries and their segments such as pre-cuts, juices, and so on. Inhibitors from products ingested by consumers are more acceptable as natural ingredients. © 2013 Institute of Food Technologists®

Electrochemical probing of high-level radioactive waste tanks containing washed sludge and precipitates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bickford, D.F.; Congdon, J.W.; Oblath, S.B.

1987-01-01

At the U.S. Department of Energy's Savannah River Plant, corrosion of carbon steel storage tanks containing alkaline, high-level radioactive waste is controlled by specification of limits on waste composition and temperature. Processes for the preparation of waste for final disposal will result in waste with low corrosion inhibitor concentrations and, in some cases, high aromatic organic concentrations, neither of which are characteristic of previous operations. Laboratory tests, conducted to determine minimum corrosion inhibitor levels indicated pitting of carbon steel near the waterline for proposed storage conditions. In situ electrochemical measurements of full-scale radioactive process demonstrations have been conducted to assessmore » the validity of laboratory tests. Probes included pH, Eh (potential relative to a standard hydrogen electrode), tank potential, and alloy coupons. In situ results are compared to those of the laboratory tests, with particular regard given to simulated solution composition.« less

pH Responsive Microcapsules for Corrosion Control

NASA Technical Reports Server (NTRS)

Calle, Luz Marina; Li, Wenyan; Muehlberg, Aaron; Boraas, Samuel; Webster, Dean; JohnstonGelling, Victoria; Croll, Stuart; Taylor, S Ray; Contu, Francesco

2008-01-01

The best coatings for corrosion protection provide not only barriers to the environment, but also a controlled release of a corrosion inhibitor, as demanded by the presence of corrosion or mechanical damage. NASA has developed pH sensitive microcapsules (patent pending) that can release their core contents when corrosion starts. The objectives of the research presented here were to encapsulate non-toxic corrosion inhibitors, to incorporate the encapsulated inhibitors into paint formulations, and to test the ability of the paints to control corrosion. Results showed that the encapsulated corrosion inhibitors, specifically Ce(NO3)3 , are effective to control corrosion over long periods of time when incorporated at relatively high pigment volume concentrations into a paint formulation.

Evaluation of serum biochemical marker concentrations and survival time in dogs with protein-losing enteropathy.

PubMed

Equilino, Mirjam; Théodoloz, Vincent; Gorgas, Daniela; Doherr, Marcus G; Heilmann, Romy M; Suchodolski, Jan S; Steiner, Jörg M; Burgener Dvm, Iwan A

2015-01-01

To evaluate serum concentrations of biochemical markers and survival time in dogs with protein-losing enteropathy (PLE). Prospective study. 29 dogs with PLE and 18 dogs with food-responsive diarrhea (FRD). Data regarding serum concentrations of various biochemical markers at the initial evaluation were available for 18 of the 29 dogs with PLE and compared with findings for dogs with FRD. Correlations between biochemical marker concentrations and survival time (interval between time of initial evaluation and death or euthanasia) for dogs with PLE were evaluated. Serum C-reactive protein concentration was high in 13 of 18 dogs with PLE and in 2 of 18 dogs with FRD. Serum concentration of canine pancreatic lipase immunoreactivity was high in 3 dogs with PLE but within the reference interval in all dogs with FRD. Serum α1-proteinase inhibitor concentration was less than the lower reference limit in 9 dogs with PLE and 1 dog with FRD. Compared with findings in dogs with FRD, values of those 3 variables in dogs with PLE were significantly different. Serum calprotectin (measured by radioimmunoassay and ELISA) and S100A12 concentrations were high but did not differ significantly between groups. Seventeen of the 29 dogs with PLE were euthanized owing to this disease; median survival time was 67 days (range, 2 to 2,551 days). Serum C-reactive protein, canine pancreatic lipase immunoreactivity, and α1-proteinase inhibitor concentrations differed significantly between dogs with PLE and FRD. Most initial biomarker concentrations were not predictive of survival time in dogs with PLE.

Toxicity evaluation of convection-enhanced delivery of small-molecule kinase inhibitors in naïve mouse brainstem.

PubMed

Zhou, Zhiping; Ho, Sharon L; Singh, Ranjodh; Pisapia, David J; Souweidane, Mark M

2015-04-01

Diffuse intrinsic pontine gliomas (DIPGs) are inoperable and lethal high-grade gliomas lacking definitive therapy. Platelet-derived growth factor receptor (PDGFR) and its downstream signaling molecules are the most commonly overexpressed oncogenes in DIPG. This study tested the effective concentration of PDGFR pathway inhibitors in cell culture and then toxicity of these small-molecule kinase inhibitors delivered to the mouse brainstem via convection-enhanced delivery (CED) for potential clinical application. Effective concentrations of small-molecule kinase inhibitors were first established in cell culture from a mouse brainstem glioma model. Sixteen mice underwent CED, a local drug delivery technique, of saline or of single and multidrug combinations of dasatinib (2 M), everolimus (20 M), and perifosine (0.63 mM) in the pons. Animals were kept alive for 3 days following the completion of infusion. No animals displayed any immediate or delayed neurological deficits postoperatively. Histological analysis revealed edema, microgliosis, acute inflammation, and/or axonal injury in the experimental animals consistent with mild acute drug toxicity. Brainstem CED of small-molecule kinase inhibitors in the mouse did not cause serious acute toxicities. Future studies will be necessary to evaluate longer-term safety to prepare for potential clinical application.

On the Error of the Dixon Plot for Estimating the Inhibition Constant between Enzyme and Inhibitor

ERIC Educational Resources Information Center

Fukushima, Yoshihiro; Ushimaru, Makoto; Takahara, Satoshi

2002-01-01

In textbook treatments of enzyme inhibition kinetics, adjustment of the initial inhibitor concentration for inhibitor bound to enzyme is often neglected. For example, in graphical plots such as the Dixon plot for estimation of an inhibition constant, the initial concentration of inhibitor is usually plotted instead of the true inhibitor…

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate

PubMed Central

Volpon, Laurent; Osborne, Michael J.; Zahreddine, Hiba; Romeo, Andrea A.; Borden, Katherine L.B.

2013-01-01

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m7G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5μM eIF4E) but much weaker affinity at more typical NMR concentrations (50-200μM). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central β-sheet and dorsal surface of eIF4E, similar to m7G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in both drug design efforts and reveal new insights into ligand eIF4E recognition. PMID:23583375

Assay format as a critical success factor for identification of novel inhibitor chemotypes of tissue-nonspecific alkaline phosphatase from high-throughput screening.

PubMed

Chung, Thomas D Y; Sergienko, Eduard; Millán, José Luis

2010-04-27

The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PP(i)), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.

Specific Inhibition of the Cyanide-insensitive Respiratory Pathway in Plant Mitochondria by Hydroxamic Acids

PubMed Central

Schonbaum, Gregory R.; Bonner, Walter D.; Storey, Bayard T.; Bahr, James T.

1971-01-01

Hydroxamic acids, R-CONHOH, are inhibitors specific to the respiratory pathway through the alternate, cyanide-insensitive terminal oxidase of plant mitochondria. The nature of the R group in these compounds affects the concentration at which the hydroxamic acids are effective, but it appears that all hydroxamic acids inhibit if high enough concentrations are used. The benzhydroxamic acids are effective at relatively low concentrations; of these, the most effective are m-chlorobenzhydroxamic acid and m-iodobenzhydroxamic acid. The concentrations required for half-maximal inhibition of the alternate oxidase pathway in mung bean (Phaseolus aureus) mitochondria are 0.03 mm for m-chlorobenzhydroxamic acid and 0.02 mm for m-iodobenzhydroxamic acid. With skunk cabbage (Symplocarpus foetidus) mitochondria, the required concentrations are 0.16 for m-chlorobenzhydroxamic acid and 0.05 for m-iodobenzhydroxamic acid. At concentrations which inhibit completely the alternate oxidase pathway, these two compounds have no discernible effect on either the respiratory pathway through cytochrome oxidase, or on the energy coupling reactions of these mitochondria. These inhibitors make it possible to isolate the two respiratory pathways and study their mode of action separately. These inhibitors also enhance an electron paramagnetic resonance signal near g = 2 in anaerobic, submitochondrial particles from skunk cabbage, which appears to be specific to the alternate oxidase and thus provides a means for its assay. PMID:5543780

Inhibitor development in non-severe haemophilia across Europe.

PubMed

Fischer, Kathelijn; Iorio, Alfonso; Lassila, Riitta; Peyvandi, Flora; Calizzani, Gabriele; Gatt, Alex; Lambert, Thierry; Windyga, Jerzy; Gilman, Estelle A; Hollingsworth, R; Makris, Michael

2015-10-01

Evidence about inhibitor formation in non-severe haemophilia and the potential role for clotting factor concentrate type is scant. It was the aim of this study to report inhibitor development in non-severe haemophilia patients enrolled in the European Haemophilia Safety Surveillance (EUHASS) study. Inhibitors are reported quarterly and total treated patients annually. Incidence rates and 95% confidence intervals (95% CI) were calculated according to diagnosis and concentrate used. Between 1-10-2008 and 31-12-2012, 68 centres reported on 7,969 patients with non-severe haemophilia A and 1,863 patients with non-severe haemophilia B. For haemophilia A, 37 inhibitors occurred in 8,622 treatment years, resulting in an inhibitor rate of 0.43/100 treatment years (95% CI 0.30-0.59). Inhibitors occurred at a median age of 35 years, after a median of 38 exposure days (EDs; P25-P75: 20-80); with 72% occurring within the first 50 EDs. In haemophilia B, one inhibitor was detected in 2,149 treatment years, resulting in an inhibitor rate of 0.05/100 years (95% CI 0.001-0.26). This inhibitor developed at the age of six years, after six EDs. The rate of inhibitors appeared similar across recombinant and plasma derived factor VIII (FVIII) concentrates. Rates for individual concentrates could not be calculated at this stage due to low number of events. In conclusion, inhibitors in non-severe haemophilia occur three times more frequently than in previously treated patients with severe haemophilia at a rate of 0.43/100 patient years (haemophilia A) and 0.05/100 years (haemophilia B). Although the majority of inhibitors developed in the first 50 EDs, inhibitor development continued with increasing exposure to FVIII.

Potentiation of carbachol-induced detrusor smooth muscle contractions by beta-adrenoceptor activation.

PubMed

Klausner, Adam P; Rourke, Keith F; Miner, Amy S; Ratz, Paul H

2009-03-15

In strips of rabbit bladder free of urothelium, the beta-adrenoceptor agonist, isoproterenol, significantly reduced basal detrusor smooth muscle tone and inhibited contractions produced by low concentrations of the muscarinic receptor agonist, carbachol. During a carbachol concentration-response curve, instead of inhibiting, isoproterenol strengthened contractions produced by high carbachol concentrations. Thus, the carbachol concentration-response curve was shifted by isoproterenol from a shallow, graded relationship, to a steep, switch-like relationship. The tyrosine kinase inhibitor, genistein, inhibited carbachol-induced contractions only in the presence of isoproterenol. Contraction produced by a single high carbachol concentration (1 microM) displayed 1 fast and 1 slow peak. In the presence of isoproterenol, the slow peak was not strengthened, but was delayed, and U-0126 (mitogen-activated protein kinase kinase inhibitor) selectively inhibited this delay concomitantly with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. Isoproterenol reduced ERK phosphorylation only in the absence of carbachol. These data support the concept that, by inhibiting weak contractions, potentiating strong contractions, and producing a more switch-like concentration-response curve, beta-adrenoceptor stimulation enhanced the effectiveness of muscarinic receptor-induced detrusor smooth muscle contraction. Moreover, beta-adrenoceptor stimulation changed the cellular mechanism by which carbachol produced contraction. The potential significance of multi-receptor and multi-cell crosstalk is discussed.

Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1.

PubMed

Chen, Allie Y; Thomas, Pei W; Stewart, Alesha C; Bergstrom, Alexander; Cheng, Zishuo; Miller, Callie; Bethel, Christopher R; Marshall, Steven H; Credille, Cy V; Riley, Christopher L; Page, Richard C; Bonomo, Robert A; Crowder, Michael W; Tierney, David L; Fast, Walter; Cohen, Seth M

2017-09-14

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC 50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1 H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Aldosterone, Parathyroid Hormone, and the Use of Renin-Angiotensin-Aldosterone System Inhibitors: The Multi-Ethnic Study of Atherosclerosis

PubMed Central

Brown, Jenifer; de Boer, Ian H.; Robinson-Cohen, Cassianne; Siscovick, David S.; Kestenbaum, Bryan; Allison, Matthew

2015-01-01

Context: Aldosterone and PTH are implicated in the pathogenesis of cardiovascular and skeletal diseases. An expanding body of evidence supports a bidirectional and positive physiologic relationship between aldosterone and PTH. Large population-based studies confirming this relationship, and whether it may be targeted as a potential method to mitigate the clinical consequences associated with excess aldosterone and PTH, are needed. Objective: We hypothesized that higher aldosterone levels would associate with higher PTH, and that the use of renin-angiotensin-aldosterone system (RAAS) inhibitors would predict lower PTH in a large, multi-ethnic, community-based cohort. Design, Setting, Participants: We conducted cross-sectional analyses of participants in the Multi-Ethnic Study of Atherosclerosis without apparent primary hyperparathyroidism or chronic kidney disease (n = 5668). We evaluated associations of RAAS inhibitor use with PTH concentration among 1888 treated hypertensive participants. We also tested associations of serum aldosterone concentration with PTH concentration among 1547 participants with these measurements. Outcome: Serum PTH concentration. Results: Higher aldosterone associated with higher PTH (β = 0.19 pg/ml per 1 ng/dl of aldosterone, P < .0001), and this finding was most pronounced among those with a primary hyperaldosteronism-like phenotype. There was a stepwise increment in PTH when comparing untreated normotensives, hypertensives using RAAS inhibitors, untreated hypertensives, and treated hypertensives using non-RAAS inhibitors (40.8, 45.0, 46.2, 47.1 pg/ml, respectively). The use of any RAAS inhibitor independently associated with lower PTH (β = −2.327 pg/ml per use of RAAS inhibitor, P = .006), when compared with the use of any non-RAAS inhibitor medication. Conclusions: Higher serum aldosterone concentration is associated with higher serum PTH concentration, and the use of RAAS inhibitors is associated with lower PTH concentration. These results extend prior evidence from observational and intervention studies suggesting a potentially important and modifiable relationship between the RAAS and PTH in humans. PMID:25412416

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection

PubMed Central

Giacobbi, Nicholas S.

2017-01-01

ABSTRACT Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. PMID:28507107

The combination of HDAC and aminopeptidase inhibitors is highly synergistic in myeloma and leads to disruption of the NFκB signalling pathway

PubMed Central

Smith, Emma M.; Zhang, Lei; Walker, Brian A.; Davenport, Emma L.; Aronson, Lauren I.; Krige, David; Hooftman, Leon; Drummond, Alan H.; Morgan, Gareth J.; Davies, Faith E.

2015-01-01

There is a growing body of evidence supporting the use of epigenetic therapies in the treatment of multiple myeloma. We show the novel HDAC inhibitor CHR-3996 induces apoptosis in myeloma cells at concentrations in the nanomolar range and with apoptosis mediated by p53 and caspase pathways. In addition, HDAC inhibitors are highly synergistic, both in vitro and in vivo, with the aminopeptidase inhibitor tosedostat (CHR-2797). We demonstrate that the basis for this synergy is a consequence of changes in the levels of NFκB regulators BIRC3/cIAP2, A20, CYLD, and IκB, which were markedly affected by the combination. When co-administered the HDAC and aminopeptidase inhibitors caused rapid nuclear translocation of NFκB family members p65 and p52, following activation of both canonical and non-canonical NFκB signalling pathways. The subsequent up-regulation of inhibitors of NFκB activation (most significantly BIRC3/cIAP2) turned off the cytoprotective effects of the NFκB signalling response in a negative feedback loop. These results provide a rationale for combining HDAC and aminopeptidase inhibitors clinically for the treatment of myeloma patients and support the disruption of the NFκB signalling pathway as a therapeutic strategy. PMID:26015393

A METHOD TO REMOVE ENVIRONMENTAL INHIBITORS PRIOR TO THE DETECTION OF WATERBORNE ENTERIC VIRUSES BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

EPA Science Inventory

A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the reverse transcription-polymerase chain reaction (RT-PCR).Environmental inhibitors, concentrated along with viruses during water sample processi...

Effect of pH and glucose on cultured human peritoneal mesothelial cells.

PubMed

Shao, J C; Yorioka, N; Nishida, Y; Yamakido, M

1999-08-01

We investigated the effects of various pH and glucose concentrations on the growth of human peritoneal mesothelial cells and on coagulation and fibrinolytic factors. Cells were cultured at various pH values in Ham's F-12 medium containing 1.0% foetal calf serum and supplemented with D-glucose or D-mannitol at various concentrations. After 4-48 h, cell proliferation and 3H-thymidine incorporation were determined. Coagulation and fibrinolytic factors were measured after 48 h. Glucose caused concentration-dependent inhibition of cell growth at all pH values, but the deleterious effect of low pH on cell proliferation was faster and stronger than that of high glucose. At a similar osmolality, mannitol caused less inhibition of cell proliferation than glucose. There was a glucose concentration-dependent increase of thrombin-antithrombin III complex production at all pH values. At pH 5.2, tissue-type plasminogen activator production was far lower than at higher pH values, and production of the plasminogen activator inhibitor showed a glucose concentration-dependent increase. At pH 6.5 or 7.3, however, the plasminogen activator inhibitor production decreased and tissue-type plasminogen activator production increased in a glucose concentration-dependent manner. Low pH and/or high glucose culture medium had an inhibitory effect on peritoneal mesothelial cells, with the effect of high glucose being partially related to hyperosmolality. These cells may modulate peritoneal coagulant and fibrinolytic activity, with the balance between coagulation and fibrinolysis being disturbed by low pH and/or high glucose.

Crystal structures of spleen tyrosine kinase in complex with novel inhibitors: structural insights for design of anticancer drugs.

PubMed

Lee, Sang Jae; Choi, Jang-Sik; Han, Byeong-Gu; Kim, Hyoun Sook; Song, Ho-Juhn; Lee, Jaekyoo; Nam, Seungyoon; Goh, Sung-Ho; Kim, Jung-Ho; Koh, Jong Sung; Lee, Byung Il

2016-10-01

Spleen tyrosine kinase (SYK) is a cytosolic nonreceptor protein tyrosine kinase that mediates key signal transduction pathways following the activation of immune cell receptors. SYK regulates cellular events induced by the B-cell receptor and Fc receptors with high intrinsic activity. Furthermore, SYK has been regarded as an attractive target for the treatment of autoimmune diseases and cancers. Here, we report the crystal structures of SYK in complex with seven newly developed inhibitors (G206, G207, O178, O194, O259, O272, and O282) to provide structural insights into which substituents of the inhibitors and binding regions of SYK are essential for lead compound optimization. Our kinase inhibitors exhibited high inhibitory activities against SYK, with half-maximal inhibitory concentrations (IC 50 ) of approximately 0.7-33 nm, but they showed dissimilar inhibitory activities against KDR, RET, JAK2, JAK3, and FLT3. Among the seven SYK inhibitors, O272 and O282 exhibited highly specific inhibitions against SYK, whereas O194 exhibited strong inhibition of both SYK and FLT3. Three inhibitors (G206, G207, and O178) more efficiently inhibited FLT3 while still substantially inhibiting SYK activity. The binding mode analysis suggested that a highly selective SYK inhibitor can be developed by optimizing the functional groups that facilitate direct interactions with Asn499. The atomic coordinates and structure factors for human SYK are in the Protein Data Bank under accession codes 4XG2 (inhibitor-free form), 4XG3 (G206), 4XG4 (G207), 5GHV (O178), 4XG6 (O194), 4XG7 (O259), 4XG8 (O272), and 4XG9 (O282). © 2016 Federation of European Biochemical Societies.

Sodium ion transport participates in non-neuronal acetylcholine release in the renal cortex of anesthetized rabbits.

PubMed

Shimizu, Shuji; Akiyama, Tsuyoshi; Kawada, Toru; Sata, Yusuke; Turner, Michael James; Fukumitsu, Masafumi; Yamamoto, Hiromi; Kamiya, Atsunori; Shishido, Toshiaki; Sugimachi, Masaru

2017-09-01

This study examined the mechanism of release of endogenous acetylcholine (ACh) in rabbit renal cortex by applying a microdialysis technique. In anesthetized rabbits, a microdialysis probe was implanted into the renal cortex and perfused with Ringer's solution containing high potassium concentration, high sodium concentration, a Na + /K + -ATPase inhibitor (ouabain), or an epithelial Na + channel blocker (benzamil). Dialysate samples were collected at baseline and during exposure to each agent, and ACh concentrations in the samples were measured by high-performance liquid chromatography. High potassium had no effect on renal ACh release. High sodium increased dialysate ACh concentrations significantly. Ouabain increased dialysate ACh concentration significantly. Benzamil decreased dialysate ACh concentrations significantly both at baseline and under high sodium. The finding that high potassium-induced depolarization does not increase ACh release suggests that endogenous ACh is released in renal cortex mainly by non-neuronal mechanism. Sodium ion transport may be involved in the non-neuronal ACh release.

Relationship between carbachol hyperstimulation-induced pancreatic intracellular trypsinogen and NF-kappa B activation in rats in vitro.

PubMed

Jiang, Chunfang; Zheng, Hai; Liu, Sunan; Fang, Kaifeng

2008-02-01

The relationship between intracellular trypsinogen activation and NF-kappa B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachol) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc) and NF-kappa B inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The activity of NF-kappa B was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-kappa B in pancreatic acinar cells treated with high concentrations of carbachol (10(-3) mol/L) in vitro was significantly decreased as compared with control group (P<0.01). The addition of 10(-2) mol/L PDTC resulted in a significant decrease of NF-kappa B activities in pancreatic acinar cells after treated with high concentrations of carbachol (10(-3) mol/L) in vitro, but the intracellular trypsinogen activity was not obviously inhibited (P>0.05). It was concluded that intracellular trypsinogen activation is likely involved in the regulation of high concentrations of carbachol-induced NF-kappa B activation in pancreatic acinar cells in vitro. NF-kappa B activation is likely not necessary for high concentrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Hydroxyl carboxylate based non-phosphorus corrosion inhibition process for reclaimed water pipeline and downstream recirculating cooling water system.

PubMed

Wang, Jun; Wang, Dong; Hou, Deyin

2016-01-01

A combined process was developed to inhibit the corrosion both in the pipeline of reclaimed water supplies (PRWS) and in downstream recirculating cooling water systems (RCWS) using the reclaimed water as makeup. Hydroxyl carboxylate-based corrosion inhibitors (e.g., gluconate, citrate, tartrate) and zinc sulfate heptahydrate, which provided Zn(2+) as a synergistic corrosion inhibition additive, were added prior to the PRWS when the phosphate (which could be utilized as a corrosion inhibitor) content in the reclaimed water was below 1.7 mg/L, and no additional corrosion inhibitors were required for the downstream RCWS. Satisfactory corrosion inhibition was achieved even if the RCWS was operated under the condition of high numbers of concentration cycles. The corrosion inhibition requirement was also met by the appropriate combination of PO4(3-) and Zn(2+) when the phosphate content in the reclaimed water was more than 1.7 mg/L. The process integrated not only water reclamation and reuse, and the operation of a highly concentrated RCWS, but also the comprehensive utilization of phosphate in reclaimed water and the application of non-phosphorus corrosion inhibitors. The proposed process reduced the operating cost of the PRWS and the RCWS, and lowered the environmental hazard caused by the excessive discharge of phosphate. Furthermore, larger amounts of water resources could be conserved as a result. Copyright © 2015. Published by Elsevier B.V.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

PubMed

Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

2014-09-01

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

Selection and use of crystallization inhibitors for matrix-type transdermal drug-delivery systems containing sex steroids.

PubMed

Lipp, R

1998-12-01

The purpose of this study was to stabilize transdermal drug-delivery systems (TDDS) highly loaded with sex steroids against recrystallization of drugs during storage. To facilitate the selection of potential crystallization inhibitors a drug-excipient interaction test was also established. Analysis of the thermal behaviour of 1:1 steroid-excipient mixtures by differential scanning calorimetry (DSC) revealed that oestradiol and gestodene interact strongly with silicone dioxide and povidones, e.g. povidone K12. The addition of povidone K12 to polyacrylate-based matrix TDDS containing either 3% oestradiol or 2% gestodene resulted in stable systems which did not recrystallize during storage at 25 degrees C for more than 5 years. Significant recrystallization was, on the other hand, observed in non-stabilized reference patches even after 1 to 2 months storage. The DSC screening model proved very effective for selection of inhibitors of the crystallization of sex steroids in matrix TDDS. The crystallization inhibitor approach is a highly versatile stabilization tool for matrix patches containing high concentrations of sex steroids.

Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell‐Derived Platelets

PubMed Central

Hirata, Shinji; Murata, Takahiko; Suzuki, Daisuke; Nakamura, Sou; Jono‐Ohnishi, Ryoko; Hirose, Hidenori; Sawaguchi, Akira; Nishimura, Satoshi; Sugimoto, Naoshi

2016-01-01

Abstract Donor‐independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ibα (GPIbα), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature‐dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen‐activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC‐derived GPIbα+ platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP‐457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP‐457 blocked GPIbα shedding from iPSC platelets at a lower half‐maximal inhibitory concentration than panmetalloproteinase inhibitor GM‐6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP‐457 exhibited improved GPIbα‐dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP‐457 exerted better hemostatic function in vivo. Our findings suggest that KP‐457, unlike GM‐6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC‐derived platelets at 37°C, which is an important step toward their clinical application. Stem Cells Translational Medicine 2017;6:720–730 PMID:28297575

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

PubMed

Geng, Tao; Mathies, Richard A

2015-01-01

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Nontoxic concentration of DNA-PK inhibitor NU7441 radio-sensitizes lung tumor cells with little effect on double strand break repair.

PubMed

Sunada, Shigeaki; Kanai, Hideki; Lee, Younghyun; Yasuda, Takeshi; Hirakawa, Hirokazu; Liu, Cuihua; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

2016-09-01

High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties

PubMed Central

Sendobry, Sandra M; Cornicelli, Joseph A; Welch, Kathryn; Bocan, Thomas; Tait, Bradley; Trivedi, Bharat K; Colbry, Norman; Dyer, Richard D; Feinmark, Steven J; Daugherty, Alan

1997-01-01

15-Lipoxygenase (15-LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15-LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol-fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties.PD 146176 inhibited rabbit reticulocyte 15-LO through a mixed noncompetitive mode with a Ki of 197 nM. The drug had minimal effects on either copper or 2,2′-azobis(2-amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki.Control New Zealand rabbits were fed a high-fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg−1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nM). During the 12 week study, there were no significant differences in weight gain, haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol.The drug plasma concentrations achieved in vivo did not inhibit low-density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176-treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP.PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15±4 to 0% (P<0.02); esterified cholesterol content was reduced from 2.1±0.7 to 0 μg mg−1 (P<0.02) in this region. Immunostainable lipid-laden macrophages present in aortic intima of control animals were totally absent in the drug-treated group.Results of these studies are consistent with a role for 15-LO in atherogenesis. PMID:9105693

Ethanol at low concentrations protects glomerular podocytes through alcohol dehydrogenase and 20-HETE.

PubMed

McCarthy, Ellen T; Zhou, Jianping; Eckert, Ryan; Genochio, David; Sharma, Rishi; Oni, Olurinde; De, Alok; Srivastava, Tarak; Sharma, Ram; Savin, Virginia J; Sharma, Mukut

2015-01-01

Clinical studies suggest cardiovascular and renal benefits of ingesting small amounts of ethanol. Effects of ethanol, role of alcohol dehydrogenase (ADH) or of 20-hydroxyeicosatetraenoic acid (20-HETE) in podocytes of the glomerular filtration barrier have not been reported. We found that mouse podocytes at baseline generate 20-HETE and express ADH but not CYP2e1. Ethanol at high concentrations altered the actin cytoskeleton, induced CYP2e1, increased superoxide production and inhibited ADH gene expression. Ethanol at low concentrations upregulated the expression of ADH and CYP4a12a. 20-HETE, an arachidonic acid metabolite generated by CYP4a12a, blocked the ethanol-induced cytoskeletal derangement and superoxide generation. Ethanol at high concentration or ADH inhibitor increased glomerular albumin permeability in vitro. 20-HETE and its metabolite produced by ADH activity, 20-carboxy-arachidonic acid, protected the glomerular permeability barrier against an ADH inhibitor, puromycin or FSGS permeability factor. We conclude that ADH activity is required for glomerular function, 20-HETE is a physiological substrate of ADH in podocytes and that podocytes are useful biosensors to understand glomeruloprotective effects of ethanol. Published by Elsevier Inc.

Ethanol at Low Concentrations Protects Glomerular Podocytes through Alcohol Dehydrogenase and 20-HETE

PubMed Central

McCarthy, Ellen T.; Zhou, Jianping; Eckert, Ryan; Genochio, David; Sharma, Rishi; Oni, Olurinde; De, Alok; Srivastava, Tarak; Sharma, Ram; Savin, Virginia J.; Sharma, Mukut

2014-01-01

Clinical studies suggest cardiovascular and renal benefits of ingesting small amounts of ethanol. Effects of ethanol, role of alcohol dehydrogenase (ADH) or of 20-hydroxyeicosatetraenoic acid (20-HETE) in podocytes of the glomerular filtration barrier have not been reported. We found that mouse podocytes at baseline generate 20-HETE and express ADH but not CYP2e1. Ethanol at high concentrations altered the actin cytoskeleton, induced CYP2e1, increased superoxide production and inhibited ADH gene expression. Ethanol at low concentrations upregulated the expression of ADH and CYP4a12a. 20-HETE, an arachidonic acid metabolite generated by CYP4a12a, blocked the ethanol-induced cytoskeletal derangement and superoxide generation. Ethanol at high concentration or ADH inhibitor increased glomerular albumin permeability in vitro. 20-HETE and its metabolite produced by ADH activity, 20-carboxy-arachidonic acid, protected the glomerular permeability barrier against an ADH inhibitor, puromycin or FSGS permeability factor. We conclude that ADH activity is required for glomerular function, 20-HETE is a physiological substrate of ADH in podocytes and that podocytes are useful biosensors to understand glomeruloprotective effects of ethanol. PMID:25447342

Curcumin Derivatives as Green Corrosion Inhibitors for α-Brass in Nitric Acid Solution

NASA Astrophysics Data System (ADS)

Fouda, A. S.; Elattar, K. M.

2012-11-01

1,7- Bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene-4-arylazo-3,5-dione I-V have been investigated as corrosion inhibitors for α-brass in 2 M nitric acid solution using weight-loss and galvanostatic polarization techniques. The efficiency of the inhibitors increases with the increase in the inhibitor concentration but decreases with a rise in temperature. The conjoint effect of the curcumin derivatives and KSCN has also been studied. The apparent activation energy ( E a*) and other thermodynamic parameters for the corrosion process have also been calculated. The galvanostatic polarization data indicated that the inhibitors were of mixed-type, but the cathode is more polarized than the anode. The slopes of the cathodic and anodic Tafel lines ( b c and b a) are maintained approximately equal for various inhibitor concentrations. However, the value of the Tafel slopes increases together as inhibitor concentration increases. The adsorption of these compounds on α-brass surface has been found to obey the Frumkin's adsorption isotherm. The mechanism of inhibition was discussed in the light of the chemical structure of the undertaken inhibitors.

Discovery of a low-systemic-exposure DGAT-1 inhibitor with a picolinoylpyrrolidine-2-carboxylic acid moiety.

PubMed

Yan, Jianwei; Wang, Gaihong; Dang, Xiangyu; Guo, Binbin; Chen, Wuhong; Wang, Ting; Zeng, Limin; Wang, Heyao; Hu, Youhong

2017-09-01

A series of diacylglycerol O-acyltransferase 1 (DGAT-1) inhibitors with a picolinoylpyrrolidine-2-carboxylic acid moiety were designed and synthesized. Of these compounds, compound 22 exhibited excellent DGAT-1-inhibitory activity (hDGAT-1 enzyme assay, 50% inhibitory concentration [IC 50 ]=3.5±0.9nM) and effectively reduced the intracellular triglyceride contents in 3T3-L1, HepG2 and Caco-2 cells. A preliminary study of the plasma and tissue distributions of compound 22 in mice revealed low plasma exposure and high concentrations in different segments of the intestine and liver, which may facilitate targeting DGAT-1. Furthermore, in an acute lipid challenge test, compound 22 showed a dose-dependent inhibitory effect on high-serum triglycerides in C57/KSJ mice induced by olive oil (1, 3, and 10mg/kg, i.g.). Copyright © 2017 Elsevier Ltd. All rights reserved.

Relationship between secretory leukocyte protease inhibitor levels in bronchoalveolar lavage fluid and postoperative pulmonary complications in patients with esophageal cancer.

PubMed

Tsukada, Katsuhiko; Miyazaki, Tatsuya; Katoh, Hiroyuki; Masuda, Norihiro; Ojima, Hitoshi; Fukuchi, Minoru; Manda, Ryokuhei; Fukai, Yasuyuki; Nakajima, Masanobu; Sohda, Makoto; Kuwano, Hiroyuki

2005-04-01

We investigated whether secretory leukocyte protease inhibitor (SLPI) is associated with pulmonary complications after esophagectomy. We measured serial changes in the SLPI concentration in the bronchoalveolar lavage fluid (BALF) of 34 patients who underwent and examined the relationship between SLPI and postoperative morbidity. Fifteen (44%) of 34 patients (high group) had a BALF SLPI concentration >90,000 pg/mL at the end of the surgery (postoperative day [POD] 0). There was no significant difference between the high group and other 19 patients (low group) with respect to age, sex, preoperative comorbid conditions, tumor stage, surgical technique, operating time, or blood loss volume. Days of intubation and pulmonary complication rate were significantly increased in the high group compared with the low group. Logistic regression analysis revealed that the BALF SLPI level on POD 0 was significant for pulmonary complications. Our results indicate that assaying SLPI levels in BALF can be useful for the prediction of pulmonary complications after esophagectomy.

Isothiazolidinone (IZD) as a phosphoryl mimetic in inhibitors of the Yersinia pestis protein tyrosine phosphatase YopH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sung-Eun; Bahta, Medhanit; Lountos, George T.

2011-07-01

The first X-ray crystal structure of the Y. pestis protein tyrosine phosphatase YopH in complex with an isothiazolidinone-based lead-fragment compound is reported. Isothiazolidinone (IZD) heterocycles can act as effective components of protein tyrosine phosphatase (PTP) inhibitors by simultaneously replicating the binding interactions of both a phosphoryl group and a highly conserved water molecule, as exemplified by the structures of several PTP1B–inhibitor complexes. In the first unambiguous demonstration of IZD interactions with a PTP other than PTP1B, it is shown by X-ray crystallography that the IZD motif binds within the catalytic site of the Yersinia pestis PTP YopH by similarly displacingmore » a highly conserved water molecule. It is also shown that IZD-based bidentate ligands can inhibit YopH in a nonpromiscuous fashion at low micromolar concentrations. Hence, the IZD moiety may represent a useful starting point for the development of YopH inhibitors.« less

A Synergistic Combination of Advanced Separation and Chemical Scale Inhibitor Technologies for Efficient Use of Imparied Water As Cooling Water in Coal-based Power Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jasbir Gill

2010-08-30

Nalco Company is partnering with Argonne National Laboratory (ANL) in this project to jointly develop advanced scale control technologies that will provide cost-effective solutions for coal-based power plants to operate recirculating cooling water systems at high cycles using impaired waters. The overall approach is to use combinations of novel membrane separations and scale inhibitor technologies that will work synergistically, with membrane separations reducing the scaling potential of the cooling water and scale inhibitors extending the safe operating range of the cooling water system. The project started on March 31, 2006 and ended in August 30, 2010. The project was amore » multiyear, multi-phase project with laboratory research and development as well as a small pilot-scale field demonstration. In Phase 1 (Technical Targets and Proof of Concept), the objectives were to establish quantitative technical targets and develop calcite and silica scale inhibitor chemistries for high stress conditions. Additional Phase I work included bench-scale testing to determine the feasibility of two membrane separation technologies (electrodialysis ED and electrode-ionization EDI) for scale minimization. In Phase 2 (Technology Development and Integration), the objectives were to develop additional novel scale inhibitor chemistries, develop selected separation processes, and optimize the integration of the technology components at the laboratory scale. Phase 3 (Technology Validation) validated the integrated system's performance with a pilot-scale demonstration. During Phase 1, Initial evaluations of impaired water characteristics focused on produced waters and reclaimed municipal wastewater effluents. Literature and new data were collected and evaluated. Characteristics of produced waters vary significantly from one site to another, whereas reclaimed municipal wastewater effluents have relatively more uniform characteristics. Assessment to date confirmed that calcite and silica/silicate are two common potential cycle-limiting minerals for using impaired waters. For produced waters, barium sulfate and calcium sulfate are two additional potential cycle-limiting minerals. For reclaimed municipal wastewater effluents, calcium phosphate scaling can be an issue, especially in the co-presence of high silica. Computational assessment, using a vast amount of Nalco's field data from coal fired power plants, showed that the limited use and reuse of impaired waters is due to the formation of deposit caused by the presence of iron, high hardness, high silica and high alkalinity in the water. Appropriate and cost-effective inhibitors were identified and developed - LL99B0 for calcite and gypsum inhibition and TX-15060 for silica inhibition. Nalco's existing dispersants HSP-1 and HSP-2 has excellent efficacy for dispersing Fe and Mn. ED and EDI were bench-scale tested by the CRADA partner Argonne National Laboratory for hardness, alkalinity and silica removal from synthetic make-up water and then cycled cooling water. Both systems showed low power consumption and 98-99% salt removal, however, the EDI system required 25-30% less power for silica removal. For Phase 2, the EDI system's performance was optimized and the length of time between clean-in-place (CIP) increased by varying the wafer composition and membrane configuration. The enhanced EDI system could remove 88% of the hardness and 99% of the alkalinity with a processing flux of 19.2 gal/hr/m{sup 2} and a power consumption of 0.54 kWh/100 gal water. Bench tests to screen alternative silica/silicate scale inhibitor chemistries have begun. The silica/silicate control approaches using chemical inhibitors include inhibition of silicic acid polymerization and dispersion of silica/silicate crystals. Tests were conducted with an initial silica concentration of 290-300 mg/L as SiO{sub 2} at pH 7 and room temperature. A proprietary new chemistry was found to be promising, compared with a current commercial product commonly used for silica/silicate control. Additional pilot cooling tower testing confirmed the bench study. We also developed a molecule to inhibit calcium carbonate precipitation and calcium sulfate precipitation at high supersaturations. During Phase 3, a long-term test of the EDI system and scale inhibitors was done at Nalco's cooling tower water testing facility, producing 850 gallons of high purity water (90+% salt removal) at a rate of 220 L/day. The EDI system's performance was stable when the salt concentration in the concentrate compartment (i.e. the EDI waste stream) was controlled and a CIP was done after every 48 hours of operation time. A combination of EDI and scale inhibitors completely eliminated blowdown discharge from the Pilot cooling Tower. The only water-consumption came from evaporation, CIP and EDI concentrate. Silica Inhibitor was evaluated in the field at a western coal fired power plant.« less

Production and Characterization of a New α-Glucosidase Inhibitory Peptide from Aspergillus oryzae N159-1

PubMed Central

Kang, Min-Gu; Yi, Sung-Hun

2013-01-01

An α-glucosidase inhibitor was developed from Aspergillus oryzae N159-1, which was screened from traditional fermented Korean foods. The intracellular concentration of the inhibitor reached its highest level when the fungus was cultured in tryptic soy broth medium at 27℃ for five days. The inhibitor was purified using a series of purification steps involving ultrafiltration, Sephadex G-25 gel permeation chromatography, strong cation exchange solid phase extraction, reverse-phase high performance liquid chromatography, and size exclusion chromatography. The final yield of the purification was 1.9%. Results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the purified α-glucosidase inhibitor was a tri-peptide, Pro-Phe-Pro, with the molecular weight of 360.1 Da. The IC50 value of the peptide against α-glucosidase activity was 3.1 mg/mL. Using Lineweaver-Burk plot analysis, the inhibition pattern indicated that the inhibitor acts as a mixed type inhibitor. PMID:24198670

Pancreatic polyamine concentrations and cholecystokinin plasma levels in rats after feeding raw or heat-inactivated soybean flour.

PubMed

Löser, C; Fölsch, U R; Mustroph, D; Cantor, P; Wunderlich, U; Creutzfeldt, W

1988-01-01

We investigated the trophic effect on the pancreas of male Wistar rats fed up to 20 days with either raw soybean flour (RSF) containing an active trypsin inhibitor or heat-inactivated soybean flour (HSF). The concentrations of the polyamines putrescine, spermidine, and spermine in the pancreas as well as cholecystokinin (CCK) concentrations in arterial and portal vein plasma were measured. Plasma CCK concentrations were measured by a sensitive radioimmunoassay specific for the sulfated region of CCK, whereas polyamine concentrations are determined by reversed phase high-performance liquid chromatography. The levels of CCK in both arterial and portal vein plasma were significantly higher in RSF- compared with HSF-fed rats, the concentration in the portal vein being twice as high compared with the aorta. A significant increase in pancreatic weight and protein content was positively correlated to an increase in putrescine and spermidine in the pancreas of RSF-fed rats compared with HSF-fed controls, whereas the spermine content did not differ between the two groups. The pancreatic DNA content in RSF-fed rats was significantly above control values of day 20 only. These data support the hypothesis that the trophic effect of soybean trypsin inhibitor on the pancreas is mediated by CCK and that polyamines might play an important role in CCK-induced pancreatic growth.

Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Espinoza, B.; Wharton, W.

1986-08-01

Cholera toxin produced a dose-dependent decrease in the restimulation of G0/G1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition inmore » the growth of sparse human fibrobasts. IBMX produced an inhibition both in the G1 and in the G2 phases of the cell cycle by a mechanism(s) that was not related to the magnitude of the increases in adenosine 3,5-cyclic monophosphate concentrations.« less

[Cellular uptake of TPS-L-carnitine synthesised as transporter-based renal targeting prodrug].

PubMed

Li, Li; Zhu, Di; Sun, Xun

2012-11-01

To synthesize transporter-based renal targeting prodrug TPS-L-Carnitine and to determine its cellular uptake in vitro. Triptolide (TP) was conjugated with L-carnitine using succinate as the linker to form TPS-L-Carnitine, which could be specifically recognized by OCTN2, a cationic transporter with high affinity to L-Carnitine and is highly expressed on the apical membrane of renal proximal tubule cells. Cellular uptake assays of the prodrug and its parent drug were performed on HK-2 cells, a human proximal tubule cell line, in different temperature, concentration and in the presence of competitive inhibitors. TPS-L-Carnitine was taken up into HK-2 cells in a saturable and temperature- and concentration-dependent manner. The uptake process could be inhibited by the competitive inhibitors. The uptake of TPS-L-Carnitine was significantly higher than that of TP at 37 degrees C in the same drug concentration. TPS-L-Carnitine was taken through endocytosis mediated by transporter. TPS-L-Carnitine provides a good renal targeting property and lays the foundation for further studies in vivo.

General Method to Determine the Flux of Charged Molecules through Nanopores Applied to β-Lactamase Inhibitors and OmpF.

PubMed

Ghai, Ishan; Pira, Alessandro; Scorciapino, Mariano Andrea; Bodrenko, Igor; Benier, Lorraine; Ceccarelli, Matteo; Winterhalter, Mathias; Wagner, Richard

2017-03-16

A major challenge in the discovery of the new antibiotics against Gram-negative bacteria is to achieve sufficiently fast permeation in order to avoid high doses causing toxic side effects. So far, suitable assays for quantifying the uptake of charged antibiotics into bacteria are lacking. We apply an electrophysiological zero-current assay using concentration gradients of β-lactamase inhibitors combined with single-channel conductance to quantify their flux rates through OmpF. Molecular dynamic simulations provide in addition details on the interactions between the nanopore wall and the charged solutes. In particular, the interaction barrier for three β-lactamase inhibitors is surprisingly as low as 3-5 kcal/mol and only slightly above the diffusion barrier of ions such as chloride. Within our macroscopic constant field model, we determine that at a zero-membrane potential a concentration gradient of 10 μM of avibactam, sulbactam, or tazobactam can create flux rates of roughly 620 molecules/s per OmpF trimer.

An instrument for automated purification of nucleic acids from contaminated forensic samples

PubMed Central

Broemeling, David J; Pel, Joel; Gunn, Dylan C; Mai, Laura; Thompson, Jason D; Poon, Hiron; Marziali, Andre

2008-01-01

Forensic crime scene sample analysis, by its nature, often deals with samples in which there are low amounts of nucleic acids, on substrates that often lead to inhibition of subsequent enzymatic reactions such as PCR amplification for STR profiling. Common substrates include denim from blue jeans, which yields indigo dye as a PCR inhibitor, and soil, which yields humic substances as inhibitors. These inhibitors frequently co-extract with nucleic acids in standard column or bead-based preps, leading to frequent failure of STR profiling. We present a novel instrument for DNA purification of forensic samples that is capable of highly effective concentration of nucleic acids from soil particulates, fabric, and other complex samples including solid components. The novel concentration process, known as SCODA, is inherently selective for long charged polymers such as DNA, and therefore is able to effectively reject known contaminants. We present an automated sample preparation instrument based on this process, and preliminary results based on mock forensic samples. PMID:18438455

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection.

PubMed

Giacobbi, Nicholas S; Sluis-Cremer, Nicolas

2017-07-01

Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. Copyright © 2017 American Society for Microbiology.

Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors

PubMed Central

Pain, Margaret; Fuller, Alexandra W.; Basore, Katherine; Pillai, Ajay D.; Solomon, Tsione; Bokhari, Abdullah A. B.; Desai, Sanjay A.

2016-01-01

Malaria parasites increase their host erythrocyte’s permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel’s structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing. PMID:26866812

Inhibition of matrix metalloproteinase-2 by PARP inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D.

2009-10-02

Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC{sub 50} values of PJ-34 and 5-AIQ were in the high micromolar range and comparablemore » to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.« less

Environmental risk assessment of three selective serotonin reuptake inhibitors in the aquatic environment: a case study including a cocktail scenario.

PubMed

Styrishave, Bjarne; Halling-Sørensen, Bent; Ingerslev, Flemming

2011-01-01

We present an environmental risk assessment of three selective serotonin reuptake inhibitors (SSRIs; citalopram, sertraline, and fluoxetine) in the aquatic environment based on two case scenarios. Abiotic and biotic degradation experiments and sorption estimates were used to predict environmental concentrations of three SSRIs from the wastewater of two psychiatric hospitals, the primary sector, and wastewater entering and leaving wastewater treatment plants (WWTPs). Assuming a sewage treatment retention time of 8 h, abiotic degradation was low, for all three SSRIs inhibitors, ranging between 0 and 2% for hydrolysis and 0 and 6% for photolysis. The biodegradation was also slow, ranging from 0 to 3% within an 8-h period. In untreated sewage, citalopram (CIT) and sertraline (SER) concentrations may be high enough to exert effects on the aquatic biota (CIT: 0.19-10.3 µg/L; SER: 0.14-17.1 µg/L). Removal of the pharmaceuticals is due primarily to sorption in the WWTP. Sertraline was estimated to have the highest concentrations in the sewage effluents, 4.4 and 19.9 ng/L for the two cases, respectively. In treated wastewater, individual SSRI concentrations are probably too low to exert effects on biota. By using concentration addition, a cocktail exposure scenario was estimated. The predicted concentration in the biota calculated from the cocktail effect was 0.05 and 0.16 nmol/g for the two cases, respectively, and SER was found to give the highest contribution to this cocktail effect. The results indicate that the concentrations in the wastewater effluents are one to two orders of magnitude lower than the concentrations likely to cause an effect in the aquatic biota. © 2010 SETAC.

Rapid, high-temperature, field test method for evaluation of geothermal calcium carbonate scale inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asperger, R.G.

1986-09-01

A new test method is described that allows the rapid field testing of calcium carbonate scale inhibitors at 500/sup 0/F (260/sup 0/C). The method evolved from use of a full-flow test loop on a well with a mass flow rate of about 1 x 10/sup 6/ lbm/hr (126 kg/s). It is a simple, effective way to evaluate the effectiveness of inhibitors under field conditions. Five commercial formulations were chosen for field evaluation on the basis of nonflowing, laboratory screening tests at 500/sup 0/F (260/sup 0/C). Four of these formulations from different suppliers controlled calcium carbonate scale deposition as measured bymore » the test method. Two of these could dislodge recently deposited scale that had not age-hardened. Performance-profile diagrams, which were measured for these four effective inhibitors, show the concentration interrelationship between brine calcium and inhibitor concentrations at which the formulations will and will not stop scale formation in the test apparatus. With these diagrams, one formulation was chosen for testing on the full-flow brine line. The composition was tested for 6 weeks and showed a dramatic decrease in the scaling occurring at the flow-control valve. This scaling was about to force a shutdown of a major, long-term flow test being done for reservoir economic evaluations. The inhibitor stopped the scaling, and the test was performed without interruption.« less

Recurrent selection to alter grain phytic acid concentration and iron bioavailability

USDA-ARS?s Scientific Manuscript database

Most of the phosphorus (P) in cereal grains is in the form of phytic acid, a potent inhibitor of iron absorption that cannot be digested by monogastric livestock or humans. High phytate content in staple crops contributes to the high incidence of iron deficiency in developing countries. Low phytic a...

Mode of action of the 2-phenylquinoline efflux inhibitor PQQ4R against Escherichia coli

PubMed Central

Machado, Diana; Fernandes, Laura; Costa, Sofia S.; Cannalire, Rolando; Manfroni, Giuseppe; Tabarrini, Oriana; Couto, Isabel; Sabatini, Stefano

2017-01-01

Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone – PQQ4R), against Escherichia coli, by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux in E. coli reducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of the E. coli inner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future adjuvants of conventional therapy against E. coli and other Gram-negative bacteria, especially their multidrug resistant forms. Their major limitation is the high toxicity for human cells at the concentrations needed to be effective against bacteria. Their future molecular optimization to improve the efflux inhibitory properties and reduce relative toxicity will optimize their potential for clinical usage against multi-drug resistant bacterial infections due to efflux. PMID:28516003

Uptake and intracellular activity of AM-1155 in phagocytic cells.

PubMed Central

Yamamoto, T; Kusajima, H; Hosaka, M; Fukuda, H; Oomori, Y; Shinoda, H

1996-01-01

The uptake and intracellular activity of AM-1155 in murine J774.1 macrophages and human polymorphonuclear leukocytes were investigated. AM-1155 penetrated phagocytic cells rapidly and reversibly, although the penetration process was not affected by metabolic inhibitors such as sodium fluoride, cyanide m-chlorophenylhydrazone, or ouabain or by nucleoside transport system inhibitors such as adenosine. The intracellular concentration-to-extracellular concentration ratio of AM-1155 in both cell types of phagocytes ranged from 5 to 7. These ratios were almost equal to those for sparfloxacin. The intracellular activity of AM-1155 in J774.1 macrophages, examined with Staphylococcus aureus 209P as a test bacterium, was dependent on the extracellular concentration. AM-1155 at a concentration of 1 microgram/ml reduced the number of viable cells of S. aureus ingested by more than 90%. The intracellular activity of AM-1155 was more potent than those of sparfloxacin, ofloxacin, ciprofloxacin, flomoxef, and erythromycin. These results suggest that the potent intracellular activity of AM-1155 might mainly be due to the high intracellular concentration and its potent in vitro activity. PMID:9124835

Identification of Inhibitors of ABCG2 by a Bioluminescence Imaging-based High-throughput Assay

PubMed Central

Zhang, Yimao; Byun, Youngjoo; Ren, Yunzhao R.; Liu, Jun O.; Laterra, John; Pomper, Martin G.

2009-01-01

ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)-based assay was developed to allow high-throughput compound screening. This assay exploits our finding that D-luciferin, the substrate of firefly luciferase (fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of D-luciferin and enhance bioluminescence signal by increasing intracellular D-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the US Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty seven compounds demonstrated BLI enhancement, a measure of anti-ABCG2 activity, of five-fold or greater, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g. mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from an FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing. PMID:19567678

The Use of Dried Blood Spots for Pharmacokinetic Monitoring of Vemurafenib Treatment in Melanoma Patients.

PubMed

Nijenhuis, Cynthia M; Huitema, Alwin D R; Marchetti, Serena; Blank, Christian; Haanen, John B A G; van Thienen, Johannes V; Rosing, Hilde; Schellens, Jan H M; Beijnen, Jos H

2016-10-01

Pharmacokinetic monitoring is increasingly becoming an important part of clinical care of tyrosine kinase inhibitor treatment. Vemurafenib is an oral tyrosine kinase inhibitor that inhibits mutated serine/threonine protein kinase B-Raf (BRAF) and is approved for the treatment of adult patients with BRAF V600 mutation-positive unresectable or metastatic melanoma. The aim of this study was to establish the relationship between dried blood spot (DBS) and plasma concentrations of vemurafenib to enable the use of DBS sampling, which is a minimally invasive form of sample collection. In total, 43 paired plasma and DBS samples (in duplicate) were obtained from 8 melanoma patients on vemurafenib therapy and were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Plasma concentrations were predicted from the DBS concentrations using 2 methods: (1) individual hematocrit correction and blood cell-to-plasma partitioning and (2) the calculated slope explaining the relationship between DBS and plasma concentrations (without individual hematocrit correction). Vemurafenib DBS concentrations and plasma concentrations showed a strong correlation (r = 0.964), and the relationship could be described by ([vemurafenib]plasma = [vemurafenib]DBS /0.64). The predicted plasma concentrations were within ±20% of the analyzed plasma concentrations in 97% and 100% of the samples for the methods with and without hematocrit correction, respectively. In conclusion, DBS concentrations and plasma concentrations of vemurafenib are highly correlated. Plasma concentrations can be predicted from DBS concentration using the blood cell-to-plasma partition and the average hematocrit value of this cohort (0.40 L/L). DBS sampling for pharmacokinetic monitoring of vemurafenib treatment can be used in clinical practice. © 2016, The American College of Clinical Pharmacology.

Glycine transporter2 inhibitors: Getting the balance right.

PubMed

Vandenberg, Robert J; Mostyn, Shannon N; Carland, Jane E; Ryan, Renae M

2016-09-01

Neurotransmitter transporters are targets for a wide range of therapeutically useful drugs. This is because they have the capacity to selectively manipulate the dynamics of neurotransmitter concentrations and thereby enhance or diminish signalling through particular brain pathways. High affinity glycine transporters (GlyTs) regulate extracellular concentrations of glycine and provide novel therapeutic targets for neurological disorders. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Inhibition of Sunn pest, Eurygaster integriceps, α-amylases by α-amylase inhibitors (T-αAI) from Triticale.

PubMed

Mehrabadi, Mohammad; Bandani, Ali R; Saadati, Fatemeh

2010-01-01

The effect of triticale α-amylases inhibitors on starch hydrolysis catalyzed by the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) midgut amylases was examined. Biochemical studgawies showed that inhibitors from Triticale (a hybrid of wheat and rye) had inhibitiory effects on E. integriceps α-amylases. The effects of the triticale α-amylase inhibitor (T-αAI) on α-amylase of E. integriceps showed a dose dependent manner of inhibition, e.g. less inhibition of enzyme activity (around 10%) with a lower dose (0.25 mg protein) and high inhibition of enzyme activity (around 80%) when a high dose of inhibitor was used (1.5 mg protein). The enzyme kinetic studies using Michaelis-Menten and Lineweaver-Burk equations showed the K(m) remained constant (0.58%) but the maximum velocity (V(max)) decreased in the presence of a crude extract of Triticale inhibitors, indicating mixed inhibition. The temperature giving 50% inactivation of enzyme (T(50)) during a 30-min incubation at pH 7.0 was 73° C. The maximum inhibitory activity was achieved at 35° C and pH 5.0. Gel assays showed the meaningful inhibition of E. integriceps α-amylases by various concentrations of Triticale inhibitors. Based on the data presented in this study, it could be said that the T-αAI has good inhibitory activity on E. integriceps gut α-amylase.

Inhibition of Sunn Pest, Eurygaster integriceps, α-Amylases by α-Amylase Inhibitors (T-αAI) from Triticale

PubMed Central

Mehrabadi, Mohammad; Bandani, Ali R.; Saadati, Fatemeh

2010-01-01

The effect of triticale α-amylases inhibitors on starch hydrolysis catalyzed by the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) midgut amylases was examined. Biochemical studgawies showed that inhibitors from Triticale (a hybrid of wheat and rye) had inhibitiory effects on E. integriceps α-amylases. The effects of the triticale α-amylase inhibitor (T-αAI) on α-amylase of E. integriceps showed a dose dependent manner of inhibition, e.g. less inhibition of enzyme activity (around 10%) with a lower dose (0.25 mg protein) and high inhibition of enzyme activity (around 80%) when a high dose of inhibitor was used (1.5 mg protein). The enzyme kinetic studies using Michaelis-Menten and Lineweaver-Burk equations showed the Km remained constant (0.58%) but the maximum velocity (Vmax) decreased in the presence of a crude extract of Triticale inhibitors, indicating mixed inhibition. The temperature giving 50% inactivation of enzyme (T50) during a 30-min incubation at pH 7.0 was 73° C. The maximum inhibitory activity was achieved at 35° C and pH 5.0. Gel assays showed the meaningful inhibition of E. integriceps α-amylases by various concentrations of Triticale inhibitors. Based on the data presented in this study, it could be said that the T-αAI has good inhibitory activity on E. integriceps gut α-amylase. PMID:21062146

Developing Hypothetical Inhibition Mechanism of Novel Urea Transporter B Inhibitor

NASA Astrophysics Data System (ADS)

Li, Min; Tou, Weng Ieong; Zhou, Hong; Li, Fei; Ren, Huiwen; Chen, Calvin Yu-Chian; Yang, Baoxue

2014-07-01

Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors.

Efficient dynamic molecular simulation using QSAR model to know inhibition activity in breast cancer medicine

NASA Astrophysics Data System (ADS)

Zharifah, A.; Kusumowardani, E.; Saputro, A.; Sarwinda, D.

2017-07-01

According to data from GLOBOCAN (IARC) at 2012, breast cancer was the highest rated of new cancer case by 43.3 % (after controlled by age), with mortality rated as high as 12.9 %. Oncology is a major field which focusing on improving the development of drug and therapeutics cancer in pharmaceutical and biotechnology companies. Nowadays, many researchers lead to computational chemistry and bioinformatic for pharmacophore generation. A pharmacophore describes as a group of atoms in the molecule which is considered to be responsible for a pharmacological action. Prediction of biological function from chemical structure in silico modeling reduces the use of chemical reagents so the risk of environmental pollution decreased. In this research, we proposed QSAR model to analyze the composition of cancer drugs which assumed to be homogenous in character and treatment. Atomic interactions which analyzed are learned through parameters such as log p as descriptors hydrophobic, n_poinas descriptor contour strength and molecular structure, and also various concentrations inhibitor (micromolar and nanomolar) from NCBI drugs bank. The differences inhibitor activity was observed by the presence of IC 50 residues value from inhibitor substances at various concentration. Then, we got a general overview of the state of safety for drug stability seen from its IC 50 value. In our study, we also compared between micromolar and nanomolar inhibitor effect from QSAR model results. The QSAR model analysis shows that the drug concentration with nanomolar is better than micromolar, related with the content of inhibitor substances concentration. This QSAR model got the equation: Log 1/IC50 = (0.284) (±0.195) logP + (0.02) (±0.012) n_poin + (-0.005) (±0.083) Inhibition10.2nanoM + (0.1) (±0.079) Inhibition30.5nanoM + (-0.016) (±0.045) Inhibition91.5nanoM + (-2.572) (±1.570) (n = 13; r = 0.813; r2 = 0.660; s = 0.764; F = 2.720; q2 = 0.660).

A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction.

PubMed

Andrade, Sheila Siqueira; Smaili, Soraya Soubhi; Monteforte, Priscila Totarelli; Miranda, Antônio; Kouyoumdjian, Maria; Sampaio, Misako Uemura; Lopes, Guiomar Silva; Oliva, Maria Luiza V

2012-09-01

BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B(2) receptors (B(2)R). BbKI was examined on smooth muscle contraction and Ca(2+) mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 μm) increased [Ca(2+)](c) and contraction, as observed with BK (2.0 μm). Not blocked by B(1) receptors (B(1)R), the BbKI agonistic effect was blocked by the B(2)R antagonist, HOE-140 (6 μm), and the involvement of B(2)R was confirmed in B(2)R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca(2+) release from internal storages, as a consequence of its interaction with B(2)R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B(2)R involvement.

The investigation of the cellular electrophysiological and antiarrhythmic effects of a novel selective sodium-calcium exchanger inhibitor, GYKB-6635, in canine and guinea-pig hearts.

PubMed

Geramipour, Amir; Kohajda, Zsófia; Corici, Claudia; Prorok, János; Szakonyi, Zsolt; Oravecz, Kinga; Márton, Zoltán; Nagy, Norbert; Tóth, András; Acsai, Károly; Virág, László; Varró, András; Jost, Norbert

2016-10-01

The sodium-calcium exchanger (NCX) is considered as the major transmembrane transport mechanism that controls Ca 2+ homeostasis. Its contribution to the cardiac repolarization has not yet been directly studied due to lack of specific inhibitors, so that an urgent need for more selective compounds. In this study, the electrophysiological effects of GYKB-6635, a novel NCX inhibitor, on the NCX, L-type calcium, and main repolarizing potassium currents as well as action potential (AP) parameters were investigated. Ion currents and AP recordings were investigated by applying the whole-cell patch clamp and standard microelectrode techniques in canine heart at 37 °C. Effects of GYKB-6635 were studied in ouabain-induced arrhythmias in isolated guinea-pig hearts. At a concentration of 1 μmol/L, GYKB significantly reduced both the inward and outward NCX currents (57% and 58%, respectively). Even at a high concentration (10 μmol/L), GYKB-6635 did not change the I CaL , the maximum rate of depolarization (dV/dt max ), the main repolarizing K + currents, and the main AP parameters. GYKB-6635 pre-treatment significantly delayed the time to the development of ventricular fibrillation (by about 18%). It is concluded that GYKB-6635 is a potent and highly selective inhibitor of the cardiac NCX and, in addition, it is suggested to also contribute to the prevention of DAD-based arrhythmias.

Mitochondrial Respiratory Chain Inhibitors Involved in ROS Production Induced by Acute High Concentrations of Iodide and the Effects of SOD as a Protective Factor

PubMed Central

Wang, Lingyan; Duan, Qi; Wang, Tingting; Ahmed, Mohamed; Zhang, Na; Li, Yongmei; Li, Lanying; Yao, Xiaomei

2015-01-01

A major source of reactive oxygen species (ROS) generation is the mitochondria. By using flow cytometry of the mitochondrial fluorescent probe, MitoSOX Red, western blot of mitochondrial ROS scavenger Peroxiredoxin (Prx) 3 and fluorescence immunostaining, ELISA of cleaved caspases 3 and 9, and TUNEL staining, we demonstrated that exposure to 100 μM KI for 2 hours significantly increased mitochondrial superoxide production and Prx 3 protein expression with increased expressions of cleaved caspases 3 and 9. Besides, we indicated that superoxide dismutase (SOD) at 1000 unit/mL attenuated the increase in mitochondrial superoxide production, Prx 3 protein expression, and lactate dehydrogenase (LDH) release and improved the relative cell viability at 100 μM KI exposure. However, SOD inhibitor diethyldithiocarbamic acid (DETC) (2 mM), Rotenone (0.5 μM), a mitochondrial complex I inhibitor, and Antimycin A (10 μM), a complex III inhibitor, caused an increase in mitochondrial superoxide production, Prx 3 protein expression, and LDH release and decreased the relative cell viability. We conclude that the inhibitors of mitochondrial respiratory chain complex I or III may be involved in oxidative stress caused by elevated concentrations of iodide, and SOD demonstrates its protective effect on the Fischer rat thyroid cell line (FRTL) cells. PMID:26294939

Berberine as a natural source inhibitor for mild steel in 1 M H 2SO 4

NASA Astrophysics Data System (ADS)

Li, Yan; Zhao, Peng; Liang, Qiang; Hou, Baorong

2005-12-01

Berberine was abstracted from coptis chinensis and its inhibition efficiency on corrosion of mild steel in 1 M H 2SO 4 was investigated through weight loss experiment, electrochemical techniques and scanning electronic microscope (SEM) with energy disperse spectrometer (EDS). The weight loss results showed that berberine is an excellent corrosion inhibitor for mild steel immersed in 1 M H 2SO 4. Potentiodynamic curves suggested that berberine suppressed both cathodic and anodic processes for its concentrations higher than 1.0 × 10 -4 M and mainly cathodic reaction was suppressed for lower concentrations. The Nyquist diagrams of impedance for mild steel in 1 M H 2SO 4 containing berberine with different concentrations showed one capacitive loop, and the polarization resistance increased with the inhibitor concentration rising. A good fit to Flory-Huggins isotherm was obtained between surface coverage degree and inhibitor concentration. The surface morphology and EDS analysis for mild steel specimens in sulfuric acid in the absence and presence of the inhibitor also proved the results obtained by the weight loss and electrochemical experiments. The correlation of inhibition effect and molecular structure of berberine was then discussed by quantum chemistry study.

Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes.

PubMed

Roger, V; Fonty, G; Komisarczuk-Bony, S; Gouet, P

1990-10-01

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R. flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic inhibitors, and lack of Na. In contrast, the attachment was affected by the removal of divalent cations (Mg and Ca), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine. Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at 100 degrees C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations (Mg and Ca), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large glycocalyx and the divalent cations Ca and Mg. Hydrophobic bonds and enzymes may also be involved.

Endothelium-dependent vasorelaxant effect of procyanidin B2 on human internal mammary artery.

PubMed

Novakovic, Aleksandra; Marinko, Marija; Jankovic, Goran; Stojanovic, Ivan; Milojevic, Predrag; Nenezic, Dragoslav; Kanjuh, Vladimir; Yang, Qin; He, Guo-Wei

2017-07-15

The aim of the present study was to investigate and characterize vasorelaxant effect of procyanidin B2 on human internal mammary artery (HIMA) as one of the mechanisms of its protective effect against vascular risk. Procyanidin B2 induced strong concentration-dependent relaxation of HIMA rings pre-contracted by phenylephrine. Pretreatment with L-NAME, a NO synthase inhibitor, hydroxocobalamin, a NO scavenger, and ODQ, an inhibitor of soluble guanylate cyclase, significantly inhibited procyanidin B2-induced relaxation of HIMA, while indomethacin, a cyclooxygenase inhibitor, considerably reduced effects of low concentrations. Among K + channel blockers, iberiotoxin, a selective blocker of large conductance Ca 2+ -activated K + channels (BK Ca ), abolished procyanidin B2-induced relaxation, glibenclamide, a selective ATP-sensitive K + (K ATP ) channels blocker, induced partial inhibition, while 4-aminopyridine, a blocker of voltage-gated K + (K V ) channels, and TRAM-34, an inhibitor of intermediate-conductance Ca 2+ -activated K + (IK Ca ) channels, slightly reduced maximal relaxation of HIMA. Further, procyanidin B2 relaxed contraction induced by phenylephrine in Ca 2+ -free Krebs solution, but had no effect on contraction induced by caffeine. Finally, thapsigargin, a sarcoplasmic reticulum Ca 2+ -ATPase inhibitor, significantly reduced relaxation of HIMA produced by procyanidin B2. These results demonstrate that procyanidin B2 produces endothelium-dependent relaxation of HIMA pre-contracted by phenylephrine. This effect is primarily the result of an increased NO synthesis and secretion by endothelial cells and partially of prostacyclin, although it involves activation of BK Ca and K ATP , as well as K V and IK Ca channels in high concentrations of procyanidin B2. Copyright © 2017 Elsevier B.V. All rights reserved.

Benzothiophene Carboxylate Derivatives as Novel Allosteric Inhibitors of Branched-chain α-Ketoacid Dehydrogenase Kinase*

PubMed Central

Tso, Shih-Chia; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L.; Qi, Xiangbing; Skvorak, Kristen J.; Dorko, Kenneth; Wallace, Amy L.; Morlock, Lorraine K.; Lee, Brendan H.; Hutson, Susan M.; Strom, Stephen C.; Williams, Noelle S.; Tambar, Uttam K.; Wynn, R. Max; Chuang, David T.

2014-01-01

The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation. BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC50 = 3.19 μm). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T½ = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations. PMID:24895126

Effects of the aspartic protease inhibitor from Lupinus bogotensis seeds on the growth and development of Hypothenemus hampei: an inhibitor showing high homology with storage proteins.

PubMed

Molina, Diana; Patiño, Luisa; Quintero, Mónica; Cortes, José; Bastos, Sara

2014-02-01

The coffee berry borer Hypothenemus hampei is a pest that causes great economic damage to coffee grains worldwide. Because the proteins consumed are digested by aspartic proteases in the insect's midgut, the inhibition of these proteases by transferring a gene encoding an aspartic protease inhibitor from Lupinus bogotensis Benth. to coffee plants could provide a promising strategy to control this pest. Five aspartic protease inhibitors from L. bogotensis (LbAPI) were accordingly purified and characterized. The gene encoding the L. bogotensis aspartic protease inhibitor (LbAPI), with the highest inhibitory activity against H. hampei, was expressed in Escherichia coli and the purified recombinant protein (rLbAPI), with a molecular mass of 15 kDa, was subsequently assessed for its ability to inhibit the aspartic protease activity present in the H. hampei midgut in vitro, as well as its effects on the growth and development of H. hampei in vivo. The in vitro experiments showed that rLbAPI was highly effective against aspartic proteases from H. hampei guts, with a half maximal inhibitory concentration (IC50) of 2.9 μg. The in vivo experiments showed that the concentration of rLbAPI (w/w) in the artificial diet necessary to cause 50% mortality (LD50) of the larvae was 0.91%. The amino acid sequence of LbAPI had high homology (52-80%) to the seed storage proteins, vicilin and β-conglutin, suggesting that this protein was generated by evolutionary events from a β-conglutin precursor. Based on these results, LbAPI may have a dual function as storage protein, and as defense protein against H. hampei. These results provide a promising alternative to obtain a coffee plant resistant to H. hampei. Copyright © 2013 Elsevier Ltd. All rights reserved.

High-fat diet-induced obesity leads to resistance to leptin-induced cardiomyocyte contractile response.

PubMed

Ren, Jun; Zhu, Bang-Hao; Relling, David P; Esberg, Lucy B; Ceylan-Isik, Asli F

2008-11-01

Levels of the obese gene product leptin are often elevated in obesity and may contribute to obesity-induced cardiovascular complications. However, the role of leptin in obesity-associated cardiac abnormalities has not been clearly defined. This study was designed to determine the influence of high-fat diet-induced obesity on cardiac contractile response of leptin. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix system in cardiomyocytes from adult rats fed low- and high-fat diets for 12 weeks. Cardiomyocyte contractile and intracellular Ca(2+) properties were examined including peak shortening, duration and maximal velocity of shortening/relengthening (TPS/TR(90), +/-dl/dt), Fura-2-fluorescence intensity change (DeltaFFI), and intracellular Ca(2+) decay rate (tau). Expression of the leptin receptor (Ob-R) was evaluated by western blot analysis. High-fat diet increased systolic blood pressure and plasma leptin levels. PS and +/-dl/dt were depressed whereas TPS and TR(90) were prolonged after high-fat diet feeding. Leptin elicited a concentration-dependent (0-1,000 nmol/l) inhibition of PS, +/-dl/dt, and DeltaFFI in low-fat but not high-fat diet-fed rat cardiomyocytes without affecting TPS and TR(90). The Janus kinase 2 (JAK2) inhibitor AG490, the mitogen-activated protein kinase (MAPK) inhibitor SB203580, and the nitric oxide synthase (NOS) inhibitor L-NAME abrogated leptin-induced cardiomyocyte contractile response in low-fat diet group without affecting the high-fat diet group. High-fat diet significantly downregulated cardiac expression of Ob-R. Elevation of extracellular Ca(2+) concentration nullified obesity-induced cardiomyocyte mechanical dysfunction and leptin-induced depression in PS. These data indicate presence of cardiac leptin resistance in diet-induced obesity possibly associated with impaired leptin receptor signaling.

Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid.

PubMed

Zhao, Xuebing; Peng, Feng; Du, Wei; Liu, Canming; Liu, Dehua

2012-08-01

Microbial lipid produced using yeast fermentation with inexpensive carbon sources such as lignocellulosic hydrolyzate can be an alternative feedstock for biodiesel production. Several inhibitors that can be generated during acid hydrolysis of lignocellulose were added solely or together into the culture medium to study their individual inhibitory actions and their synergistic effects on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides. When the inhibitors were present in isolation in the medium, to obtain a high cell biomass accumulation, the concentrations of formic acid, acetic acid, furfural and vanillin should be lower than 2, 5, 0.5 and 1.5 g/L, respectively. However, the synergistic effects of these compounds could dramatically decrease the minimum critical inhibitory concentrations leading to significant growth and lipid production inhibitions. Unlike the above-cited inhibitors, sodium lignosulphonate had no negative influence on biomass accumulation when its concentration was in the range of 0.5-2.0 g/L; in effect, it was found to facilitate cell growth and sugar-to-lipid conversion. The fatty acid compositional profile of the yeast lipid was in the compositional range of various plant oils and animal tallow. Finally, the crude yeast lipid from bagasse hydrolyzate could be well converted into fatty acid methyl ester (FAME, biodiesel) by enzymatic transesterification in a tert-butanol system with biodiesel yield of 67.2% and lipid-to-biodiesel conversion of 88.4%.

Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

PubMed

Vézina, C; Carcao, M; Infante-Rivard, C; Lillicrap, D; Stain, A M; Paradis, E; Teitel, J; Rivard, G E

2014-11-01

The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg(-1)  day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure. © 2014 John Wiley & Sons Ltd.

Estimating Margin of Exposure to Thyroid Peroxidase Inhibitors Using High-throughput In Vitro Data, High-throughput Exposure Modeling, and Physiologically-Based Pharmacokinetic/Pharmacodynamic Modeling

EPA Science Inventory

Some pharmaceuticals and environmental chemicals bind the thyroid peroxidase (TPO) enzyme and disrupt thyroid hormone production. The potential for TPO inhibition is a function of both the binding affinity and concentration of the chemical within the thyroid gland. The former can...

Discovery of N-(3-(5-((3-acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide (CHMFL-BTK-01) as a highly selective irreversible Bruton's tyrosine kinase (BTK) inhibitor.

PubMed

Liang, Qianmao; Chen, Yongfei; Yu, Kailin; Chen, Cheng; Zhang, Shouxiang; Wang, Aoli; Wang, Wei; Wu, Hong; Liu, Xiaochuan; Wang, Beilei; Wang, Li; Hu, Zhenquan; Wang, Wenchao; Ren, Tao; Zhang, Shanchun; Liu, Qingsong; Yun, Cai-Hong; Liu, Jing

2017-05-05

Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC 50 : 7 nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35) = 0.00) among 468 kinases/mutants at the concentration of 1 μM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC 50 : <30 nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Discovery of Novel Bruton's Tyrosine Kinase (BTK) Inhibitors Bearing a N,9-Diphenyl-9H-purin-2-amine Scaffold.

PubMed

Ge, Yang; Jin, Yue; Wang, Changyuan; Zhang, Jianbin; Tang, Zeyao; Peng, Jinyong; Liu, Kexin; Li, Yanxia; Zhou, Youwen; Ma, Xiaodong

2016-12-08

Based on the pyrimidine skeleton of EGFR T790M inhibitors, a series of N ,9-diphenyl-9 H -purin-2-amine derivatives were identified as effective BTK inhibitors. Among these compounds, inhibitors 10d , 10i , and 10j , possessing IC 50 values of 0.5, 0.5, and 0.4 nM, displayed anti-BTK kinase activity that was as potent as the reference compounds. In particular, compound 10j suppressed the proliferation of two typical B-cell leukemia cell lines expressing high levels of BTK with concentrations of 7.75 and 12.6 μM. The activity of the subject compound as determined by the CCK-8 method and apoptosis analysis validated that inhibitor 1 0j is slightly more potent than AVL-292 and ibrutinib. The results of these experimental explorations suggested that 10j could serve as a valuable molecule for control of leukemia pending further developments.

New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase: design, synthesis, crystal structures, and biological evaluation.

PubMed

Zidar, Nace; Tomašić, Tihomir; Šink, Roman; Kovač, Andreja; Patin, Delphine; Blanot, Didier; Contreras-Martel, Carlos; Dessen, Andréa; Premru, Manica Müller; Zega, Anamarija; Gobec, Stanislav; Mašič, Lucija Peterlin; Kikelj, Danijel

2011-11-01

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Identification and Characterization of Inhibitors of West Nile Virus

PubMed Central

Puig-Basagoiti, Francesc; Qing, Min; Dong, Hongping; Zhang, Bo; Zou, Gang; Yuan, Zhiming

2011-01-01

Although flaviviruses cause significant human diseases, no antiviral therapy is currently available for clinical treatment of these pathogens. To identify flavivirus inhibitors, we performed a high-throughput screening of compound libraries using cells containing luciferase-reporting replicon of West Nile viruses (WNV). Five novel small molecular inhibitors of WNV were identified from libraries containing 96,958 compounds. The inhibitors suppress epidemic strain of WNV in cell culture, with EC50 (50% effective concentration) values of <10 µM and TI (therapeutic index) values of >10. Viral titer reduction assays, using various flaviviruses and nonflaviviruses, showed that the compounds have distinct antiviral spectra. Mode-of-action analysis showed that the inhibitors block distinct steps of WNV replication: four compounds inhibit viral RNA syntheses, while the other compound suppresses both viral translation and RNA syntheses. Biochemical enzyme assays showed that two compounds selectively inhibit viral RNA-dependent RNA polymerase (RdRp), while another compound specifically inhibits both RdRp and methyltransferase. The identified compounds could potentially be developed for treatment of flavivirus infections. PMID:19501258

Detoxification of acidic catalyzed hydrolysate of Kappaphycus alvarezii (cottonii).

PubMed

Meinita, Maria Dyah Nur; Hong, Yong-Ki; Jeong, Gwi-Taek

2012-01-01

Red seaweed, Kappaphycus alvarezii, holds great promise for use in biofuel production due to its high carbohydrate content. In this study, we investigated the effect of fermentation inhibitors to the K. alvarezii hydrolysate on cell growth and ethanol fermentation. In addition, detoxification of fermentation inhibitors was performed to decrease the fermentation inhibitory effect. 5-Hydroxymethylfurfural and levulinic acid, which are liberated from acidic hydrolysis, was also observed in the hydrolysate of K. alvarezii. These compounds inhibited ethanol fermentation. In order to remove these inhibitors, activated charcoal and calcium hydroxide were introduced. The efficiency of activated charcoals was examined and over-liming was used to remove the inhibitors. Activated charcoal was found to be more effective than calcium hydroxide to remove the inhibitors. Detoxification by activated charcoal strongly improved the fermentability of dilute acid hydrolysate in the production of bioethanol from K. alvarezii with Saccharomyces cerevisiae. The optimal detoxifying conditions were found to be below an activated charcoal concentration of 5%.

Effects of sample handling methods on substance P concentrations and immunoreactivity in bovine blood samples.

PubMed

Mosher, Ruby A; Coetzee, Johann F; Allen, Portia S; Havel, James A; Griffith, Gary R; Wang, Chong

2014-02-01

To determine the effects of protease inhibitors and holding times and temperatures before processing on the stability of substance P in bovine blood samples. Blood samples obtained from a healthy 6-month-old calf. Blood samples were dispensed into tubes containing exogenous substance P and 1 of 6 degradative enzyme inhibitor treatments: heparin, EDTA, EDTA with 1 of 2 concentrations of aprotinin, or EDTA with 1 of 2 concentrations of a commercially available protease inhibitor cocktail. Plasma was harvested immediately following collection or after 1, 3, 6, 12, or 24 hours of holding at ambient (20.3° to 25.4°C) or ice bath temperatures. Total substance P immunoreactivity was determined with an ELISA; concentrations of the substance P parent molecule, a metabolite composed of the 9 terminal amino acids, and a metabolite composed of the 5 terminal amino acids were determined with liquid chromatography-tandem mass spectrometry. Regarding blood samples processed immediately, no significant differences in substance P concentrations or immunoreactivity were detected among enzyme inhibitor treatments. In blood samples processed at 1 hour of holding, substance P parent molecule concentration was significantly lower for ambient temperature versus ice bath temperature holding conditions; aprotinin was the most effective inhibitor of substance P degradation at the ice bath temperature. The ELISA substance P immunoreactivity was typically lower for blood samples with heparin versus samples with other inhibitors processed at 1 hour of holding in either temperature condition. Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.

Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia Pestis Outer Protein H (YopH) Phosphatase†, §, ¶

PubMed Central

Bahta, Medhanit; Lountos, George T.; Dyas, Beverly; Kim, Sung-Eun; Ulrich, Robert G.; Waugh, David S.; Burke, Terrence R.

2011-01-01

Our current study reports the first KM optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (KM = 80 μM was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime-ligation. A co-crystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime derivatives. By this process, a potent (IC50 = 190 nM) and non-promiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a non-cytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH. PMID:21443195

An overview of the new frontiers in the treatment of atherogenic dyslipidemias.

PubMed

Rached, F H; Chapman, M J; Kontush, A

2014-07-01

Cardiovascular diseases (CVDs) are the leading cause of morbidity/mortality worldwide. Dyslipidemia is a major risk factor for premature atherosclerosis and CVD. Lowering low-density-lipoprotein cholesterol (LDL-C) levels is well established as an intervention for the reduction of CVDs. Statins are the first-line drugs for treatment of dyslipidemia, but they do not address all CVD risk. Development of novel therapies is ongoing and includes the following: (i) reduction of LDL-C concentrations using antibodies to proprotein convertase subtilisin/kexin-9, antisense oligonucleotide inhibitors of apolipoprotein B production, microsomal transfer protein (MTP) inhibitors, and acyl-coenzyme A cholesterol acyl transferase inhibitors; (ii) reduction in levels of triglyceride-rich lipoproteins with ω-3 fatty acids, MTP inhibitors, and diacylglycerol acyl transferase-1 inhibitors; and (iii) increase of high-density-lipoprotein (HDL) cholesterol levels, HDL particle numbers, and/or HDL functionality using cholesteryl ester transfer protein inhibitors, HDL-derived agents, apolipoprotein AI mimetic peptides, and microRNAs. Large prospective outcome trials of several of these emerging therapies are under way, and thrilling progress in the field of lipid management is anticipated.

Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase

PubMed Central

Babaoglu, Kerim; Simeonov, Anton; Irwin, John J.; Nelson, Michael E.; Feng, Brian; Thomas, Craig J.; Cancian, Laura; Costi, M. Paola; Maltby, David A.; Jadhav, Ajit; Inglese, James; Austin, Christopher P.; Shoichet, Brian K.

2009-01-01

High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate “hit lists”; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against β-lactamase using quantitative HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting β-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 µM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens. PMID:18333608

Biological evaluation of some uracil derivatives as potent glutathione reductase inhibitors

NASA Astrophysics Data System (ADS)

Güney, Murat; Ekinci, Deniz; Ćavdar, Huseyin; Şentürk, Murat; Zilbeyaz, Kani

2016-04-01

Discovery of glutathione reductase (GR) inhibitors has become very popular recently due to antimalarial and anticancer activities. In this study, GR inhibitory capacities of some uracil derivatives (UDCs) (1-4) were reported. Some commercially available molecules (5-6) were also tested for comparison reasons. The novel UDCs were obtained in high yields using simple chemical procedures and exhibited much potent inhibitory activities against GR at low nanomolar concentrations with IC50 values ranging from 2.68 to 166.6 nM as compared with well-known agents.

Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

PubMed

Förstner, Philip; Bayer, Fabienne; Kalu, Nnanya; Felsen, Susanne; Förtsch, Christina; Aloufi, Abrar; Ng, David Y W; Weil, Tanja; Nestorovich, Ekaterina M; Barth, Holger

2014-07-14

Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.

Cationic PAMAM Dendrimers as Pore-Blocking Binary Toxin Inhibitors

PubMed Central

2015-01-01

Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria. PMID:24954629

Novel Inhibitors of Mycobacterium tuberculosis dTDP-6-deoxy-L-lyxo-4-hexulose Reductase (RmlD) Identified by Virtual Screening

PubMed Central

Wang, Yi; Hess, Tamara Noelle; Jones, Victoria; Zhou, Joe Zhongxiang; McNeil, Michael R.; McCammon, J. Andrew

2011-01-01

The complex and highly impermeable cell wall of Mycobacterium tuberculosis (Mtb) is largely responsible for the ability of the mycobacterium to resist the action of chemical therapeutics. An L-rhamnosyl residue, which occupies an important anchoring position in the Mtb cell wall, is an attractive target for novel anti-tuberculosis drugs. In this work, we report a virtual screening (VS) study targeting Mtb dTDP-deoxy-L-lyxo-4-hexulose reductase (RmlD), the last enzyme in the L-rhamnosyl synthesis pathway. Through two rounds of VS, we have identified four RmlD inhibitors with half inhibitory concentrations of 0.9-25 μM, and whole-cell minimum inhibitory concentrations of 20-200 μg/ml. Compared with our previous high throughput screening targeting another enzyme involved in L-rhamnosyl synthesis, virtual screening produced higher hit rates, supporting the use of computational methods in future anti-tuberculosis drug discovery efforts. PMID:22014548

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study.

PubMed

Cheng, Mengxia; Chen, Zilin

2017-08-01

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis-Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half-maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

PubMed

Marley, K; Maier, C S; Helfand, S C

2012-09-01

Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

Synthesis and study of thiocarbonate derivatives of choline as potential inhibitors of acetylcholinesterase.

PubMed

Boyle, N A; Talesa, V; Giovannini, E; Rosi, G; Norton, S J

1997-09-12

Fourteen alkyl and aryl thiocarbonate derivatives of choline were synthesized and studied as potential inhibitors of acetylcholinesterase (AChE). Twelve of the compounds inhibited AChEs derived from calf forebrain, human red blood cells, and octopus brain ranging from low to moderately high inhibition potency. The concentration of each inhibitory compound giving 50% inhibition of enzyme activity (IC50 values, which ranged from 1 x 10(-2) to 8 x 10(-7) M) was determined and is reported; inhibitor constants (Ki values) for the most inhibitory compounds, (1-pentylthiocarbonyl)choline chloride and (1-heptylthiocarbonyl)choline chloride, were calculated from kinetic data and are also reported. The inhibitors are competitive with substrate, and they are not hydrolyzed by the AChE activities. Certain of these new compounds may provide direction for the development of new drugs that have anticholinesterase activity and may be used for the treatment of Alzheimer's disease.

Identification of Broad-Based HIV-1 Protease Inhibitors From Combinatorial Libraries

PubMed Central

Chang, Max W.; Giffin, Michael J.; Muller, Rolf; Savage, Jeremiah; Lin, Ying C.; Hong, Sukwon; Jin, Wei; Whitby, Landon R.; Elder, John H.; Boger, Dale L.; Torbett, Bruce E.

2011-01-01

Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature, infectious viral particle. Advances in chemical synthesis have led to the development of new chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified that inhibits wild type protease, as well as a drug-resistant protease with 6 mutations. Moreover, analysis of this compound suggests an allosteric, non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy. PMID:20507280

Effect of lignocellulose-derived inhibitors on the growth and D-lactic acid production of Sporolactobacillus inulinus YBS1-5.

PubMed

Bai, Zhongzhong; Gao, Zhen; He, Bingfang; Wu, Bin

2015-10-01

The impact of lignocellulose-derived inhibitors on the cell growth and D-lactic production of Sporolactobacillus inulinus YBS1-5 was investigated. At high concentrations, both furans and phenolics, such as furfural, HMF, syringaldehyde and vanillin, affected cell growth and D-lactic acid production and syringaldehyde exhibited the highest. Further experiments showed that only vanillin caused cellular membrane damage. Based on the Biolog approach, in vivo studies on intact S. inulinus cells indicated that phenolics had a stronger inhibitory effect than furan derivatives on the metabolic activity of the concerned substrates related with the key enzymes of D-lactic acid fermentation. The direct in vitro inhibitory effect of the model compounds on the four key enzymes displayed similar patterns. Syringaldehyde was the strongest inhibitor. In general, comparison with published results for other microorganisms indicated that strain YBS1-5 was a robust microorganism against inhibitors of lignocellulose hydrolysate. Notably, in concentrated corn stover hydrolysate, S. inulinus YBS1-5 produced 70.7 g/L D-lactic acid, which was 87.7 % of the yield from the control experiment. However, the fermentation time was prolonged 36 h. In order to improve fermentation rate, a detoxification technology or more robust mutant to phenolics especially syringaldehyde should be developed.

Depolymerized products of lambda-carrageenan as a potent angiogenesis inhibitor.

PubMed

Chen, Haimin; Yan, Xiaojun; Lin, Jing; Wang, Feng; Xu, Weifeng

2007-08-22

Since angiogenesis is involved in initiating and promoting several diseases such as cancer and cardiovascular events, this study was designed to evaluate the anti-angiogenesis of low-molecular-weight (LMW), highly sulfated lambda-carrageenan oligosaccharides (lambda-CO) obtained by carrageenan depolymerization, by CAM (chick chorioallantoic membrane) model and human umbilical vein endothelial cells (HUVECs). Significant inhibition of vessel growth was observed at 200 microg/pellet. A histochemistry assay also revealed a decrease of capillary plexus and connective tissue in lambda-CO treated samples. lambda-CO inhibited the viability of cells at the high concentration of 1 mg/mL, whereas it affected the cell survival slightly (>95%) at a low concentration (<250 microg/mL), and HUVEC is the most sensitive to lambda-CO among three kinds of cells. Furthermore, the inhibitory action of lambda-CO was also observed in the endothelial cell invasion and migration at relatively low concentration (150-300 microg/mL), through down-regulation of intracellular matrix metalloproteinases (MMP-2) expression on endothelial cells. Taken together, these findings demonstrate that lambda-CO is a potential angiogenesis inhibitor with combined effects of inhibiting invasion, migration, and proliferation.

Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein

PubMed Central

Rehm, Markus; Huber, Heinrich J; Dussmann, Heiko; Prehn, Jochen H M

2006-01-01

Activation of effector caspases is a final step during apoptosis. Single-cell imaging studies have demonstrated that this process may occur as a rapid, all-or-none response, triggering a complete substrate cleavage within 15 min. Based on biochemical data from HeLa cells, we have developed a computational model of apoptosome-dependent caspase activation that was sufficient to remodel the rapid kinetics of effector caspase activation observed in vivo. Sensitivity analyses predicted a critical role for caspase-3-dependent feedback signalling and the X-linked-inhibitor-of-apoptosis-protein (XIAP), but a less prominent role for the XIAP antagonist Smac. Single-cell experiments employing a caspase fluorescence resonance energy transfer substrate verified these model predictions qualitatively and quantitatively. XIAP was predicted to control this all-or-none response, with concentrations as high as 0.15 μM enabling, but concentrations >0.30 μM significantly blocking substrate cleavage. Overexpression of XIAP within these threshold concentrations produced cells showing slow effector caspase activation and submaximal substrate cleavage. Our study supports the hypothesis that high levels of XIAP control caspase activation and substrate cleavage, and may promote apoptosis resistance and sublethal caspase activation in vivo. PMID:16932741

Inhibition of Cell Wall-Associated Enzymes in Vitro and in Vivo with Sugar Analogs

PubMed Central

Nagahashi, Gerald; Tu, Shu-I; Fleet, George; Namgoong, Sun K.

1990-01-01

Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with β-glucosidase, β-GIcNAcase, and α-mannosidase, respectively. Swainsonine specifically inhibited α-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited β-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised. PMID:16667291

Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function. [UV radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minor, P.D.; Dimmock, N.J.

1977-05-15

Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and ..cap alpha..-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at highmore » concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent.« less

Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases by 2-thiohydantoin compounds

PubMed Central

Kogiso, Mari; Yao, Yuan; Zhou, Chao; Li, Xiao-Nan; Song, Yongcheng

2015-01-01

Somatic mutations of isocitrate dehydrogenase 1 (IDH1) at R132 are frequently found in certain cancers such as glioma. With losing the activity of wild-type IDH1, the R132H and R132C mutant proteins can reduce α-ketoglutaric acid (α-KG) to D-2-hydroxyglutaric acid (D2HG). The resulting high concentration of D2HG inhibits many α-KG-dependent dioxygenases, including histone demethylases, to cause broad histone hypermethylation. These aberrant epigenetic changes are responsible for initiation of these cancers. We report the synthesis, structure activity relationships, enzyme kinetics and binding thermodynamics of a novel series of 2-thiohydantoin and related compounds, among which several compounds are potent inhibitors of mutant IDH1 with Ki as low as 420 nM. X-ray crystal structures of IDH1(R132H) in complex with two inhibitors are reported, showing their inhibitor-protein interactions. These compounds can decrease the cellular concentration of D2HG, reduce the levels of histone methylation, and suppress proliferation of stem-like cancer cells in BT142 glioma with IDH1 R132H mutation. PMID:26280302

Assessment of urinary inhibitor or promoter activity in uric acid nephrolithiasis

PubMed Central

Doizi, Steeve; Rodgers, Kathy; Poindexter, John; Sakhaee, Khashayar; Maalouf, Naim M.

2017-01-01

Purpose To assess the presence of a reduced inhibitor activity or an increased promoter activity in urine of idiopathic uric acid stone formers (IUASF) compared to non-stone formers (NSF) independent of urinary pH. Methods 30 IUASF, 9 obese NSF and 12 lean NSF collected 24-hour urine under metabolic diet. Three urine aliquots per subject were used to assess spontaneous nucleation (SN, de novo crystal formation), crystal growth (CG) using a 0.1 mg/mL seed of anhydrous uric acid (UA) and steady state (SS) of UA solubility using a 5 mg/mL seed of UA (assessing maximum amount of UA dissolvable in urine). All experiments were conducted for 6 hours at a constant pH of 5.0. UA concentration was measured in filtered aliquots at 0, 3 and 6 hours. Results At baseline, 24-hour urinary pH was significantly lower and UA saturation significantly higher in IUASF. No significant SN occurred and a similar SS UA concentration was reached in the three groups. IUASF and lean NSF displayed a similar decrease in UA concentration during CG, while obese NSF started with higher UA concentration and consequently displayed higher magnitude of decrease in UA concentration for CG. Conclusions This study suggests that there is no significant difference between IUASF and NSF in terms of promoter or inhibitor activity in whole urine against UA stone formation when urine pH is maintained constant. The findings suggest that UA stone formation is dictated by a high urinary saturation with respect to UA, driven primarily by a low urine pH. PMID:26723865

Study of caffeine as corrosion inhibitors of carbon steel in chloride solution containing hydrogen sulfide using electrochemical impedance spectroscopy (EIS)

NASA Astrophysics Data System (ADS)

Solehudin, Agus; Berman, Ega Taqwali; Nurdin, Isdiriayani

2015-09-01

The corrosion behaviour of steel surface in the absence and presence of caffeine in 3.5% NaCl solution containing dissolved H2S gas is studied using electrochemical impedance spectroscopy (EIS). The experimental results of carbon steel corrosion in 3.5% NaCl solution containing 500 mg/l H2S at different caffeine concentrations showed that corrosion rate of carbon steel decreases with increasing of caffeine concentrations from 0 to 0,1 mmol/l. Whereas, the corrosion rate increase with increasing of caffeine concentrations from 1 to 10 mmol/l. It is clear that no inhibition efficiency increases with increasing inhibitor concentration. The optimum value of inhibition efficiency was 90% at a caffeine concentration of 0.1 mmol/l. This suggests that caffeine's performance as a corrosion inhibitor is more effective at a concentration of 0.1 mmol/l.

Comparative Study on Corrosion Protection of Reinforcing Steel by Using Amino Alcohol and Lithium Nitrite Inhibitors

PubMed Central

Lee, Han-Seung; Ryu, Hwa-Sung; Park, Won-Jun; Ismail, Mohamed A.

2015-01-01

In this study, the ability of lithium nitrite and amino alcohol inhibitors to provide corrosion protection to reinforcing steel was investigated. Two types of specimens—reinforcing steel and a reinforced concrete prism that were exposed to chloride ion levels resembling the chloride attack environment—were prepared. An autoclave accelerated corrosion test was then conducted. The variables tested included the chloride-ion concentration and molar ratios of anti-corrosion ingredients in a CaOH2-saturated aqueous solution that simulated a cement-pore solution. A concentration of 25% was used for the lithium nitrite inhibitor LiNO2, and an 80% solution of dimethyl ethanolamine ((CH3)2NCH2CH2OH, hereinafter DMEA) was used for the amino alcohol inhibitor. The test results indicated that the lithium nitrite inhibitor displayed anti-corrosion properties at a molar ratio of inhibitor of ≥0.6; the amino alcohol inhibitor also displayed anti-corrosion properties at molar ratios of inhibitor greater than approximately 0.3. PMID:28787936

Structure-activity relationship of cyanine tau aggregation inhibitors

PubMed Central

Chang, Edward; Congdon, Erin E.; Honson, Nicolette S.; Duff, Karen E.; Kuret, Jeff

2009-01-01

A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3’-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (≥300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity. PMID:19432420

Small Molecule Ice Recrystallization Inhibitors Enable Freezing of Human Red Blood Cells with Reduced Glycerol Concentrations

PubMed Central

Capicciotti, Chantelle J.; Kurach, Jayme D. R.; Turner, Tracey R.; Mancini, Ross S.; Acker, Jason P.; Ben, Robert N.

2015-01-01

In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at −80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells. PMID:25851700

Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations.

PubMed

Capicciotti, Chantelle J; Kurach, Jayme D R; Turner, Tracey R; Mancini, Ross S; Acker, Jason P; Ben, Robert N

2015-04-08

In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at -80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells.

A novel inhibitor of Lactobacillus biofilms prevents stuck fermentations in a shake flask model

USDA-ARS?s Scientific Manuscript database

Yeast ethanol fermentations contain contaminating bacteria and yeast, with Lactobacilli being a frequent contaminant. These bacteria tolerate the low pH and high ethanol concentrations present in the fermentation, and can decrease the ethanol yield. Fermentations are routinely treated with antibioti...

Study of benzotriazole as corrosion inhibitors of carbon steel in chloride solution containing hydrogen sulfide using electrochemical impedance spectroscopy (EIS)

NASA Astrophysics Data System (ADS)

Solehudin, Agus; Nurdin, Isdiriayani

2014-03-01

Corrosion and inhibition studies on API 5LX65 carbon steel in chloride solution containing various concentrations of benzotriazole has been conducted at temperature of 70°C using Electrochemical Impedance Spectroscopy (EIS). Corroded carbon steel surface with and without inhibitor have been observed using X-ray Diffraction (XRD), Scanning Electron Microscope (SEM), and Energy Dispersive Spectroscopy (EDS). The objectives of this research are to study the performance of benzotriazole as corrosion inhibitors. The experimental results of carbon steel corrosion in 3.5% NaCl solution containing 500 mg/l H2S at different BTAH concentrations showed that corrosion rate of carbon steel decreases with increasing of BTAH concentrations from 0 to 10 mmol/l. The inhibition efficiency of BTAH was found to be affected by its concentration. The optimum efficiency obtained of BTAH is 93% at concentration of 5 mmol/l. The result of XRD and EDS analysis reveal the iron sulfide (FeS) formation on corroded carbon steel surface without inhibitor. The EDS spectrum show the Nitrogen (N) bond on carbon steel surface inhibited by BTAH.

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

PubMed Central

Wang, Yiming; Gratzke, Christian; Tamalunas, Alexander; Wiemer, Nicolas; Ciotkowska, Anna; Rutz, Beata; Waidelich, Raphaela; Strittmatter, Frank; Liu, Chunxiao; Stief, Christian G.; Hennenberg, Martin

2016-01-01

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions. PMID:27071060

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate.

PubMed

Wang, Yiming; Gratzke, Christian; Tamalunas, Alexander; Wiemer, Nicolas; Ciotkowska, Anna; Rutz, Beata; Waidelich, Raphaela; Strittmatter, Frank; Liu, Chunxiao; Stief, Christian G; Hennenberg, Martin

2016-01-01

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients' adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1-10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics.

PubMed

Liu, Qingsong; Kirubakaran, Sivapriya; Hur, Wooyoung; Niepel, Mario; Westover, Kenneth; Thoreen, Carson C; Wang, Jinhua; Ni, Jing; Patricelli, Matthew P; Vogel, Kurt; Riddle, Steve; Waller, David L; Traynor, Ryan; Sanda, Takaomi; Zhao, Zheng; Kang, Seong A; Zhao, Jean; Look, A Thomas; Sorger, Peter K; Sabatini, David M; Gray, Nathanael S

2012-03-23

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

The effect of functionalized polycarboxylate structures as corrosion inhibitors in a simulated concrete pore solution

NASA Astrophysics Data System (ADS)

Fazayel, A. S.; Khorasani, M.; Sarabi, A. A.

2018-05-01

In this study, the effects of polycarboxylate derivatives with different comonomers and functional groups on the control or reduction of corrosion in steel specimens were evaluated through electrochemical impedance spectroscopy (EIS) and potentiodynamic analysis. A highly alkaline contaminated concrete pore solution (CPS) containing chlorides was used to simulate the pitting corrosion, and according to the results, the mechanism of inhibitive action was determined. Both the inhibition efficiency and pitting corrosion inhibition of methacrylate-copolymers were in the order of poly methacrylate-co acrylamide > poly methacrylate-co-2-acrylamido-2 methylpropane sulfonic acid > poly methacrylate-co-hydroxyethyl methacrylate. In addition, the corrosion potential of steel specimens in all studied concentrations of NaCl with different concentrations of polymethacrylate-co acrylamide (as the best inhibitor in this study) in saturated Ca(OH)2 solution showed almost an identical trend. Polymethacrylic acid-co-acrylamide showed a 92.35% inhibitor efficiency in the saturated Ca(OH)2 solution containing 1.8 wt.% chlorides and could effectively reduce the corrosion rate. Even at 3.5 wt.% of NaCl, this inhibitor could remarkably reduce the destructive effect of chloride ion attacks on the steel surface and passive film. The inhibition effect of these polymeric inhibitors seemed to be due to the formation of a barrier layer on the metal surface, approved by the well-known adsorption mechanism of organic molecules at the metal/solution interface. The results of SEM, EDS and AFM investigations were also in agreement with the outcomes of electrochemical studies.

A 2,4-dichlorophenoxyacetic acid analog screened using a maize coleoptile system potentially inhibits indole-3-acetic acid influx in Arabidopsis thaliana

PubMed Central

Suzuki, Hiromi; Matano, Naoyuki; Nishimura, Takeshi; Koshiba, Tomokazu

2014-01-01

Studies using inhibitors of indole-3-acetic acid (IAA) transport, not only for efflux but influx carriers, provide many aspects of auxin physiology in plants. 1-Naphtoxyacetic acid (1-NOA), an analog of the synthetic auxin 1-N-naphtalene acetic acid (NAA), inhibits the IAA influx carrier AUX1. However, 1-NOA also shows auxin activity because of its structural similarity to NAA. In this study, we have identified another candidate inhibitor of the IAA influx carrier. The compound, “7-B3; ethyl 2-[(2-chloro-4-nitrophenyl)thio]acetate,” is a 2,4-dichlorophenoxyacetic acid (2,4-D) analog. At high concentrations (> 300 µM), 7-B3 slightly reduced IAA transport and tropic curvature of maize coleoptiles, whereas lower concentrations had almost no effect. We have analyzed the effects of 7-B3 on Arabidopsis thaliana seedlings. 7-B3 rescued the 2,4-D-inhibited root elongation, but not the NAA-inhibited root elongation. The effect of 7-B3 was weaker than that of 1-NOA. Both 1-NOA and 7-B3 inhibited DR5::GUS expression induced by IAA and 2,4-D, but not that induced by NAA. At high concentrations, 1-NOA exhibited auxin activity, but 7-B3 did not. Furthermore, 7-B3 inhibited apical hook formation in etiolated seedlings more effectively than 1-NOA did. These results indicate that 7-B3 is a potential inhibitor of IAA influx that has almost no effect on IAA efflux or auxin signaling. PMID:24800738

Use of Gelidium amansii as a promising resource for bioethanol: a practical approach for continuous dilute-acid hydrolysis and fermentation.

PubMed

Park, Jeong-Hoon; Hong, Ji-Yeon; Jang, Hyun Chul; Oh, Seung Geun; Kim, Sang-Hyoun; Yoon, Jeong-Jun; Kim, Yong Jin

2012-03-01

A facile continuous method for dilute-acid hydrolysis of the representative red seaweed species, Gelidium amansii was developed and its hydrolysate was subsequently evaluated for fermentability. In the hydrolysis step, the hydrolysates obtained from a batch reactor and a continuous reactor were systematically compared based on fermentable sugar yield and inhibitor formation. There are many advantages to the continuous hydrolysis process. For example, the low melting point of the agar component in G. amansii facilitates improved raw material fluidity in the continuous reactor. In addition, the hydrolysate obtained from the continuous process delivered a high sugar and low inhibitor concentration, thereby leading to both high yield and high final ethanol titer in the fermentation process. Copyright © 2011 Elsevier Ltd. All rights reserved.

In Vitro and In Vivo Effects of α-Amylase Inhibitor From Avena sativa Seeds on Life History and Physiological Characteristics of Sitotroga cerealella (Lepidoptera: Gelechiidae)

PubMed Central

Borzoui, Ehsan; Nouri-Ganbalani, Gadir

2017-01-01

Abstract The inhibitory effects of Avena sativa L. seed extract were studied on life history and some physiological aspects of Sitotroga cerealella (Olivier; Lepidoptera: Gelechiidae). The inhibition of α-amylase activity in vitro by A. sativa proteinaceous extract suggested its potential antimetabolic effect on S. cerealella larvae. Although, chronic ingestion of A. sativa inhibitor (I10: 0.108 mg protein/artificial seed) did not show significant reduction of the growth and development of S. cerealella. However, a delay in the developmental time of immature stages was detected when S. cerealella larvae were continuously fed on I30 and I50 concentrations (0.429 and 1.11 mg protein/artificial seed, respectively) of the inhibitor. The highest realized fecundity was recorded for females which came from larvae fed on I10 concentration (102.46 ± 2.50 eggs/female), and the lowest fecundity was observed for females which came from larvae fed on I50 concentration (31.64 ± 3.17 eggs/female). The lightest weight of pupae of S. cerealella was observed on I50 concentration (2.76 ± 0.07 mg). The lowest glycogen and lipid contents of the pupae were detected on I50 concentration (50.00 ± 3.53 and 289.57 ± 29.00 µg/pupa, respectively). The lower survival rate of pupae at low temperature indicated that S. cerealella fed on I50 concentration of the inhibitor was less cold tolerant than control insects. The inhibitory studies indicated that A. sativa proteinaceous extract is a good candidate as an inhibitor of the α-amylase of this pest. This inhibitor can be expressed in genetically engineered plants to confer resistance to S. cerealella. PMID:29099952

Evaluation of the efficacy and safety of rivaroxaban using a computer model for blood coagulation.

PubMed

Burghaus, Rolf; Coboeken, Katrin; Gaub, Thomas; Kuepfer, Lars; Sensse, Anke; Siegmund, Hans-Ulrich; Weiss, Wolfgang; Mueck, Wolfgang; Lippert, Joerg

2011-04-22

Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Union and several other countries for the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery and is in advanced clinical development for the treatment of thromboembolic disorders. Its mechanism of action is antithrombin independent and differs from that of other anticoagulants, such as warfarin (a vitamin K antagonist), enoxaparin (an indirect thrombin/Factor Xa inhibitor) and dabigatran (a direct thrombin inhibitor). A blood coagulation computer model has been developed, based on several published models and preclinical and clinical data. Unlike previous models, the current model takes into account both the intrinsic and extrinsic pathways of the coagulation cascade, and possesses some unique features, including a blood flow component and a portfolio of drug action mechanisms. This study aimed to use the model to compare the mechanism of action of rivaroxaban with that of warfarin, and to evaluate the efficacy and safety of different rivaroxaban doses with other anticoagulants included in the model. Rather than reproducing known standard clinical measurements, such as the prothrombin time and activated partial thromboplastin time clotting tests, the anticoagulant benchmarking was based on a simulation of physiologically plausible clotting scenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivity for tissue factor concentration inducing clotting, and a steep concentration-effect relationship, rapidly flattening towards higher inhibitor concentrations, both suggesting a broad therapeutic window. The predicted dosing window is highly accordant with the final dose recommendation based upon extensive clinical studies.

Inhibition of MRSA and of Clostridium difficile by durancin 61A: synergy with bacteriocins and antibiotics.

PubMed

Hanchi, Hasna; Hammami, Riadh; Gingras, Hélène; Kourda, Rim; Bergeron, Michel G; Ben Hamida, Jeannette; Ouellette, Marc; Fliss, Ismail

2017-03-01

The aim of this study was to evaluate the efficacy of durancin 61A alone or in combination with nisin, pediocin PA-1, reuterin, microcin J25, vancomycin or tetracycline as an inhibitor of resistant clinical pathogens and to shed light on its mode of action. Durancin and reuterin were effective inhibitors of Clostridium difficile, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus. The combination of durancin and reuterin was highly synergistic against C. difficile (fractional inhibitory concentration index = 0.2). Durancin/vancomycin combination was synergistic against S. aureus ATCC ® 700699 (fractional inhibitory concentration index = 0.3). Conclusion & future perspective: Durancin 61A alone or combined with other bacteriocins or antibiotics may therefore provide a possible therapeutic option for the treatment of infections by these pathogens.

Circulating plasma cholesteryl ester transfer protein activity and blood pressure tracking in the community

USDA-ARS?s Scientific Manuscript database

Clinical trials using cholesteryl ester transfer protein (CETP) inhibitors to raise high-density lipoprotein cholesterol (HDL-C) concentrations reported an 'off-target' blood pressure (BP) raising effect. We evaluated the relations of baseline plasma CETP activity and longitudinal BP change. One tho...

Elevated cell proliferation and VEGF production by high-glucose conditions in Müller cells involve XIAP

PubMed Central

Sun, Y; Wang, D; Ye, F; Hu, D-N; Liu, X; Zhang, L; Gao, L; Song, E; Zhang, D Y

2013-01-01

Purpose Müller cells have important roles in the pathogenesis of diabetic retinopathy by promoting cell proliferation and inducing the production of vascular endothelial growth factor (VEGF) under hyperglycemic conditions. The objective of this study was to determine the potential mechanism of Müller cell proliferation and VEGF production due to high-glucose conditions. Methods Primary cultured rat Müller cells were incubated with medium containing variable concentrations of glucose and/or embelin, a specific inhibitor of X-linked inhibitor of apoptosis protein (XIAP), for 72 h. The proliferation of Müller cells was assessed by the MTT assay. The expression and/or phosphorylation of 146 proteins were assessed using protein pathway array. Results High concentrations of glucose-induced Müller cell proliferation and altered expression and/or phosphorylation of 47 proteins that have been identified to have key roles in several important signaling pathways (XIAP, VEGF, HIF1α, NFκB, etc) and are involved in the regulation of cell survival, proliferation, or apoptosis. However, Müller cell alterations induced by high-glucose conditions were counteracted by the XIAP inhibitor embelin, and 26 proteins/phosphorylations (out of 47) were restored to their normal levels. Nine proteins, including NFκB p65, p-p38, tumor necrosis factor-α, urokinase-type plasminogen activator, CREB, IL-1β, HCAM, estrogen receptor-α, and p-Stat3, were involved in regulatory networks between XIAP and VEGF. Conclusions The current study suggests that XIAP may be a potential regulator that can mediate a series of pathological changes induced by high-glucose conditions in Müller cells. Therefore, embelin could be a potential agent for the prevention and treatment of diabetic retinopathy. PMID:23928877

Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier.

PubMed

Hildyard, John C W; Ammälä, Carina; Dukes, Iain D; Thomson, Stephen A; Halestrap, Andrew P

2005-01-01

Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K(i) values <0.1 muM. Inhibitor titrations of pyruvate-dependent respiration by heart mitochondria gave values (+/-S.E.) for the concentration of inhibitor binding sites (pmol per mg protein) and their K(i) (nM) of 56.0+/-0.9 and 0.057+/-0.010 nM for the more hydrophobic GW604714X; for GW450863X the values were 59.9+/-4.6 and 0.60+/-0.12 nM. [(3)H]-methoxy-GW450863X binding was also used to determine the MPC content of the heart, kidney, liver and brain mitochondria giving values of 56, 40, 26 and 20 pmol per mg protein respectively. Binding to yeast mitochondria was <10% of that in rat liver mitochondria, consistent with the slow rate of pyruvate transport into yeast mitochondria. [(3)H]-methoxy-GW450863X binding was inhibited by GW604714X and by the established MPC inhibitor, UK5099. The absorbance spectra of GW450863X and GW604714X were markedly changed by the addition of beta-mercaptoethanol suggesting that the novel inhibitors, like alpha-cyanocinnamate, possess an activated double bond that attacks a critical cysteine residue on the MPC. However, no labelled protein was detected following SDS-PAGE suggesting that the covalent modification is reversible. GW604714X and GW450863X inhibited l-lactate transport by the plasma membrane monocarboxylate transporter MCT1, but at concentrations more than four orders of magnitude greater than the MPC.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs.

PubMed

Chen, Xiaole; Wang, He; Shen, Yue; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

2016-08-19

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to "shotgun" clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin. Copyright © 2016 Elsevier Inc. All rights reserved.

Pharmacologic targeting of bacterial β-glucuronidase alleviates nonsteroidal anti-inflammatory drug-induced enteropathy in mice.

PubMed

LoGuidice, Amanda; Wallace, Bret D; Bendel, Lauren; Redinbo, Matthew R; Boelsterli, Urs A

2012-05-01

Small intestinal mucosal injury is a frequent adverse effect caused by nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms are not completely understood, but topical (luminal) effects have been implicated. Many carboxylic acid-containing NSAIDs, including diclofenac (DCF), are metabolized to acyl glucuronides (AGs), and/or ether glucuronides after ring hydroxylation, and exported into the biliary tree. In the gut, these conjugates are cleaved by bacterial β-glucuronidase, releasing the potentially harmful aglycone. We first confirmed that DCF-AG was an excellent substrate for purified Escherichia coli β-D-glucuronidase. Using a previously characterized novel bacteria-specific β-glucuronidase inhibitor (Inhibitor-1), we then found that the enzymatic hydrolysis of DCF-AG in vitro was inhibited concentration dependently (IC₅₀ ∼164 nM). We next hypothesized that pharmacologic inhibition of bacterial β-glucuronidase would reduce exposure of enterocytes to the aglycone and, as a result, alleviate enteropathy. C57BL/6J mice were administered an ulcerogenic dose of DCF (60 mg/kg i.p.) with or without oral pretreatment with Inhibitor-1 (10 μg per mouse, b.i.d.). Whereas DCF alone caused the formation of numerous large ulcers in the distal parts of the small intestine and increased (2-fold) the intestinal permeability to fluorescein isothiocyanate-dextran, Inhibitor-1 cotreatment significantly alleviated mucosal injury and reduced all parameters of enteropathy. Pharmacokinetic profiling of DCF plasma levels in mice revealed that Inhibitor-1 coadministration did not significantly alter the C(max), half-life, or area under the plasma concentration versus time curve of DCF. Thus, highly selective pharmacologic targeting of luminal bacterial β-D-glucuronidase by a novel class of small-molecule inhibitors protects against DCF-induced enteropathy without altering systemic drug exposure.

Pharmacologic Targeting of Bacterial β-Glucuronidase Alleviates Nonsteroidal Anti-Inflammatory Drug-Induced Enteropathy in Mice

PubMed Central

LoGuidice, Amanda; Wallace, Bret D.; Bendel, Lauren; Redinbo, Matthew R.

2012-01-01

Small intestinal mucosal injury is a frequent adverse effect caused by nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms are not completely understood, but topical (luminal) effects have been implicated. Many carboxylic acid-containing NSAIDs, including diclofenac (DCF), are metabolized to acyl glucuronides (AGs), and/or ether glucuronides after ring hydroxylation, and exported into the biliary tree. In the gut, these conjugates are cleaved by bacterial β-glucuronidase, releasing the potentially harmful aglycone. We first confirmed that DCF-AG was an excellent substrate for purified Escherichia coli β-d-glucuronidase. Using a previously characterized novel bacteria-specific β-glucuronidase inhibitor (Inhibitor-1), we then found that the enzymatic hydrolysis of DCF-AG in vitro was inhibited concentration dependently (IC50 ∼164 nM). We next hypothesized that pharmacologic inhibition of bacterial β-glucuronidase would reduce exposure of enterocytes to the aglycone and, as a result, alleviate enteropathy. C57BL/6J mice were administered an ulcerogenic dose of DCF (60 mg/kg i.p.) with or without oral pretreatment with Inhibitor-1 (10 μg per mouse, b.i.d.). Whereas DCF alone caused the formation of numerous large ulcers in the distal parts of the small intestine and increased (2-fold) the intestinal permeability to fluorescein isothiocyanate-dextran, Inhibitor-1 cotreatment significantly alleviated mucosal injury and reduced all parameters of enteropathy. Pharmacokinetic profiling of DCF plasma levels in mice revealed that Inhibitor-1 coadministration did not significantly alter the Cmax, half-life, or area under the plasma concentration versus time curve of DCF. Thus, highly selective pharmacologic targeting of luminal bacterial β-d-glucuronidase by a novel class of small-molecule inhibitors protects against DCF-induced enteropathy without altering systemic drug exposure. PMID:22328575

Engineering wild-type robust Pediococcus acidilactici strain for high titer L- and D-lactic acid production from corn stover feedstock.

PubMed

Yi, Xia; Zhang, Peng; Sun, Jiaoe; Tu, Yi; Gao, Qiuqiang; Zhang, Jian; Bao, Jie

2016-01-10

Pediococcus acidilactici TY112 producing L-lactic acid and P. acidilactici ZP26 producing D-lactic acid, were engineered from the wild-type P. acidilactici DQ2 by ldhD or ldh gene disruption, and the robustness of the wild-type strain to the inhibitors derived from lignocellulose pretreatment was maintained well. In simultaneous saccharification and fermentation (SSF), 77.66 g L(-1) of L-lactic acid and 76.76 g L(-1) of D-lactic acid were obtained at 25% (w/w) solids content of dry dilute acid pretreated and biodetoxified corn stover feedstock. L- and D-Lactic acid yield and productivity were highly dependent on the inhibitor removal extent due to the significant down-regulation on the expressions of ldh and ldhD encoding lactate dehydrogenase by inhibitor, especially syringaldehyde and vanillin at the low concentrations. This study provided a prototype of industrial process for high titer L- and D-lactic acid production from lignocellulose feedstock. Copyright © 2015 Elsevier B.V. All rights reserved.

CT-721, a Potent Bcr-Abl Inhibitor, Exhibits Excellent In Vitro and In Vivo Efficacy in the Treatment of Chronic Myeloid Leukemia

PubMed Central

Sun, Yinghui; Zhao, Na; Wang, Huan; Wu, Qiong; Han, Yunqi; Liu, Qichao; Wu, Mangang; Liu, Yuliang; Kong, Fansheng; Wang, He; Sun, Ying; Sun, Deguang; Jing, Lutao; Tang, Guojing; Hu, Yuandong; Xiao, Dengming; Luo, Hong; Han, Yongxin; Peng, Yong

2017-01-01

Kinase inhibitors that target Bcr-Abl are highly effective in the treatment of chronic myeloid leukemia (CML). However, these inhibitors are often invalidated due to the drug resistance. Therefore, the discovery and development of novel Bcr-Abl inhibitors is required to overwhelm the drug resistance in the treatment of CML resistant to the currently used first-line Bcr-Abl inhibitors. Herein we have described a newly developed Bcr-Abl inhibitor CT-721, which displayed potent inhibitory effects on wild-type and T315I mutant Bcr-Abl. It functioned as a typically ATP-competitive inhibitor, superior to other existing Bcr-Abl inhibitors. CT-721 also demonstrated time-dependent inhibition of Bcr-Abl activation and the resultant downstream signaling transduction pathways in Bcr-Abl positive cells. Furthermore, CT-721 induced cell apoptosis and cell cycle arrest, and efficaciously inhibited tumor growth in Bcr-Abl-expressed K562 and KU812 xenograft models in a mechanism-based manner. Further PK/PD studies revealed a positive in vivo correlation between the compound concentration and inhibition of Bcr-Abl activity. Taken together, CT-721 is a potent and time-dependent Bcr-Abl kinase inhibitor, and has shown strong in vitro and in vivo anti-CML activities with a favorable pharmacokinetic profile, differentiating it from other Bcr-Abl kinase inhibitors already approved and current in development for the treatment of CML. PMID:28928866

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

PubMed

Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2013-03-01

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy. Georg Thieme Verlag KG Stuttgart · New York.

High-Throughput Screening and Quantitative Chemical Ranking for Sodium-Iodide Symporter Inhibitors in ToxCast Phase I Chemical Library.

PubMed

Wang, Jun; Hallinger, Daniel R; Murr, Ashley S; Buckalew, Angela R; Simmons, Steven O; Laws, Susan C; Stoker, Tammy E

2018-05-01

Thyroid uptake of iodide via the sodium-iodide symporter (NIS) is the first step in the biosynthesis of thyroid hormones that are critical for health and development in humans and wildlife. Despite having long been a known target of endocrine disrupting chemicals such as perchlorate, information regarding NIS inhibition activity is still unavailable for the vast majority of environmental chemicals. This study applied a previously validated high-throughput approach to screen for NIS inhibitors in the ToxCast phase I library, representing 293 important environmental chemicals. Here 310 blinded samples were screened in a tiered-approach using an initial single-concentration (100 μM) radioactive-iodide uptake (RAIU) assay, followed by 169 samples further evaluated in multi-concentration (0.001 μM-100 μM) testing in parallel RAIU and cell viability assays. A novel chemical ranking system that incorporates multi-concentration RAIU and cytotoxicity responses was also developed as a standardized method for chemical prioritization in current and future screenings. Representative chemical responses and thyroid effects of high-ranking chemicals are further discussed. This study significantly expands current knowledge of NIS inhibition potential in environmental chemicals and provides critical support to U.S. EPA's Endocrine Disruptor Screening Program (EDSP) initiative to expand coverage of thyroid molecular targets, as well as the development of thyroid adverse outcome pathways (AOPs).

Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia pestis Outer Protein H (YopH) Phosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bahta, Medhanit; Lountos, George T.; Dyas, Beverly

Our current study reports the first K{sub M} optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (K{sub M} = 80 {micro}M) was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime ligation. A cocrystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employedmore » for the design of furanyl-based oxime derivatives. By this process, a potent (IC{sub 50} = 190 nM) and nonpromiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a noncytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH.« less

A real-time high-throughput fluorescence assay for sphingosine kinases

PubMed Central

Lima, Santiago; Milstien, Sheldon; Spiegel, Sarah

2014-01-01

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format. PMID:24792926

Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach.

PubMed

Cui, Huaqing; Kamal, Zeeshan; Ai, Teng; Xu, Yanli; More, Swati S; Wilson, Daniel J; Chen, Liqiang

2014-10-23

Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD(+)-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene-benzamides and -nicotinamides. Biochemical evaluation of these two series provided structure-activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor against NAD(+). Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel structure merits further exploration.

Design and synthesis of sulfonamide-substituted diphenylpyrimidines (SFA-DPPYs) as potent Bruton's tyrosine kinase (BTK) inhibitors with improved activity toward B-cell lymphoblastic leukemia.

PubMed

Liu, He; Qu, Menghua; Xu, Lina; Han, Xu; Wang, Changyuan; Shu, Xiaohong; Yao, Jihong; Liu, Kexin; Peng, Jinyong; Li, Yanxia; Ma, Xiaodong

2017-07-28

A new series of diphenylpyrimidine derivatives (SFA-DPPYs) were synthesized by introducing a functional sulfonamide into the C-2 aniline moiety of pyrimidine template, and then were biologically evaluated as potent Bruton's tyrosine kinase (BTK) inhibitors. Among these molecules, inhibitors 10c, 10i, 10j and 10k displayed high potency against the BTK enzyme, with IC 50 values of 1.18 nM, 0.92 nM, 0.42 nM and 1.05 nM, respectively. In particular, compound 10c could remarkably inhibit the proliferation of the B lymphoma cell lines at concentrations of 6.49 μM (Ramos cells) and 13.2 μM (Raji cells), and was stronger than the novel agent spebrutinib. In addition, the inhibitory potency toward the normal PBMC cells showed that inhibitor 10c possesses low cell cytotoxicity. All these explorations indicated that molecule 10c could serve as a valuable inhibitor for B-cell lymphoblastic leukemia treatment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

PubMed

Heightman, Tom D; Berdini, Valerio; Braithwaite, Hannah; Buck, Ildiko M; Cassidy, Megan; Castro, Juan; Courtin, Aurélie; Day, James E H; East, Charlotte; Fazal, Lynsey; Graham, Brent; Griffiths-Jones, Charlotte M; Lyons, John F; Martins, Vanessa; Muench, Sandra; Munck, Joanne M; Norton, David; O'Reilly, Marc; Palmer, Nick; Pathuri, Puja; Reader, Michael; Rees, David C; Rich, Sharna J; Richardson, Caroline; Saini, Harpreet; Thompson, Neil T; Wallis, Nicola G; Walton, Hugh; Wilsher, Nicola E; Woolford, Alison J-A; Cooke, Michael; Cousin, David; Onions, Stuart; Shannon, Jonathan; Watts, John; Murray, Christopher W

2018-05-31

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf.

PubMed

Tao, Yi; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

2013-06-27

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography-mass spectrometry. Three flavonoids including quercetin-3-O-β-D-arabinopyranosyl-(1→2)-β-D-galactopyranoside, quercetin-3-O-β-D-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources. Copyright © 2013 Elsevier B.V. All rights reserved.

An alternative assay to discover potential calmodulin inhibitors using a human fluorophore-labeled CaM protein.

PubMed

González-Andrade, Martín; Figueroa, Mario; Rodríguez-Sotres, Rogelio; Mata, Rachel; Sosa-Peinado, Alejandro

2009-04-01

This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change ( approximately 80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC(50) values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.

Distinguishing defensive characteristics in the phloem of ash species resistant and susceptible to emerald ash borer.

PubMed

Cipollini, Don; Wang, Qin; Whitehill, Justin G A; Powell, Jeff R; Bonello, Pierluigi; Herms, Daniel A

2011-05-01

We examined the extent to which three Fraxinus cultivars and a wild population that vary in their resistance to Emerald Ash Borer (EAB) could be differentiated on the basis of a suite of constitutive chemical defense traits in phloem extracts. The EAB-resistant Manchurian ash (F. mandshurica, cv. Mancana) was characterized by having a rapid rate of wound browning, a high soluble protein concentration, low trypsin inhibitor activities, and intermediate levels of peroxidase activity and total soluble phenolic concentration. The EAB-susceptible white ash (F. americana, cv. Autumn Purple) was characterized by a slow wound browning rate and low levels of peroxidase activity and total soluble phenolic concentrations. An EAB-susceptible green ash cultivar (F. pennsylvanica, cv. Patmore) and a wild accession were similar to each other on the basis of several chemical defense traits, and were characterized by high activities of peroxidase and trypsin inhibitor, a high total soluble phenolic concentration, and an intermediate rate of wound browning. Lignin concentration and polyphenol oxidase activities did not differentiate resistant and susceptible species. Of 33 phenolic compounds separated by HPLC and meeting a minimum criterion for analysis, nine were unique to Manchurian ash, five were shared among all species, and four were found in North American ashes and not in the Manchurian ash. Principal components analysis revealed clear separations between Manchurian, white, and green ashes on the basis of all phenolics, as well as clear separations on the basis of quantities of phenolics that all species shared. Variation in some of these constitutive chemical defense traits may contribute to variation in resistance to EAB in these species.

Study of benzotriazole as corrosion inhibitors of carbon steel in chloride solution containing hydrogen sulfide using electrochemical impedance spectroscopy (EIS)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solehudin, Agus, E-mail: asolehudin@upi.edu; Nurdin, Isdiriayani

2014-03-24

Corrosion and inhibition studies on API 5LX65 carbon steel in chloride solution containing various concentrations of benzotriazole has been conducted at temperature of 70°C using Electrochemical Impedance Spectroscopy (EIS). Corroded carbon steel surface with and without inhibitor have been observed using X-ray Diffraction (XRD), Scanning Electron Microscope (SEM), and Energy Dispersive Spectroscopy (EDS). The objectives of this research are to study the performance of benzotriazole as corrosion inhibitors. The experimental results of carbon steel corrosion in 3.5% NaCl solution containing 500 mg/l H{sub 2}S at different BTAH concentrations showed that corrosion rate of carbon steel decreases with increasing of BTAHmore » concentrations from 0 to 10 mmol/l. The inhibition efficiency of BTAH was found to be affected by its concentration. The optimum efficiency obtained of BTAH is 93% at concentration of 5 mmol/l. The result of XRD and EDS analysis reveal the iron sulfide (FeS) formation on corroded carbon steel surface without inhibitor. The EDS spectrum show the Nitrogen (N) bond on carbon steel surface inhibited by BTAH.« less

Biochemical and Pharmacological Characterizations of ESI-09 Based EPAC Inhibitors: Defining the ESI-09 “Therapeutic Window”

PubMed Central

Zhu, Yingmin; Chen, Haijun; Boulton, Stephen; Mei, Fang; Ye, Na; Melacini, Giuseppe; Zhou, Jia; Cheng, Xiaodong

2015-01-01

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce “protein denaturation”. Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks. PMID:25791905

Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 "therapeutic window".

PubMed

Zhu, Yingmin; Chen, Haijun; Boulton, Stephen; Mei, Fang; Ye, Na; Melacini, Giuseppe; Zhou, Jia; Cheng, Xiaodong

2015-03-20

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce "protein denaturation". Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

Serum α1-proteinase inhibitor concentrations in dogs with exocrine pancreatic disease, chronic hepatitis or proteinuric chronic kidney disease.

PubMed

Heilmann, R M; Grützner, N; Hokamp, J A; Lidbury, J A; Xenoulis, P G; Suchodolski, J S; Nabity, M B; Cianciolo, R; Steiner, J M

2018-06-01

Serum canine α 1 -proteinase inhibitor (cα 1 -PI) concentrations were evaluated in dogs with pancreatitis (n=24), exocrine pancreatic insufficiency (EPI; n=29), chronic hepatitis (CH; n=11) or proteinuric chronic kidney disease (CKD-P; n=61) to determine whether systemic proteinase/proteinase-inhibitor balance is altered in these conditions. Dogs with CKD-P had significantly lower cα 1 -PI concentrations than dogs with pancreatitis, EPI or CH; 16% of dogs with CKD-P had serum cα 1 -PI concentrations below the reference interval. Serum and urine cα 1 -PI concentrations were inversely correlated in dogs with CKD-P, but not in dogs with CH. This suggests that renal loss of cα 1 -PI contributes to decreased serum concentrations in dogs with CKD-P, while hepatic cα 1 -PI synthesis with CH either is not compromised or is counterbalanced by extrahepatic production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Exposure‐Response Model of Subcutaneous C1‐Inhibitor Concentrate to Estimate the Risk of Attacks in Patients With Hereditary Angioedema

PubMed Central

Tortorici, Michael A.; Pawaskar, Dipti; Pragst, Ingo; Machnig, Thomas; Hutmacher, Matthew; Zuraw, Bruce; Cicardi, Marco; Craig, Timothy; Longhurst, Hilary; Sidhu, Jagdev

2018-01-01

Subcutaneous C1‐inhibitor (HAEGARDA, CSL Behring), is a US Food and Drug Administration (FDA)‐approved, highly concentrated formulation of a plasma‐derived C1‐esterase inhibitor (C1‐INH), which, in the phase III Clinical Studies for Optimal Management in Preventing Angioedema with Low‐Volume Subcutaneous C1‐inhibitor Replacement Therapy (COMPACT) trial, reduced the incidence of hereditary angioedema (HAE) attacks when given prophylactically. Data from the COMPACT trial were used to develop a repeated time‐to‐event model to characterize the timing and frequency of HAE attacks as a function of C1‐INH activity, and then develop an exposure–response model to assess the relationship between C1‐INH functional activity levels (C1‐INH(f)) and the risk of an attack. The C1‐INH(f) values of 33.1%, 40.3%, and 63.1% were predicted to correspond with 50%, 70%, and 90% reductions in the HAE attack risk, respectively, relative to no therapy. Based on trough C1‐INH(f) values for the 40 IU/kg (40.2%) and 60 IU/kg (48.0%) C1‐INH (SC) doses, the model predicted that 50% and 67% of the population, respectively, would see at least a 70% decrease in the risk of an attack. PMID:29316335

Cartilage degradation biomarkers predict efficacy of a novel, highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: confirmation by multivariate analysis that modulation of type II collagen and aggrecan degradation peptides parallels pathologic changes.

PubMed

Settle, Steven; Vickery, Lillian; Nemirovskiy, Olga; Vidmar, Tom; Bendele, Alison; Messing, Dean; Ruminski, Peter; Schnute, Mark; Sunyer, Teresa

2010-10-01

To demonstrate that the novel highly selective matrix metalloproteinase 13 (MMP-13) inhibitor PF152 reduces joint lesions in adult dogs with osteoarthritis (OA) and decreases biomarkers of cartilage degradation. The potency and selectivity of PF152 were evaluated in vitro using 16 MMPs, TACE, and ADAMTS-4 and ADAMTS-5, as well as ex vivo in human cartilage explants. In vivo effects were evaluated at 3 concentrations in mature beagles with partial medial meniscectomy. Gross and histologic changes in the femorotibial joints were evaluated using various measures of cartilage degeneration. Biomarkers of cartilage turnover were examined in serum, urine, or synovial fluid. Results were analyzed individually and in combination using multivariate analysis. The potent and selective MMP-13 inhibitor PF152 decreased human cartilage degradation ex vivo in a dose-dependent manner. PF152 treatment of dogs with OA reduced cartilage lesions and decreased biomarkers of type II collagen (type II collagen neoepitope) and aggrecan (peptides ending in ARGN or AGEG) degradation. The dose required for significant inhibition varied with the measure used, but multivariate analysis of 6 gross and histologic measures indicated that all doses differed significantly from vehicle but not from each other. Combined analysis of cartilage degradation markers showed similar results. This highly selective MMP-13 inhibitor exhibits chondroprotective effects in mature animals. Biomarkers of cartilage degradation, when evaluated in combination, parallel the joint structural changes induced by the MMP-13 inhibitor. These data support the potential therapeutic value of selective MMP-13 inhibitors and the use of a set of appropriate biomarkers to predict efficacy in OA clinical trials.

Effect of proton pump inhibitors on the serum concentrations of the selective serotonin reuptake inhibitors citalopram, escitalopram, and sertraline.

PubMed

Gjestad, Caroline; Westin, Andreas A; Skogvoll, Eirik; Spigset, Olav

2015-02-01

The selective serotonin reuptake inhibitors (SSRIs) citalopram, escitalopram, and sertraline are all metabolized by the cytochrome P-450 isoenzyme CYP2C19, which is inhibited by the proton pump inhibitors (PPIs) omeprazole, esomeprazole, lansoprazole, and pantoprazole. The aim of the present study was to evaluate the effect of these PPIs on the serum concentrations of citalopram, escitalopram, and sertraline. Serum concentrations from patients treated with citalopram, escitalopram, or sertraline were obtained from a routine therapeutic drug monitoring database, and samples from subjects concomitantly using PPIs were identified. Dose-adjusted SSRI serum concentrations were calculated to compare data from those treated and those not treated with PPIs. Citalopram concentrations were significantly higher in patients treated with omeprazole (+35.3%; P < 0.001), esomeprazole (+32.8%; P < 0.001), and lansoprazole (+14.7%; P = 0.043). Escitalopram concentrations were significantly higher in patients treated with omeprazole (+93.9%; P < 0.001), esomeprazole (+81.8%; P < 0.001), lansoprazole (+20.1%; P = 0.008), and pantoprazole (+21.6%; P = 0.002). Sertraline concentrations were significantly higher in patients treated with esomeprazole (+38.5%; P = 0.0014). The effect of comedication with PPIs on the serum concentration of SSRIs is more pronounced for omeprazole and esomeprazole than for lansoprazole and pantoprazole, and escitalopram is affected to a greater extent than are citalopram and sertraline. When omeprazole or esomeprazole are used in combination with escitalopram, a 50% dose reduction of the latter should be considered.

Propylthiouracil, Perchlorate, and Thyroid-Stimulating Hormone Modulate High Concentrations of Iodide Instigated Mitochondrial Superoxide Production in the Thyroids of Metallothionein I/II Knockout Mice

PubMed Central

Duan, Qi; Wang, Tingting; Zhang, Na; Perera, Vern; Liang, Xue; Abeysekera, Iruni Roshanie

2016-01-01

Background Increased oxidative stress has been suggested as one of the underlying mechanisms in iodide excess-induced thyroid disease. Metallothioneins (MTs) are regarded as scavengers of reactive oxygen species (ROS) in oxidative stress. Our aim is to investigate the effects of propylthiouracil (PTU), a thyroid peroxidase inhibitor, perchlorate (KClO4), a competitive inhibitor of iodide transport, and thyroid stimulating hormone (TSH) on mitochondrial superoxide production instigated by high concentrations of iodide in the thyroids of MT-I/II knockout (MT-I/II KO) mice. Methods Eight-week-old 129S7/SvEvBrd-Mt1tm1Bri Mt2tm1Bri/J (MT-I/II KO) mice and background-matched wild type (WT) mice were used. Results By using a mitochondrial superoxide indicator (MitoSOX Red), lactate dehydrogenase (LDH) release, and methyl thiazolyl tetrazolium (MTT) assay, we demonstrated that the decreased relative viability and increased LDH release and mitochondrial superoxide production induced by potassium iodide (100 µM) can be relieved by 300 µM PTU, 30 µM KClO4, or 10 U/L TSH in the thyroid cell suspensions of both MT-I/II KO and WT mice (P<0.05). Compared to the WT mice, a significant decrease in the relative viability along with a significant increase in LDH release and mitochondrial superoxide production were detected in MT-I/II KO mice(P<0.05). Conclusion We concluded that PTU, KClO4, or TSH relieved the mitochondrial oxidative stress induced by high concentrations of iodide in the thyroids of both MT-I/II KO and WT mice. MT-I/II showed antioxidant effects against high concentrations of iodide-induced mitochondrial superoxide production in the thyroid. PMID:26754589

Alterations in receptor expression or agonist concentration change the pathways gastrin-releasing peptide receptor uses to regulate extracellular signal-regulated kinase.

PubMed

Chen, Pei-Wen; Kroog, Glenn S

2004-12-01

G protein-coupled receptors activate extracellular signal-regulated kinases (ERKs) via different pathways in different cell types. In this study, we demonstrate that gastrin-releasing peptide receptor (GRPr) regulates ERK through multiple pathways in a single cell type depending upon receptor expression and agonist concentration. We examined stably transfected BALB/c 3T3 fibroblasts expressing GRPr constructs at different levels and treated the cells with several concentrations of bombesin (BN, a GRPr agonist) to activate a variable number of GRPr per cell. GRPr induced two waves of ERK activation and one wave of ERK inhibition. One wave of activation required an intact GRPr carboxyl-terminal domain (CTD). It peaked 6 min after addition of high BN concentration ([BN]) in cells with high GRPr expression. Another wave of activation was CTD-independent. It peaked 2 to 4 min after BN addition in cells when [BN] and/or GRPr expression were lower. The early wave of ERK activation was more sensitive than the later one to pretreatment with Bisindolylmaleimide I (GF 109203X) (a protein kinase C inhibitor) or hypertonic sucrose. Because these two waves of activation differ in time course, dose-response curve, requirement for GRPr CTD, and sensitivity to inhibitors, they result from different signaling pathways. A third pathway in these cells inhibited ERK phosphorylation 2 min after addition of high [BN] in cells with high GRPr expression. Furthermore, a GRPr-expressing human duodenal cancer cell line showed differential sensitivity to GF 109203X throughout BN-induced ERK activation, indicating that GRPr may activate ERK via multiple pathways in cells expressing endogenous GRPr.

Effect of protease inhibitors on angiotensin-converting enzyme activity in human T-lymphocytes.

PubMed

Petrov, V; Fagard, R; Lijnen, P

2000-05-01

The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with ophtaldialdehyde. The effect of various concentrations (10(-9) to 10(-3) mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphocytes.

Rho-kinase inhibitors augment the inhibitory effect of propofol on rat bronchial smooth muscle contraction.

PubMed

Hanazaki, Motohiko; Yokoyama, Masataka; Morita, Kiyoshi; Kohjitani, Atsushi; Sakai, Hiroyasu; Chiba, Yoshihiko; Misawa, Miwa

2008-06-01

Airway smooth muscle contraction is not caused by the increase in intracellular Ca(2+) ([Ca(2+)](i)) alone because agonist stimulation increases tension at the same [Ca(2+)](i) (increase in Ca(2+) sensitivity). The small G protein Rho A and Rho-kinase (ROCK) play important roles in the regulation of Ca(2+) sensitivity. In this study, we investigated the effects of three ROCK inhibitors (fasudil, Y-27632, and H-1152) on rat airway smooth muscle contraction and the effects of ROCK inhibitors on propofol-induced bronchodilatory effects. Ring strips from intrapulmonary bronchus of male Wistar rats were placed in 400-microL organ baths containing Krebs-Henseleit solution. After obtaining stable contraction with 30 microM acetylcholine, (1) propofol (1 microM-1 mM) was cumulatively applied; (2) cumulative doses of Y-27632 (0.01-300 microM), fasudil (0.01-100 microM), or H-1152 (0.01-100 microM) were applied; (3) propofol (1 microM-1 mM), with Y-27632, fasudil or H-1152 (0.03 microM or 0.1 microM), was cumulatively applied. (1) Propofol produced concentration-dependent relaxation of rat bronchial smooth muscle. (2) All ROCK inhibitors produced concentration-dependent relaxation. (3) 0.03 microM Y-27632 and fasudil had no significant effect on the concentration-response curve for propofol, while 0.1 microM of both agents significantly shifted concentration-response curves to the left and decreased EC(50). H-1152 (both 0.03 microM and 0.1 microM) significantly sifted the concentration-response curve for propofol to the left and decreased EC(50). ROCK inhibitors, especially H-1152, can attenuate the contraction of rat airway smooth muscle. The combined use of ROCK inhibitors and propofol causes greater relaxation.

Potent histone deacetylase inhibitors built from trichostatin A and cyclic tetrapeptide antibiotics including trapoxin

PubMed Central

Furumai, Ryohei; Komatsu, Yasuhiko; Nishino, Norikazu; Khochbin, Saadi; Yoshida, Minoru; Horinouchi, Sueharu

2001-01-01

Trichostatin A (TSA) and trapoxin (TPX) are potent inhibitors of histone deacetylases (HDACs). TSA is proposed to block the catalytic reaction by chelating a zinc ion in the active-site pocket through its hydroxamic acid group. On the other hand, the epoxyketone is suggested to be the functional group of TPX capable of alkylating the enzyme. We synthesized a novel TPX analogue containing a hydroxamic acid instead of the epoxyketone. The hybrid compound cyclic hydroxamic acid-containing peptide (CHAP) 1 inhibited HDAC1 at low nanomolar concentrations. The HDAC1 inhibition by CHAP1 was reversible as it was by TSA, in contrast to the irreversible inhibition by TPX. CHAP with an aliphatic chain length of five, which corresponded to that of acetylated lysine, was stronger than those with other lengths. These results suggest that TPX is a substrate mimic and that the replacement of the epoxyketone with the hydroxamic acid converted TPX to an inhibitor chelating the zinc like TSA. Interestingly, HDAC6, but not HDAC1 or HDAC4, was resistant to TPX and CHAP1, whereas TSA inhibited these HDACs to a similar extent. HDAC6 inhibition by TPX at a high concentration was reversible, probably because HDAC6 is not alkylated by TPX. We further synthesized the counterparts of all known naturally occurring cyclic tetrapeptides containing the epoxyketone. HDAC1 was highly sensitive to all these CHAPs much more than HDAC6, indicating that the structure of the cyclic tetrapeptide framework affects the target enzyme specificity. These results suggest that CHAP is a unique lead to develop isoform-specific HDAC inhibitors. PMID:11134513

Comparison of the effects of acute fluvoxamine and desipramine administration on melatonin and cortisol production in humans.

PubMed Central

Skene, D J; Bojkowski, C J; Arendt, J

1994-01-01

1. Acute administration of the specific serotonin uptake inhibitor, fluvoxamine (100 mg at 16.00 h), markedly increased nocturnal plasma melatonin concentrations, with high levels extending into the morning hours. 2. Acute administration of the noradrenaline uptake inhibitor, desipramine (DMI) (100 mg at 16.00 h), increased evening plasma melatonin concentrations. 3. Both drug treatments increased the duration of melatonin secretion, fluvoxamine significantly delaying the offset time and DMI significantly advancing the onset time. 4. The stimulatory effect of DMI on plasma melatonin was mirrored by increased urinary 6-sulphatoxymelatonin (aMT6s) excretion. 5. On the contrary, there was no correlation between plasma melatonin and urinary aMT6s concentrations following fluvoxamine treatment, suggesting that fluvoxamine may inhibit the metabolism of melatonin. 6. Treatment with DMI increased plasma cortisol concentrations in the evening and early morning, treatment with fluvoxamine increased plasma cortisol at 03.00 h, 10.00 h and 11.00 h. 7. The drug treatments affected different aspects of the nocturnal plasma melatonin profile suggesting that the amplitude of the melatonin rhythm may depend upon serotonin availability and/or melatonin metabolism whilst the onset of melatonin production depends upon noradrenaline availability. PMID:8186063

Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

PubMed

Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

2003-09-01

The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

PubMed

Yu, Peter Y; Gardner, Heather L; Roberts, Ryan; Cam, Hakan; Hariharan, Seethalakshmi; Ren, Ling; LeBlanc, Amy K; Xiao, Hui; Lin, Jiayuh; Guttridge, Denis C; Mo, Xiaokui; Bennett, Chad E; Coss, Christopher C; Ling, Yonghua; Phelps, Mitch A; Houghton, Peter; London, Cheryl A

2017-01-01

STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations.

PubMed

Xu, Junnan; Song, Dan; Bai, Qiufang; Zhou, Lijun; Cai, Liping; Hertz, Leif; Peng, Liang

2014-01-13

This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

Novel Design Strategy for Checkpoint Kinase 2 Inhibitors Using Pharmacophore Modeling, Combinatorial Fusion, and Virtual Screening

PubMed Central

Wang, Yen-Ling

2014-01-01

Checkpoint kinase 2 (Chk2) has a great effect on DNA-damage and plays an important role in response to DNA double-strand breaks and related lesions. In this study, we will concentrate on Chk2 and the purpose is to find the potential inhibitors by the pharmacophore hypotheses (PhModels), combinatorial fusion, and virtual screening techniques. Applying combinatorial fusion into PhModels and virtual screening techniques is a novel design strategy for drug design. We used combinatorial fusion to analyze the prediction results and then obtained the best correlation coefficient of the testing set (r test) with the value 0.816 by combining the BesttrainBesttest and FasttrainFasttest prediction results. The potential inhibitors were selected from NCI database by screening according to BesttrainBesttest + FasttrainFasttest prediction results and molecular docking with CDOCKER docking program. Finally, the selected compounds have high interaction energy between a ligand and a receptor. Through these approaches, 23 potential inhibitors for Chk2 are retrieved for further study. PMID:24864236

Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo.

PubMed

Thurber, Greg M; Yang, Katy S; Reiner, Thomas; Kohler, Rainer H; Sorger, Peter; Mitchison, Tim; Weissleder, Ralph

2013-01-01

Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.

Discovery of chiral dihydropyridopyrimidinones as potent, selective and orally bioavailable inhibitors of AKT.

PubMed

Parthasarathy, Saravanan; Henry, Kenneth; Pei, Huaxing; Clayton, Josh; Rempala, Mark; Johns, Deidre; De Frutos, Oscar; Garcia, Pablo; Mateos, Carlos; Pleite, Sehila; Wang, Yong; Stout, Stephanie; Condon, Bradley; Ashok, Sheela; Lu, Zhohai; Ehlhardt, William; Raub, Tom; Lai, Mei; Geeganage, Sandaruwan; Burkholder, Timothy P

2018-06-01

During the course of our research efforts to develop potent and selective AKT inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones (DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against ROCK 2 . A key challenge in this program was the poor physicochemical properties of the initial lead compound 5. Integration of structure-based drug design and physical properties-based design resulted in replacement of a highly hydrophobic poly fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound 6 with much improved physicochemical properties. Subsequent SAR studies led to the synthesis of new pyran analog 7 with improved cell potency. Further optimization of pharmacokintetics properties by increasing permeability with appropriate fluorinated alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the phosphorylation of GSK3β in vivo and had robust, dose and concentration dependent efficacy in the U87MG tumor xenograft model. Copyright © 2018 Elsevier Ltd. All rights reserved.

Use of a Cyclooxygenase-2 Inhibitor Does Not Inhibit Platelet Activation or Growth Factor Release From Platelet-Rich Plasma.

PubMed

Ludwig, Hilary C; Birdwhistell, Kate E; Brainard, Benjamin M; Franklin, Samuel P

2017-12-01

It remains unestablished whether use of cyclooxygenase (COX)-2 inhibitors impairs platelet activation and anabolic growth factor release from platelets in platelet-rich plasma (PRP). The purpose of this study was to assess the effects of a COX-2 inhibitor on platelet activation and anabolic growth factor release from canine PRP when using a clinically applicable PRP activator and to determine whether a 3-day washout would be sufficient to abrogate any COX-2 inhibitor-related impairment on platelet function. Controlled laboratory study. Ten healthy dogs underwent blood collection and PRP preparation. Dogs were then administered a COX-2 inhibitor for 7 days, after which PRP preparation was repeated. The COX-2 inhibitor was continued for 4 more days and PRP preparation performed a third time, 3 days after discontinuation of the COX-2 inhibitor. Immediately after PRP preparation, the PRP was divided into 4 aliquots: 2 unactivated and 2 activated using human γ-thrombin (HGT). One activated and 1 unactivated sample were assessed using flow cytometry for platelet expression of CD62P and platelet-bound fibrinogen using the canine activated platelet-1 (CAP1) antibody. The 2 remaining samples were centrifuged and the supernatant assayed for transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), and thromboxane B2 (TXB2) concentrations. Differences in platelet activation and TGF-β1, PDGF-BB, and TXB2 concentrations over the 3 study weeks were evaluated using a 1-way repeated-measures ANOVA, and comparisons between activated and unactivated samples within a study week were assessed with paired t tests. There were no statistically significant ( P > .05) effects of the COX-2 inhibitor on percentage of platelets positive for CD62P or CAP1 or on concentrations of TGF-β1, PDGF-BB, or TXB2. All unactivated samples had low levels of activation or growth factor concentrations and significantly ( P < .05) greater activation and growth factor concentrations in HGT-activated samples. This COX-2 inhibitor did not impair platelet activation, growth factor release, or TXB2 production in this canine PRP when using HGT as an activator. Studies are warranted to determine whether COX-2 inhibitors affect platelet activation and growth factor release from human PRPs. These results suggest that there is no need to withhold a COX-2 inhibitor before PRP preparation, particularly if thrombin is going to be used to activate the PRP. This is clinically relevant information because many patients who are candidates for PRP therapy for treatment of musculoskeletal injury are also using COX-2 inhibitors.

Potency of a tau fibrillization inhibitor is influenced by its aggregation state

PubMed Central

Congdon, Erin E.; Necula, Mihaela; Blackstone, Robert D.; Kuret, Jeff

2007-01-01

Tau fibrillization is a potential therapeutic target for Alzheimer’s and other neurodegenerative diseases. Several small molecule inhibitors of tau aggregation have been developed for this purpose. One of them, 3,3′-bis(β-hydroxyethyl)-9-ethyl-5,5′-dimethoxythiacarbocyanine iodide (N744), is a cationic thiacarbocyanine dye that inhibits recombinant tau filament formation when present at submicromolar concentrations. To prepare dosing regimens for testing N744 activity in biological models, its full concentration-effect relationship in the range 0.01 – 60 μM was examined in vitro by electron microscopy and laser light scattering methods. Results revealed that N744 concentration dependence was biphasic, with fibrillization inhibitory activity appearing at submicromolar concentration, but with relief of inhibition and increases in fibrillization apparent above 10 μM. Therefore, fibrillization was inhibited ≥50% only over a narrow concentration range, which was further reduced by filament stabilizing modifications such as tau pseudophosphorylation. N744 inhibitory activity also was paralleled by changes in its aggregation state, with dimer predominating at inhibitory concentrations and large dye aggregates appearing at high concentrations. Ligand dimerization was promoted by the presence of tau protein, which lowered the equilibrium dissociation constant for dimerization more than an order of magnitude relative to controls. The results suggest that ligand aggregation may play an important role in both inhibitory and disinhibitory phases of the concentration-effect curve, and may lead to complex dose response relationships in model systems. PMID:17559794

Effect of the scale inhibitor on ion content in reverse osmosis system for seawater desalination

NASA Astrophysics Data System (ADS)

Gao, Yuhua; Liu, Zhenfa; Zhang, Lihui; Li, Haihua

2017-09-01

A scale inhibitor was synthesized from polysuccinimide with 2-aminoethanesulfonic acid and aspartic acid. The effect of scale inhibitor on ion content in reverse osmosis system for seawater desalination was studied. The results showed that the ion content of permeate water is lower with the scale inhibitor added in RO system for seawater desalination than without scale inhibitor. On the contrary, the ion content of concentrate water is higher when with scale inhibitor in RO system.

In Vitro and In Vivo Effects of α-Amylase Inhibitor From Avena sativa Seeds on Life History and Physiological Characteristics of Sitotroga cerealella (Lepidoptera: Gelechiidae).

PubMed

Borzoui, Ehsan; Nouri-Ganbalani, Gadir; Naseri, Bahram

2017-11-01

The inhibitory effects of Avena sativa L. seed extract were studied on life history and some physiological aspects of Sitotroga cerealella (Olivier; Lepidoptera: Gelechiidae). The inhibition of α-amylase activity in vitro by A. sativa proteinaceous extract suggested its potential antimetabolic effect on S. cerealella larvae. Although, chronic ingestion of A. sativa inhibitor (I10: 0.108 mg protein/artificial seed) did not show significant reduction of the growth and development of S. cerealella. However, a delay in the developmental time of immature stages was detected when S. cerealella larvae were continuously fed on I30 and I50 concentrations (0.429 and 1.11 mg protein/artificial seed, respectively) of the inhibitor. The highest realized fecundity was recorded for females which came from larvae fed on I10 concentration (102.46 ± 2.50 eggs/female), and the lowest fecundity was observed for females which came from larvae fed on I50 concentration (31.64 ± 3.17 eggs/female). The lightest weight of pupae of S. cerealella was observed on I50 concentration (2.76 ± 0.07 mg). The lowest glycogen and lipid contents of the pupae were detected on I50 concentration (50.00 ± 3.53 and 289.57 ± 29.00 µg/pupa, respectively). The lower survival rate of pupae at low temperature indicated that S. cerealella fed on I50 concentration of the inhibitor was less cold tolerant than control insects. The inhibitory studies indicated that A. sativa proteinaceous extract is a good candidate as an inhibitor of the α-amylase of this pest. This inhibitor can be expressed in genetically engineered plants to confer resistance to S. cerealella. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

Tautomerism of N-(3,4-dichlorophenyl)-1H-indazole-5-carboxamide - A new selective, highly potent and reversible MAO-B inhibitor

NASA Astrophysics Data System (ADS)

Tzvetkov, Nikolay T.; Stammler, Hans-Georg; Antonov, Liudmil

2017-12-01

The tautomeric properties of an N-(3,4-dichlorophenyl)-1H-indazole-5-carboxamide (NTZ-1006, 2) derivative, developed as highly potent, reversible and selective MAO-B inhibitor useful for the treatment of Parkinson's disease (PD) and other neurological disorders, have been studied both experimentally and theoretically. The theoretical data (M06-2X, B3LYP and MP2-4 quantum chemical calculations) have shown that due to aromaticity reasons the 1H tautomer strongly dominates over the 2H form. There are no substantial spectral changes by changing the solvent and the concentration, which leads to a conclusion that compound 2 exists in solution as 1H tautomer and its tautomerism is not influenced by the solvents and the concentration. The results are in line with the understanding for the tautomerism of 1H-indazole and shows that substitution at the C5 position in the indazole unit does not influence the tautomeric state. The isolated crystal structure of 2 is in an excellent agreement with the computation in respect of the most stable tautomer. Combined single X-ray/molecular modeling studies including HYdrogen-DEsolvation (HYDE) analysis provided not only insights into the enzyme-inhibitor interaction within the binding site of the human MAO-B isoform, but also a valuable information regarding the most stable 1H-indazole tautomeric form of NTZ-1006 that contributes to its high potency against hMAO-B enzyme (IC50 0.586 nm) and selectivity (>17000-fold) over the hMAO-A isoenzyme.

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

PubMed

Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

2016-01-01

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

Enhancement of corrosion resistance of carbon steel by Dioscorea Hispida starch in NaCl

NASA Astrophysics Data System (ADS)

Zulhusni, M. D. M.; Othman, N. K.; Lazim, Azwan Mat

2015-09-01

Starch is a one of the most abundant natural product in the world and has the potential as corrosion inhibitor replacing harmful synthetic chemical based corrosion inhibitor. This research was aimed to examines the potential of starch extracted from local Malaysian wild yam (Dioscorea hispida), as corrosion inhibitor to carbon steel in NaCl media replicating sea water. By using gravimetric test and analysis, in which the carbon steel specimens were immersed in NaCl media for 24, 48 and 60 hours with the starch as corrosion inhibitor. the corrosion rate (mmpy) and inhibition efficiencies (%) was calculated. The results obtained showed decrease in corrosion rate as higher concentration of starch was employed. The inhibition efficiencies also shows an increasing manner up to 95.97 % as the concentration of the inhibitor increased.

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

PubMed

Heng, Sabrina; Stieglitz, Kimberly A; Eldo, Joby; Xia, Jiarong; Cardia, James P; Kantrowitz, Evan R

2006-08-22

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

Studies on 2-phenylquinoline Staphylococcus aureus NorA efflux pump inhibitors: New insights on the C-6 position.

PubMed

Felicetti, Tommaso; Cannalire, Rolando; Nizi, Maria Giulia; Tabarrini, Oriana; Massari, Serena; Barreca, Maria Letizia; Manfroni, Giuseppe; Schindler, Bryan D; Cecchetti, Violetta; Kaatz, Glenn W; Sabatini, Stefano

2018-06-06

The alarming and rapid spread of antimicrobial resistance among bacteria represents a high risk for global health. Targeting factors involved in resistance to restore the activity of failing antibiotics is a promising strategy to overcome this urgent medical need. Efflux pump inhibitors are able to increase antibiotic concentrations in bacteria, thus they can be considered true antimicrobial resistance breakers. In this work, continuing our studies on inhibitors of the Staphylococcus aureus NorA pump, we designed, synthesized and biologically evaluated novel 2-phenylquinoline derivatives starting from our hits 1 and 2. Two of the synthesized compounds (6 and 7) bearing a C-6 benzyloxy group showed the best NorA inhibition activity, thereby providing an excellent starting point to direct future chemical optimizations. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Enhanced ethanol fermentation by engineered Saccharomyces cerevisiae strains with high spermidine contents.

PubMed

Kim, Sun-Ki; Jo, Jung-Hyun; Jin, Yong-Su; Seo, Jin-Ho

2017-05-01

Construction of robust and efficient yeast strains is a prerequisite for commercializing a biofuel production process. We have demonstrated that high intracellular spermidine (SPD) contents in Saccharomyces cerevisiae can lead to improved tolerance against various fermentation inhibitors, including furan derivatives and acetic acid. In this study, we examined the potential applicability of the S. cerevisiae strains with high SPD contents under two cases of ethanol fermentation: glucose fermentation in repeated-batch fermentations and xylose fermentation in the presence of fermentation inhibitors. During the sixteen times of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivities than a control strain with lower SPD contents. Specifically, at the sixteenth fermentation, the ethanol productivity of a S. cerevisiae strain with twofold higher SPD content was 31% higher than that of the control strain. When the SPD content was elevated in an engineered S. cerevisiae capable of fermenting xylose, the resulting S. cerevisiae strain exhibited much 40-50% higher ethanol productivities than the control strain during the fermentations of synthetic hydrolysate containing high concentrations of fermentation inhibitors. These results suggest that the strain engineering strategy to increase SPD content is broadly applicable for engineering yeast strains for robust and efficient production of ethanol.

Subatomic and atomic crystallographic studies of aldose reductase: implications for inhibitor binding.

PubMed

Podjarny, A; Cachau, R E; Schneider, T; Van Zandt, M; Joachimiak, A

2004-04-01

The determination of several of aldose reductase-inhibitor complexes at subatomic resolution has revealed new structural details, including the specific interatomic contacts involved in inhibitor binding. In this article, we review the structures of the complexes of ALR2 with IDD 594 (resolution: 0.66 angstrom, IC50 (concentration of the inhibitor that produced half-maximal effect): 30 nM, space group: P2(1)), IDD 393 (resolution: 0.90 angstrom, IC50: 6 nM, space group: P1), fidarestat (resolution: 0.92 angstrom, IC50: 9 nM, space group: P2(1)) and minalrestat (resolution: 1.10 angstrom, IC50: 73 nM, space group: P1). The structures are compared and found to be highly reproductible within the same space group (root mean square (RMS) deviations: 0.15 approximately 0.3 angstrom). The mode of binding of the carboxylate inhibitors IDD 594 and IDD 393 is analysed. The binding of the carboxylate head can be accurately determined by the subatomic resolution structures, since both the protonation states and the positions of the atoms are very precisely known. The differences appear in the binding in the specificity pocket. The high-resolution structures explain the differences in IC50, which are confirmed both experimentally by mass spectrometry measures of VC50 and theoretically by free energy perturbation calculations. The binding of the cyclic imide inhibitors fidarestat and minalrestat is also described, focusing on the observation of a Cl(-) ion which binds simultaneously with fidarestat. The presence of this anion, binding also to the active site residue His110, leads to a mechanism in which the inhibitor can bind in a neutral state and then become charged inside the active site pocket. This mechanism can explain the excellent in vivo properties of cyclic imide inhibitors. In summary, the complete and detailed information supplied by the subatomic resolution structures can explain the differences in binding energy of the different inhibitors.

Studies on the impact, detection, and control of microbiology influenced corrosion related to pitting failures in the Russian oil and gas industry. Final CRADA report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ehst, D.

2006-09-30

The objectives of the Project are: (1) to design effective anti-corrosion preparations (biocides, inhibitors, penetrants and their combinations) for gas- and oil-exploration industries; (2) to study a possibility of development of environmentally beneficial ('green') biocides and inhibitors of the new generation; (3) to develop chemical and microbiological methods of monitoring of sites at risk of corrosion; and (4) to evaluate potentialities in terms of technology, raw materials and material and technical basis to set up a production of effective anti-corrosion preparations of new generation in Russia. During the four years of the project 228 compounds and formulations were synthesized andmore » studied in respect to their corrosion inhibiting activity. A series of compounds which were according to the Bubble tests more efficient (by a factor of 10-100) than the reference inhibitor SXT-1102, some possessing the similar activity or slightly better activity than new inhibitor ??-1154? (company ONDEO/Nalco). Two synthetic routes for the synthesis of mercaptopyrimidines as perspective corrosion inhibitors were developed. Mercaptopyrimidine derivatives can be obtained in one or two steps from cheap and easily available precursors. The cost for their synthesis is not high and can be further reduced after the optimization of the production processes. A new approach for lignin utilization was proposed. Water-soluble derivative of lignin can by transformed to corrosion protective layer by its electropolymerization on a steel surface. Varying lignosulfonates from different sources, as well as conditions of electrooxidation we proved, that drop in current at high anodic potentials is due to electropolymerization of lignin derivative at steel electrode surface. The electropolymerization potential can be sufficiently decreased by an increase in ionic strength of the growing solution. The lignosulfonate electropolymerization led to the considerable corrosion protection effect of carbon steel. More than three times decrease of corrosion rate on steel surface was observed after lignosulfonate electropolymerization, exceeding protective effect of standard commercially available corrosion inhibitor. Solikamsky lignin could be a promising candidate as a base for the development of the future green corrosion inhibitor. A protective effect of isothiazolones in compositions with other biocides and inhibitors was investigated. Additionally to high biocidal properties, combination of kathon 893 and copper sulfate may also produce a strong anticorrosion effect depending on concentrations of the biocides. Based on its joint biocidal and anticorrosion properties, this combination can be recommended for protection of pipelines against carbon dioxide-induced corrosion. By means of linear polarization resistance test, corrosion properties of biocides of different classes were studied. Isothiazolones can be recommended for treating oil-processing waters in Tatarstan to curb carbon dioxide - induced corrosion. A laboratory research on evaluation of the efficiency of biocides, inhibitors and penetrants by biological and physical-and-chemical methods has been carried out. It was shown that action of corrosion inhibitors and biocides strongly depends on character of their interaction with mineral substances available in waters on oil-exploration sites. It was found that one of approaches to designing environmentally safe ('green') antimicrobial formulations may be the use of synergetic combinations, which allow one to significantly decrease concentrations of biocides. It was shown that the efficacy of biocides and inhibitors depends on physicochemical characteristics of the environment. Anticorrosion and antimicrobial effects of biocides and inhibitors depended in much on the type of medium and aeration regimen. Effects of different biocides, corrosion inhibitors. penetrants and their combinations on the biofilm were investigated. It has been shown that minimal inhibiting concentrations of the reagents for the biofilm are much higher than those for aquatic microorganisms. Results obtained from the research in stationary conditions have been confirmed with data from experiments carried out in hydrodynamic conditions. New approaches to the investigation of biocorrosive processes on the basis of bioluminescent method of intracellular ATP determination have been developed. Approaches and methods developed on the basis of bioluminescent method could significantly simplify the analysis of biocorrosion processes and allow to conduct the analysis directly under the field conditions in situ. An express method to assess biogenic sulfate reduction in soil and water samples has been elaborated. The method intends for field application and allows one to no-problem assess action of such harmful and corrosion provoking microorganisms, as sulfate-reducing bacteria.« less

Aldosterone response to angiotensin II during hypoxemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colice, G.L.; Ramirez, G.

1986-07-01

Exercise stimulates the renin-angiotensin-aldosterone system (RAAS). However, increases in plasma aldosterone concentrations (PAC) are suppressed when exercise is performed at high altitude or under hypoxemic conditions. As the angiotensin-II response to high-altitude exercise is normal, it is speculated that an inhibitor, discharged during hypoxemia, acted to suppress angiotensin-II-mediated aldosterone release. A study was conducted to test this hypothesis, taking into account the measurement of the aldosterone response to exogenous angiotensin II during normoxemia and hypoxemia. It was found that the dose-response curve of PAC to angiotensin II was not significantly inhibited by the considered model of hypoxemia. The hypoxemia-mediated releasemore » of an angiotensin II inhibitor does, therefore, not explain the previous observations of PAC suppression during hypoxemic exercise. 28 references.« less

Recombinant factor VIIa (eptacog alfa): a review of its use in congenital hemophilia with inhibitors, acquired hemophilia, and other congenital bleeding disorders.

PubMed

Croom, Katherine F; McCormack, Paul L

2008-01-01

Recombinant factor VIIa (NovoSeven; also known as recombinant activated factor VII or eptacog alfa) is structurally similar to human plasma-derived coagulation factor VIIa, but is manufactured using DNA biotechnology. Recombinant factor VIIa interacts with thrombin-activated platelets to produce a thrombin burst leading to accelerated fibrin clot formation localized to the site of vascular injury. It is approved in many countries for use as an intravenous hemostatic agent in patients with congenital hemophilia with inhibitors, and also for acquired hemophilia, factor VII deficiency, and Glanzmann thrombasthenia in some countries. Studies have shown it to be effective and generally well tolerated when used intravenously to treat bleeding episodes or provide hemostatic cover during surgery in patients with congenital hemophilia with inhibitors, acquired hemophilia, factor VII deficiency or Glanzmann thrombasthenia. Based on available data, its efficacy in terms of patient-assessed response may be similar to that of activated prothrombin complex concentrate (aPCC), but treatment with a single 270 microg/kg dose of recombinant factor VIIa might reduce the need for rescue therapy compared with aPCC. Recombinant factor VIIa is not immunogenic in patients with hemophilia, does not produce an anamnestic response in hemophilia patients with inhibitors, and has very low thrombogenicity. It is recommended in guidelines as the treatment of choice for bleeds in patients with hemophilia B with high-responding inhibitors and for patients with factor VII deficiency, and is also a first-line therapeutic option for high-responder hemophilia A patients with inhibitors and those with acquired hemophilia. Cost data from pharmacoeconomic analyses support its use in hemophilia patients with inhibitors. Thus, recombinant factor VIIa is a valuable treatment option for patients with these rare, but potentially serious, bleeding disorders.

Comparative technoeconomic analysis of a softwood ethanol process featuring posthydrolysis sugars concentration operations and continuous fermentation with cell recycle.

PubMed

Schneiderman, Steven J; Gurram, Raghu N; Menkhaus, Todd J; Gilcrease, Patrick C

2015-01-01

Economical production of second generation ethanol from Ponderosa pine is of interest due to widespread mountain pine beetle infestation in the western United States and Canada. The conversion process is limited by low glucose and high inhibitor concentrations resulting from conventional low-solids dilute acid pretreatment and enzymatic hydrolysis. Inhibited fermentations require larger fermentors (due to reduced volumetric productivity) and low sugars lead to low ethanol titers, increasing distillation costs. In this work, multiple effect evaporation (MEE) and nanofiltration (NF) were evaluated to concentrate the hydrolysate from 30 g/l to 100, 150, or 200 g/l glucose. To ferment this high gravity, inhibitor containing stream, traditional batch fermentation was compared with continuous stirred tank fermentation (CSTF) and continuous fermentation with cell recycle (CSTF-CR). Equivalent annual operating cost (EAOC = amortized capital + yearly operating expenses) was used to compare these potential improvements for a local-scale 5 MGY ethanol production facility. Hydrolysate concentration via evaporation increased EAOC over the base process due to the capital and energy intensive nature of evaporating a very dilute sugar stream; however, concentration via NF decreased EAOC for several of the cases (by 2 to 15%). NF concentration to 100 g/l glucose with a CSTF-CR was the most economical option, reducing EAOC by $0.15 per gallon ethanol produced. Sensitivity analyses on NF options showed that EAOC improvement over the base case could still be realized for even higher solids removal requirements (up to two times higher centrifuge requirement for the best case) or decreased NF performance. © 2015 American Institute of Chemical Engineers.

Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways.

PubMed Central

Mateo, Jesús; García-Lecea, Marta; Cadenas, Susana; Hernández, Carlos; Moncada, Salvador

2003-01-01

Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance. PMID:14531732

Demonstration of Aflatoxin Inhibitory Activity in a Cotton Seed Coat Xylan

PubMed Central

Mellon, J. E.; Cotty, P. J.; Godshall, M. A.; Roberts, E.

1995-01-01

An inhibitor of aflatoxin biosynthesis localized in the seed coats of developing cotton was partially purified and characterized. Aqueous extracts from 25-day postanthesis seed coat tissue inhibited aflatoxin (B(inf1)) production in liquid cultures of Aspergillus flavus AF13. Inhibition was concentration dependent, with a 50% effective dose of 173 (mu)g of crude extract per ml of medium. The inhibitor was neutral in charge. Two active fractions were obtained from crude preparations by gel filtration chromatography (BioGel P-100). The purest fraction eluted in the void volume. Carbohydrate composition analysis of this void volume inhibitor indicated a composition of xylose (>90%) and mannose. Aflatoxin production in vitro was inversely related to inhibitor concentration in the fermentation medium (log of aflatoxin versus log of [inhibitor]; r(sup2) = 0.82; P < 0.002). The void volume inhibitor had a 50% effective dose of 6.2 (mu)g/ml, a 28-fold purification of the inhibitor material. These data support the hypothesis that seed coat inhibitory activity is associated with a cottonseed-specific xylan. PMID:16535194

[Concentration of cysteine proteinase inhibitors in urine, amniotic fluid and serum from women in pregnancy complicated by EPH-gestosis].

PubMed

Karmowski, A; Sobiech, K A; Kertyńska, I; Terpiłowski, L; Słowińska-Lisowska, M; Pałczyński, B; Malik, B

2000-10-01

Cysteine proteinase inhibitors (IPC) concentration was measured by the modified Barrett method using papaine in urine, amniotic fluid and serum obtained from the healthy labored women and from labored women in pregnancy complicated by EPH-gestosis. It was noticed the statistically significant increase in the IPC concentration in the material from the pregnant women with EPH-gestosis comparing to the women, which pregnancy had the physiologically normal course.

Inhibition of Growth by Combined Treatment with Inhibitors of Lactate Dehydrogenase and either Phenformin or Inhibitors of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3.

PubMed

Lea, Michael A; Guzman, Yolanda; Desbordes, Charles

2016-04-01

Enhanced glycolysis in cancer cells presents a target for chemotherapy. Previous studies have indicated that proliferation of cancer cells can be inhibited by treatment with phenformin and with an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB) namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work, the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) were investigated. The inhibitors of lactate dehydrogenase (LDHA) studied in order of half-maximal inhibitory concentrations were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. In colonic and bladder cancer cells, additive growth inhibitory effects were seen with the LDHA inhibitors, of which NHI-2 was effective at the lowest concentrations. Growth inhibition was generally greater with PFK15 than with PQP. The increased acidification of the culture medium and glucose uptake caused by phenformin was blocked by combined treatment with PFKFB3 or LDHA inhibitors. The results suggest that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation and may mitigate lactic acidosis caused by phenformin when used as a single agent. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Localized CO2 corrosion of carbon steel with different microstructures in brine solutions with an imidazoline-based inhibitor

NASA Astrophysics Data System (ADS)

Zhang, Huan-huan; Pang, Xiaolu; Gao, Kewei

2018-06-01

CO2 corrosion behavior of carbon steel with different microstructures (H steel: coarse laminar pearlite; T steel: globular and shot rod shaped pearlite) was analyzed in 3 wt.% NaCl solution at 60 °C with imidazoline-based inhibitor by electrochemical and weight loss methods. Electrochemical measurements showed that, compared to H steel, the inhibitor film adsorbed on T steel had a higher pitting corrosion resistance and the inhibition efficiency for T steel was larger at each concentration of inhibitor. Weight loss results exhibited that both steels suffered general corrosion in absence of inhibitor; however, localized corrosion was observed on the samples with insufficient concentration of inhibitor. H steel suffered more severe localized corrosion than T steel, it was related to that H steel had a higher density of dislocations in the pearlite area and the larger driving force for galvanic corrosion. The localized corrosion on H steel mainly distributed on the laminar pearlite area.

Effect of Proton Pump Inhibitors on the Serum Concentrations of the Selective Serotonin Reuptake Inhibitors Citalopram, Escitalopram, and Sertraline

PubMed Central

Gjestad, Caroline; Westin, Andreas A.; Skogvoll, Eirik

2015-01-01

Background: The selective serotonin reuptake inhibitors (SSRIs) citalopram, escitalopram, and sertraline are all metabolized by the cytochrome P-450 isoenzyme CYP2C19, which is inhibited by the proton pump inhibitors (PPIs) omeprazole, esomeprazole, lansoprazole, and pantoprazole. The aim of the present study was to evaluate the effect of these PPIs on the serum concentrations of citalopram, escitalopram, and sertraline. Methods: Serum concentrations from patients treated with citalopram, escitalopram, or sertraline were obtained from a routine therapeutic drug monitoring database, and samples from subjects concomitantly using PPIs were identified. Dose-adjusted SSRI serum concentrations were calculated to compare data from those treated and those not treated with PPIs. Results: Citalopram concentrations were significantly higher in patients treated with omeprazole (+35.3%; P < 0.001), esomeprazole (+32.8%; P < 0.001), and lansoprazole (+14.7%; P = 0.043). Escitalopram concentrations were significantly higher in patients treated with omeprazole (+93.9%; P < 0.001), esomeprazole (+81.8%; P < 0.001), lansoprazole (+20.1%; P = 0.008), and pantoprazole (+21.6%; P = 0.002). Sertraline concentrations were significantly higher in patients treated with esomeprazole (+38.5%; P = 0.0014). Conclusions: The effect of comedication with PPIs on the serum concentration of SSRIs is more pronounced for omeprazole and esomeprazole than for lansoprazole and pantoprazole, and escitalopram is affected to a greater extent than are citalopram and sertraline. When omeprazole or esomeprazole are used in combination with escitalopram, a 50% dose reduction of the latter should be considered. PMID:24887634

Simultaneous determination of the HIV nucleoside analogue reverse transcriptase inhibitors lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by reversed phase high performance liquid chromatography.

PubMed

Verweij-van Wissen, C P W G M; Aarnoutse, R E; Burger, D M

2005-02-25

A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a SymmetryShield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015-5 mg/l for all five NRTIs. The average accuracies for the assay were 92-102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.

[Effect of total ischemia and 3',5'-cAMP on the activity of the thermostable cytoplasmic inhibitor of Ca2+ ion transport in rat heart mitochondria].

PubMed

Turakulov, Ia Kh; Luchenko, M B; Gaĭnutdinov, M Kh; Abidov, A A

1985-01-01

Activity of cytoplasmic inhibitor of Ca2+ transport in rat heart mitochondria was studied after total ischemia and incubation of heart homogenates with cAMP. Distinct inactivation of the inhibitor occurred under these conditions. The decrease of the inhibitor activity in ischemic myocardium appears to serve as a compensatory mechanism: 1. pyruvate dehydrogenase and the enzymes of tricarboxylic acid cycle were activated due to increase in Ca2+ concentration in mitochondria, 2. as a result of Ca2+ accumulation in mitochondria the elevated concentration of Ca2+ was decreased in myoplasm, which developed after impairment of plasmatic membranes and of sarcoplasmic reticulum membranes.

Application of a cosmetic additive as an eco-friendly inhibitor for mild steel corrosion in HCl solution.

PubMed

Liao, Liu Li; Mo, Shi; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing

2016-07-15

The use of the cosmetic ingredient cocamidopropylamine oxide (CAO) to inhibit the corrosion of steel in 0.5mol/LHCl is investigated. Electrochemical and weight loss methods were used to evaluate the inhibiting effect of CAO and the influences of inhibitor concentration and temperature were determined. It was found that CAO acted as a mix-type inhibitor and was adsorbed chemically onto the steel in HCl solution, and the maximum inhibition efficiency was found at critical micelle concentration (CMC) of CAO in tested corrosive media. Moreover, it was speculated that relationships of the two adsorption sites of the inhibitor and steel surface were different. Copyright © 2016 Elsevier Inc. All rights reserved.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

Aromatase inhibitor (anastrozole) affects growth of endometrioma cells in culture.

PubMed

Badawy, Shawky Z A; Brown, Shereene; Kaufman, Lydia; Wojtowycz, Martha A

2015-05-01

To study the effects of aromatase inhibitor (anastrozole) on the growth and estradiol secretion of endometrioma cells in culture. Endometrioma cells are grown in vitro until maximum growth before used in this study. This was done in the research laboratory for tissue culture, in an academic hospital. Testosterone at a concentration of 10 μg/mL was added as a substrate for the intracellular aromatase. In addition, aromatase inhibitor was added at a concentration of 200 and 300 μg/mL. The effect on cell growth and estradiol secretion is evaluated using Student's t-test. The use of testosterone increased estradiol secretion by endometrioma cells in culture. The use of aromatase inhibitor significantly inhibited the growth of endometrioma cells, and estradiol secretion. Aromatase inhibitor (anastrozole) may be an effective treatment for endometriosis due to inhibition of cellular aromatase. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Corrosion mitigation of N-(2-hydroxy-3-trimethyl ammonium)propyl chitosan chloride as inhibitor on mild steel.

PubMed

Sangeetha, Y; Meenakshi, S; SairamSundaram, C

2015-01-01

The biopolymer N-(2-hydroxy-3-trimethyl ammonium)propyl chitosan chloride (HTACC) was synthesised and its influence as a novel corrosion inhibitor on mild steel in 1M HCl was studied using gravimetric and electrochemical experiments. The compound obtained was characterised using FTIR and NMR studies. The inhibition efficiency increased with the increase in concentration and reached a maximum of 98.9% at 500 ppm concentration. Polarisation studies revealed that HTACC acts both as anodic and cathodic inhibitor. Electrochemical impedance studies confirmed that the inhibition is through adsorption on the metal surface. The extent of inhibition exhibits a negative trend with increase in temperature. Langmuir isotherm provides the best description on the adsorption nature of the inhibitor. SEM analysis indicated the presence of protective film formed by the inhibitor on the metal surface. Copyright © 2014 Elsevier B.V. All rights reserved.

Nicotinamide Ribosyl Uptake Mutants in Haemophilus influenzae

PubMed Central

Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiβ, Anita; Hilpert, Anna-Karina; Reidl, Joachim

2003-01-01

The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease. PMID:12933892

Synthesis and characterization of a novel organic corrosion inhibitor for mild steel in 1 M hydrochloric acid

NASA Astrophysics Data System (ADS)

Ahmed, Mohammed H. Othman; Al-Amiery, Ahmed A.; Al-Majedy, Yasmin K.; Kadhum, Abdul Amir H.; Mohamad, Abu Bakar; Gaaz, Tayser Sumer

2018-03-01

The synthesis and characterization of a novel organic corrosion inhibitor (4-(3-mercapto-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)phenol), for mild steel in 1 M hydrochloric acid (HCl) has been successfully reported for the first time. The inhibitor evaluated as corrosion inhibitor for mild steel in 1 M of Hydrochloric acid solution using electrochemical impedance spectroscopy (EIS), and electrochemical frequency modulation (EFM) measurement techniques. Changes in the impedance parameters suggested an adsorption of the inhibitor onto the mild steel surface, leading to the formation of protective films. The results show that the inhibition efficiencies increased with increasing the concentrations of the inhibitors and decreased with increasing temperature. The maximum inhibition efficiency up to 67% at the maximum concentration 0.5 mM. This shows that those inhibitors are effective in helping to reduce and slowing down the corrosion process that occurs to mild steel with a hydrochloric acid solution by providing an organic inhibitor for the mild steel that can be weakened by increasing the temperature. The adsorption process of the synthesized organic inhibitor depends on its electronic characteristics in addition to steric effects and the nature of metal surface, temperature degree and the varying degrees of surface-site activity. The synthesized inhibitor molecules were absorbed by metal surface and follow Langmuir isotherms.

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

PubMed

Franco, Octávio L; dos Santos, Roseane C; Batista, João A N; Mendes, Ana Cristina M; de Araújo, Marcus Aurélio M; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima; de Freitas, Sonia M

2003-06-01

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.

Stable, concentrated solutions of high molecular weight polyaniline and articles therefrom

DOEpatents

Mattes, Benjamin R.; Wang, Hsing-Lin

2000-01-01

Stable, concentrated solutions of high molecular weight polyaniline. In order to process high quality fibers and other articles possessing good mechanical properties, it is known that solution concentrations of the chosen polymer should be in the range from 15-30% (w/w). Moreover, it is desirable to use the highest molecular weight consistent with the solubility properties of the polymer. However, such solutions are inherently unstable, forming gels before processing can be achieved. The present invention describes the addition gel inhibitors (GIs) to the polymer solution, thereby permitting high concentrations (>15% (w/w)) of high molecular weight ((M.sub.w)>120,000, and (M.sub.n)>30,000) emeraldine base (EB) polyaniline to be dissolved. Secondary amines have been used for this purpose in concentrations which are small compared to those which might otherwise be used in a cosolvent role therefor. The resulting solutions are useful for generating excellent fibers, films, coatings and other objects, since the solutions are stable for significant time periods, and the GIs are present in too small concentrations to cause polymer deterioration. It is demonstrated that the GIs found to be useful do not act as cosolvents, and that gelation times of the solutions are directly proportional to the concentration of GI. In particular, there is a preferred concentration of GI, which if exceeded causes structural and electrical conductivity degradation of resulting articles. Heating of the solutions significantly improves solubility.

Validation of N-myristoyltransferase as Potential Chemotherapeutic Target in Mammal-Dwelling Stages of Trypanosoma cruzi

PubMed Central

Herrera, Linda J.; Brand, Stephen; Santos, Andres; Nohara, Lilian L.; Harrison, Justin; Norcross, Neil R.; Thompson, Stephen; Smith, Victoria; Lema, Carolina; Varela-Ramirez, Armando; Gilbert, Ian H.; Almeida, Igor C.; Maldonado, Rosa A.

2016-01-01

Background Trypanosoma cruzi causes Chagas disease, an endemic and debilitating illness in Latin America. Lately, owing to extensive population movements, this neglected tropical disease has become a global health concern. The two clinically available drugs for the chemotherapy of Chagas disease have rather high toxicity and limited efficacy in the chronic phase of the disease, and may induce parasite resistance. The development of new anti-T. cruzi agents is therefore imperative. The enzyme N-myristoyltransferase (NMT) has recently been biochemically characterized, shown to be essential in Leishmania major, Trypanosoma brucei, and T. cruzi¸ and proposed as promising chemotherapeutic target in these trypanosomatids. Methodology/Principal Findings Here, using high-content imaging we assayed eight known trypanosomatid NMT inhibitors, against mammal-dwelling intracellular amastigote and trypomastigote stages and demonstrated that three of them (compounds 1, 5, and 8) have potent anti-proliferative effect at submicromolar concentrations against T. cruzi, with very low toxicity against human epithelial cells. Moreover, metabolic labeling using myristic acid, azide showed a considerable decrease in the myristoylation of proteins in parasites treated with NMT inhibitors, providing evidence of the on-target activity of the inhibitors. Conclusions/Significance Taken together, our data point out to the potential use of NMT inhibitors as anti-T. cruzi chemotherapy. PMID:27128971

Small molecule inhibitors of human adipocyte fatty acid binding protein (FABP4).

PubMed

Zhang, Mingming; Zhu, Weiliang; Li, Yingxia

2014-06-01

Fatty acid binding protein 4 (FABP4) is expressed in adipocytes and macrophages, and modulates inflammatory and metabolic response. Studies in FABP4-deficient mice have shown that this lipid carrier has a significant role within the field of metabolic syndrome, inflammation and atherosclerosis; thus, its inhibition may open up new opportunities to develop novel therapeutic agents. A number of potent small molecule inhibitors of FABP4 have been identified and found to have the potential to prevent and treat metabolic diseases such as type-2 diabetes and atherosclerosis. Due to the ubiquity of endogenous fatty acids and the high intracellular concentration of FABP4, the inhibitors need to have significantly greater intrinsic potency than endogenous fatty acids. Furthermore, heart-type FABP (FABP3), which is expressed in both heart and skeletal muscle, is involved in active fatty acid metabolism where it transports fatty acids from the cell membrane to mitochondria for oxidation. However, FABP3 shares high overall sequence identity and similar 3D structure with FABP4, but has a potential problem with selectivity. In this review, we would like to analyze the main inhibitors that have appeared in the literature in the last decade, focusing on chemical structures, biological properties, selectivity and structure-activity relationships.

Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

PubMed

Hartwig, Thomas; Corvalan, Claudia; Best, Norman B; Budka, Joshua S; Zhu, Jia-Ying; Choe, Sunghwa; Schulz, Burkhard

2012-01-01

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

PubMed

Zech, Stephan G; Kohlmann, Anna; Zhou, Tianjun; Li, Feng; Squillace, Rachel M; Parillon, Lois E; Greenfield, Matthew T; Miller, David P; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Xu, Yongjin; Miret, Juan J; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2016-01-28

Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

Thermoresponsive Microcarriers for Smart Release of Hydrate Inhibitors under Shear Flow.

PubMed

Lee, Sang Seok; Park, Juwoon; Seo, Yutaek; Kim, Shin-Hyun

2017-05-24

The hydrate formation in subsea pipelines can cause oil and gas well blowout. To avoid disasters, various chemical inhibitors have been developed to prevent or delay the hydrate formation and growth. Nevertheless, direct injection of the inhibitors results in environmental contamination and cross-suppression of inhibition performance in the presence of other inhibitors against corrosion and/or formation of scale, paraffin, and asphaltene. Here, we suggest a new class of microcarriers that encapsulate hydrate inhibitors at high concentration and release them on demand without active external triggering. The key to the success in microcarrier design lies in the temperature dependence of polymer brittleness. The microcarriers are microfluidically created to have an inhibitor-laden water core and polymer shell by employing water-in-oil-in-water (W/O/W) double-emulsion drops as a template. As the polymeric shell becomes more brittle at a lower temperature, there is an optimum range of shell thickness that renders the shell unstable at temperature responsible for hydrate formation under a constant shear flow. We precisely control the shell thickness relative to the radius by microfluidics and figure out the optimum range. The microcarriers with the optimum shell thickness are selectively ruptured by shear flow only at hydrate formation temperature and release the hydrate inhibitors. We prove that the released inhibitors effectively retard the hydrate formation without reduction of their performance. The microcarriers that do not experience the hydration formation temperature retain the inhibitors, which can be easily separated from ruptured ones for recycling by exploiting the density difference. Therefore, the use of microcarriers potentially minimizes the environmental damages.

Isolation of a small molecule inhibitor of DNA base excision repair

PubMed Central

Madhusudan, Srinivasan; Smart, Fiona; Shrimpton, Paul; Parsons, Jason L.; Gardiner, Laurence; Houlbrook, Sue; Talbot, Denis C.; Hammonds, Timothy; Freemont, Paul A.; Sternberg, Michael J. E.; Dianov, Grigory L.; Hickson, Ian D.

2005-01-01

The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3′-phosphodiesterase and 3′-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy. PMID:16113242

Exposure-Response Model of Subcutaneous C1-Inhibitor Concentrate to Estimate the Risk of Attacks in Patients With Hereditary Angioedema.

PubMed

Zhang, Ying; Tortorici, Michael A; Pawaskar, Dipti; Pragst, Ingo; Machnig, Thomas; Hutmacher, Matthew; Zuraw, Bruce; Cicardi, Marco; Craig, Timothy; Longhurst, Hilary; Sidhu, Jagdev

2018-03-01

Subcutaneous C1-inhibitor (HAEGARDA, CSL Behring), is a US Food and Drug Administration (FDA)-approved, highly concentrated formulation of a plasma-derived C1-esterase inhibitor (C1-INH), which, in the phase III Clinical Studies for Optimal Management in Preventing Angioedema with Low-Volume Subcutaneous C1-inhibitor Replacement Therapy (COMPACT) trial, reduced the incidence of hereditary angioedema (HAE) attacks when given prophylactically. Data from the COMPACT trial were used to develop a repeated time-to-event model to characterize the timing and frequency of HAE attacks as a function of C1-INH activity, and then develop an exposure-response model to assess the relationship between C1-INH functional activity levels (C1-INH(f)) and the risk of an attack. The C1-INH(f) values of 33.1%, 40.3%, and 63.1% were predicted to correspond with 50%, 70%, and 90% reductions in the HAE attack risk, respectively, relative to no therapy. Based on trough C1-INH(f) values for the 40 IU/kg (40.2%) and 60 IU/kg (48.0%) C1-INH (SC) doses, the model predicted that 50% and 67% of the population, respectively, would see at least a 70% decrease in the risk of an attack. © 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

PubMed

Davis, Mindy I; Patrick, Stephen L; Blanding, Walker M; Dwivedi, Varun; Suryadi, Jimmy; Golden, Jennifer E; Coussens, Nathan P; Lee, Olivia W; Shen, Min; Boxer, Matthew B; Hall, Matthew D; Sharlow, Elizabeth R; Drew, Mark E; Morris, James C

2016-10-01

Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

PubMed

Pieters, Marlien; Barnard, Sunelle A; Loots, Du Toit; Rijken, Dingeman C

2017-01-01

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.

Antimicrobial 2-hydroxyisocaproic acid and chlorhexidine resist inactivation by dentine.

PubMed

Sakko, M; Tjäderhane, L; Sorsa, T; Hietala, P; Rautemaa, R

2016-04-01

To compare the antibacterial activity of 2-hydroxyisocaproic acid (HICA) with currently used root canal medicaments and to examine their interactions with potential inhibitors in nutrient-deficient and nutrient-rich conditions. First, the antibacterial activity of single concentrations of HICA, calcium hydroxide solution or slurry, chlorhexidine digluconate or acetate was tested against Enterococcus faecalis with and without potential inhibitors: dentine powder (DP), hydroxyapatite or bovine serum albumin, in a low concentration of peptone water. Relative viable counts were determined by culture at 1, 24 and 48 h. In the second set of experiments, the activity of three concentrations of HICA was evaluated against two isolates of E. faecalis with and without potential inhibitors in nutrient-rich thioglycollate broth using a modification of a standard microdilution method. The minimum bactericidal concentration was determined by culture at 1, 24 and 48 h. Concentrations of ≥33 mg mL(-1) of HICA were found to be bactericidal against E. faecalis in both nutrient-deficient and nutrient-rich environments at 24- to 48-h incubation, whereas the initial activity of Ca(OH)2 slurry was lost at 48-h incubation. HICA tolerated well all tested potential inhibitors up to 19 mg mL(-1) . DP concentrations higher than this inhibited its activity in a dose-dependent manner in both environments. DP demonstrated moderate antibacterial activity, and it enhanced the otherwise limited activity of Ca(OH)2 slurry and solution. DP did not impact on the activity of chlorhexidine. These results support the long-term antibacterial activity of HICA and indicate its tolerance to clinically relevant concentrations of dentine and other inhibitors commonly present in the root canal system. Therefore, HICA may have potential as an interappointment medication in the treatment of root canal infections. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Novel DDR Processing of Corn Stover Achieves High Monomeric Sugar Concentrations from Enzymatic Hydrolysis (230 g/L) and High Ethanol Concentration (10% v/v) During Fermentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xiaowen; Jennings, Ed; Shekiro, Joe

Distilling and purifying ethanol, butanol, and other products from second and later generation lignocellulosic biorefineries adds significant capital and operating cost for biofuels production. The energy costs associated with distillation affects plant gate and life cycle analysis costs. Lower titers in fermentation due to lower sugar concentrations from pretreatment increase both energy and production costs. In addition, higher titers decrease the volumes required for enzymatic hydrolysis and fermentation vessels. Therefore, increasing biofuels titers has been a research focus in renewable biofuels production for several decades. In this work, we achieved over 200 g/L of monomeric sugars after high solids enzymaticmore » hydrolysis using the novel deacetylation and disc refining (DDR) process on corn stover. The high sugar concentrations and low chemical inhibitor concentrations from the DDR process allowed ethanol titers as high as 82 g/L in 22 hours, which translates into approximately 10 vol% ethanol. To our knowledge, this is the first time that 10 vol% ethanol in fermentation derived from corn stover without any sugar concentration or purification steps has been reported. Techno-economic analysis shows the higher titer ethanol achieved from the DDR process could significantly reduce the minimum ethanol selling price from cellulosic biomass.« less

Kinetics of Corrosion Inhibition of Aluminum in Acidic Media by Water-Soluble Natural Polymeric Pectates as Anionic Polyelectrolyte Inhibitors.

PubMed

Hassan, Refat M; Zaafarany, Ishaq A

2013-06-17

Corrosion inhibition of aluminum (Al) in hydrochloric acid by anionic polyeletrolyte pectates (PEC) as a water-soluble natural polymer polysaccharide has been studied using both gasometric and weight loss techniques. The results drawn from these two techniques are comparable and exhibit negligible differences. The inhibition efficiency was found to increase with increasing inhibitor concentration and decrease with increasing temperature. The inhibition action of PEC on Al metal surface was found to obey the Freundlich isotherm. Factors such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and temperature affecting the corrosion rates were examined. The kinetic parameters were evaluated and a suitable corrosion mechanism consistent with the kinetic results is discussed in the paper.

Kinetics of Corrosion Inhibition of Aluminum in Acidic Media by Water-Soluble Natural Polymeric Pectates as Anionic Polyelectrolyte Inhibitors

PubMed Central

Hassan, Refat M.; Zaafarany, Ishaq A.

2013-01-01

Corrosion inhibition of aluminum (Al) in hydrochloric acid by anionic polyeletrolyte pectates (PEC) as a water-soluble natural polymer polysaccharide has been studied using both gasometric and weight loss techniques. The results drawn from these two techniques are comparable and exhibit negligible differences. The inhibition efficiency was found to increase with increasing inhibitor concentration and decrease with increasing temperature. The inhibition action of PEC on Al metal surface was found to obey the Freundlich isotherm. Factors such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and temperature affecting the corrosion rates were examined. The kinetic parameters were evaluated and a suitable corrosion mechanism consistent with the kinetic results is discussed in the paper. PMID:28809282

Virodhamine relaxes the human pulmonary artery through the endothelial cannabinoid receptor and indirectly through a COX product

PubMed Central

Kozłowska, H; Baranowska, M; Schlicker, E; Kozłowski, M; Laudañski, J; Malinowska, B

2008-01-01

Background and purpose: The endocannabinoid virodhamine is a partial agonist at the cannabinoid CB1 receptor and a full agonist at the CB2 receptor, and relaxes rat mesenteric arteries through endothelial cannabinoid receptors. Its concentration in the periphery exceeds that of the endocannabinoid anandamide. Here, we examined the influence of virodhamine on the human pulmonary artery. Experimental approach: Isolated human pulmonary arteries were obtained during resections for lung carcinoma. Vasorelaxant effects of virodhamine were examined on endothelium-intact vessels precontracted with 5-HT or KCl. Key results: Virodhamine, unlike WIN 55,212-2, relaxed 5-HT-precontracted vessels concentration dependently. The effect of virodhamine was reduced by endothelium denudation, two antagonists of the endothelial cannabinoid receptor, cannabidiol and O-1918, and a high concentration of the CB1 receptor antagonist rimonabant (5 μM), but only slightly attenuated by the NOS inhibitor L-NAME and not affected by a lower concentration of rimonabant (100 nM) or by the CB2 and vanilloid receptor antagonists SR 144528 and capsazepine, respectively. The COX inhibitor indomethacin and the fatty acid amide hydrolase inhibitor URB597 and combined administration of selective blockers of small (apamin) and intermediate and large (charybdotoxin) conductance Ca2+-activated K+ channels attenuated virodhamine-induced relaxation. The vasorelaxant potency of virodhamine was lower in KCl- than in 5-HT-precontracted preparations. Conclusions and implications: Virodhamine relaxes the human pulmonary artery through the putative endothelial cannabinoid receptor and indirectly through a COX-derived vasorelaxant prostanoid formed from the virodhamine metabolite, arachidonic acid. One or both of these mechanisms may stimulate vasorelaxant Ca2+-activated K+ channels. PMID:18806815

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

PubMed

Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

2016-01-01

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effect of SMURF2 Targeting on Susceptibility to MEK Inhibitors in Melanoma

PubMed Central

2013-01-01

Background The mitogen-activated protein–kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas. MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy. Methods We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth. Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression. Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression. RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish. All statistical tests were two-sided. Results Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF. High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity. Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor–induced apoptosis. Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells. Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005). Conclusions Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors. PMID:23250956

Simple and simultaneous determination of the hiv-protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir plus M8 nelfinavir metabolite and the nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma by reversed-phase liquid chromatography.

PubMed

Poirier, Jean-Marie; Robidou, Pascal; Jaillon, Patrice

2005-04-01

Several studies suggest that therapeutic drug monitoring of protease inhibitors and nonnucleoside reverse transcriptase inhibitors may contribute to the clinical outcome of HIV-infected patients. Because of the growing number of antiretroviral drugs and of drug combinations than can be administered to these patients, an accurate high-performance liquid chromatographic (HPLC) method allowing the simultaneous determination of these drugs may be useful. To date, the authors present the first simultaneous HPLC determination of the new protease inhibitor atazanavir with all the others currently in use (M8 nelfinavir metabolite included) and the 2 widely used nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine. This simple HPLC method allows the analysis all these drugs at a single ultraviolet wavelength following a 1-step liquid-liquid extraction procedure. A 500-muL plasma sample was spiked with internal standard and subjected to liquid-liquid extraction using by diethyl ether at pH 10. HPLC was performed using a Symmetry Shield RP18 and gradient elution. All the drugs of interest and internal standard were detected with ultraviolet detection at 210 nm. Calibration curves were linear in the range 50-10,000 ng/mL. The observed concentrations of the quality controls at plasma concentrations ranging from 50 to 5000 ng/mL for these drugs showed that the overall accuracy varied from 92% to 104% and 92% to 106% for intraday and day-to-day analysis, respectively. No metabolites of the assayed compounds or other drugs commonly coadministered to HIV-positive patients were found to coelute with the drugs of interest or with the internal standard. This assay was developed for the purpose of therapeutic monitoring (TDM) in HIV-infected patients.

Pharmacokinetic drivers of toxicity for basic molecules: Strategy to lower pKa results in decreased tissue exposure and toxicity for a small molecule Met inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diaz, Dolores, E-mail: diaz.dolores@gene.com; Ford, Kevin A.; Hartley, Dylan P.

Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstratedmore » by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd > 3 l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd = 1.0 l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins. -- Highlights: ► Lower pKa for a small molecule: reduced tissue drug levels and toxicity. ► New analysis tools to assess electrostatic effects and ionization are presented. ► Chemical and PK drivers of toxicity can be leveraged to improve safety.« less

The effects of composting on the nutritional composition of fibrous bio-regenerative life support systems (BLSS) plant waste residues and its impact on the growth of Nile tilapia ( Oreochromis niloticus)

NASA Astrophysics Data System (ADS)

Gonzales, John M.; Lowry, Brett A.; Brown, Paul B.; Beyl, Caula A.; Nyochemberg, Leopold

2009-04-01

Utilization of bio-regenerative life support systems (BLSS) plant waste residues as a nutritional source by Nile tilapia ( Oreochromis niloticus) has proven problematic as a result of high concentrations of fibrous compounds in the plant waste residues. Nutritional improvement of plant waste residues by composting with the oyster mushroom ( Pleurotus ostreatus), and the effects on growth and nutrient utilization of Nile tilapia fed such residues were evaluated. Five Nile tilapia (mean weight = 70.9 ± 3.1 g) were stocked in triplicate aquaria and fed one of two experimental diets, cowpea (CP) and composted cowpea (CCP), twice daily for a period of 8 weeks. Composting of cowpea residue resulted in reduced concentrations of nitrogen-free extract, hemi-cellulose and trypsin inhibitor activity, though trypsin inhibitor activity remained high. Composting did not reduce crude fiber, lignin, or cellulose concentrations in the diet. No significant differences ( P < 0.05) were observed in weight gain, specific growth rate, survival rate, daily consumption, and food conversion ratio between tilapia fed CP and CCP. These results suggest that P. ostreatus is not a suitable candidate for culture in conjunction with the culture of Nile tilapia. Additional work is needed to determine what, if any, benefit can be obtained from incorporating composted residue as feed for Nile tilapia.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, Harry A.; Gadbois, Donna M.; Tobey, Robert A.; Bradbury, E. Morton

1993-01-01

A G.sub.1 phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G.sub.1 phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G.sub.1 cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G.sub.1 phase, suggesting that such G.sub.1 phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

1993-02-09

A G[sub 1] phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G[sub 1] phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G[sub 1] cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G[sub 1] phase, suggesting that such G[sub 1] phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Relationship between Paronychia and Drug Concentrations of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

PubMed

Masago, Katsuhiro; Irie, Kei; Fujita, Shiro; Imamichi, Fumiko; Okada, Yutaka; Katakami, Nobuyuki; Fukushima, Shoji; Yatabe, Yasushi

2018-06-14

The purpose of the study was to evaluate the site of paronychia in patients with non-small cell lung cancer harboring an epidermal growth factor receptor (EGFR) gene activating mutation who were treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). The study included 55 patients with non-small-cell lung cancer who were treated with an EGFR TKIs. Resulting all toxicities were graded using the Common Terminology Criteria for Adverse Events version 4.0 system. Drug concentrations were determined with use of a quantum triple-quadrupole mass spectrometer and dried blood spots testing. Paronychia most commonly occurred in the thumb and the big toe. There was no correlation between the severity of paronychia and the drug concentration of each EGFR TKI at the site of paronychia. The mean penetration rates of the drug from plasma to the tip of the finger and toe were 74.1% (erlotinib), 82.2% (gefitinib), and 99.9% (afatinib). High concentrations of an EGFR TKI at the affected site did not play a role in the onset mechanism of paronychia. Therefore, educating patients about ways to avoid compression may be a better approach to managing this adverse event than reducing the dose of the EGFR-TKI or stopping treatment. © 2018 S. Karger AG, Basel.

Study on tea leaves extract as green corrosion inhibitor of mild steel in hydrochloric acid solution

NASA Astrophysics Data System (ADS)

Hamdan, A. B.; Suryanto; Haider, F. I.

2018-01-01

Corrosion inhibitor from extraction of plant has been considered as the most preferable and most chosen technique to prevent corrosion of metal in acidic medium because of the environmental friendly factor. In this study, black tea leaves extraction was tested as corrosion inhibitor for mild steel in 0.1M of hydrochloric acid (HCl) with the absence and presence of corrosion inhibitor. The efficiency and effectiveness of black tea as corrosion inhibitor was tested by using corrosion weight loss measurement experiment was carried out with varies parameters which with different concentration of black tea extract solution. The extraction of black tea solution was done by using aqueous solvent method. The FT-IR result shows that black tea extract containing compounds such as catechin, caffeine and tannins that act as anti-corrosive reagents and responsible to enhance the effectiveness of black tea extract as corrosion inhibitor by forming the hydrophobic thin film through absorption process. As a result of weight loss measurement, it shows that loss in weight of mild steel reduces as the concentration of inhibitor increases. The surface analysis was done on the mild steel samples by using SEM.

Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity.

PubMed

Fiene, Amelie; Baqi, Younis; Lecka, Joanna; Sévigny, Jean; Müller, Christa E

2015-01-07

The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z'-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z'-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.

Differential inhibition of activity, activation and gene expression of MMP-9 in THP-1 cells by azithromycin and minocycline versus bortezomib: A comparative study

PubMed Central

Knoops, Sofie; Aldinucci Buzzo, João L.; Boon, Lise; Martens, Erik; Opdenakker, Ghislain; Kolaczkowska, Elzbieta

2017-01-01

Gelatinase B or matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) is increased in inflammatory processes and cancer, and is associated with disease progression. In part, this is due to MMP-9-mediated degradation of extracellular matrix, facilitating influx of leukocytes into inflamed tissues and invasion or metastasis of cancer cells. MMP-9 is produced as proMMP-9 and its propeptide is subsequently removed by other proteases to generate proteolytically active MMP-9. The significance of MMP-9 in pathologies triggered the development of specific inhibitors of this protease. However, clinical trials with synthetic inhibitors of MMPs in the fight against cancer were disappointing. Reports on active compounds which inhibit MMP-9 should be carefully examined in this regard. In a considerable set of recent publications, two antibiotics (minocycline and azythromycin) and the proteasome inhibitor bortezomib, used in cancers, were reported to inhibit MMP-9 at different stages of its expression, activation or activity. The current study was undertaken to compare and to verify the impact of these compounds on MMP-9. With exception of minocycline at high concentrations (>100 μM), the compounds did not affect processing of proMMP-9 into MMP-9, nor did they affect direct MMP-9 gelatinolytic activity. In contrast, azithromycin specifically reduced MMP-9 mRNA and protein levels without affecting NF-κB in endotoxin-challenged monocytic THP-1 cells. Bortezomib, although being highly toxic, had no MMP-9-specific effects but significantly upregulated cyclooxygenase-2 (COX-2) activity and PGE2 levels. Overall, our study clarified that azithromycin decreased the levels of MMP-9 by reduction of gene and protein expression while minocycline inhibits proteolytic activity at high concentrations. PMID:28369077

Regulation of Na+-K+-2Cl− cotransport by protein phosphorylation in ferret erythrocytes

PubMed Central

Flatman, Peter W; Creanor, James

1999-01-01

Na+-K+-2Cl− cotransport in ferret erythrocytes was measured as the bumetanide-sensitive uptake of 86Rb. The resting cotransport rate was high but could be increased threefold by treating erythrocytes with calyculin A, a potent inhibitor of serine/threonine phosphatases. Twenty nanomolar was sufficient to maximally and rapidly (within 4 min) stimulate transport. The effects of several kinase inhibitors were tested. High concentrations of K-252a, K-252b, calphostin C and hypericin caused less than 20 % inhibition. Staurosporine (IC50, 0.06 μm) and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; IC50, 2.5 μm) were more potent but still only partially (40–50 %) inhibited transport, an effect mimicked by reducing ionized intracellular Mg2+ concentration to submicromolar levels. Genistein may inhibit all transport at a sufficiently high dose (IC50, 0.36 mM) perhaps by directly inhibiting the transporter. Staurosporine, PP1 and the removal of Mg2+ all prevented subsequent stimulation by calyculin A, and all inhibited calyculin-stimulated transport by 20–30 %. The effects of staurosporine, PP1 and Mg2+ removal were not additive. The phosphatase that dephosphorylates the cotransporter is probably Mg2+ (or possibly Ca2+ or Mn2+) sensitive and not the target for calyculin A. The data suggest that this phosphatase is inhibited by phosphorylation, and that it is the regulation of this process which is affected by calyculin A and the kinase inhibitors tested here. Phosphorylation of the phosphatase is probably regulated by members of the Src family of tyrosine kinases. PMID:10358111

High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity

PubMed Central

Akhtar, Naureen; Karabika, Eugenia; Kinghorn, James R.; Glass, Anthony D.M.; Unkles, Shiela E.

2015-01-01

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters. PMID:25855763

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis.

PubMed

Price, Samuel J; Pangloli, Philipus; Krishnan, Hari B; Dia, Vermont P

2016-12-01

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) concentrations on the stability of lunasin against pepsin-pancreatin hydrolysis (PPH). In addition, the effect of KTI mutation on lunasin stability against PPH was determined. LEP were produced by calcium and pH precipitation methods of 30% aqueous ethanol extract from defatted soybean flour. LEP, lunasin-enriched commercially available products and KTI control and mutant flours underwent PPH and samples were taken after pepsin and pepsin-pancreatin hydrolysis. The concentrations of BBI, KTI, and lunasin all decreased after hydrolysis, but they had varying results. BBI concentration ranged from 167.5 to 655.8μg/g pre-hydrolysis and 171.5 to 250.1μg/g after hydrolysis. KTI concentrations ranged from 0.3 to 122.3μg/g pre-hydrolysis and 9.0 to 18.7μg/g after hydrolysis. Lunasin concentrations ranged from 8.5 to 71.0μg/g pre-hydrolysis and 4.0 to 13.2μg/g after hydrolysis. In all products tested, lunasin concentration after PPH significantly correlated with BBI and KTI concentrations. Mutation in two KTI isoforms led to a lower concentration of lunasin after PPH. This is the first report on the potential role of KTI in lunasin stability against PPH and must be considered in designing lunasin-enriched products that could potentially survive digestion after oral ingestion. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors.

PubMed

Chen, Rong; Zhou, Jingjing; Qin, Lingyun; Chen, Yao; Huang, Yongqi; Liu, Huili; Su, Zhengding

2017-06-27

In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 μM. The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library, including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

Activation of neurokinin NK(2) receptors by tachykinin peptides causes contraction of uterus in pregnant women near term.

PubMed

Patak, E N; Ziccone, S; Story, M E; Fleming, A J; Lilley, A; Pennefather, J N

2000-06-01

The aim of this study was firstly to elucidate whether the mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)-regulated contractility of myometrium obtained from near-term pregnant women, and secondly to investigate the receptor subtype(s) responsible. In the presence of peptidase inhibitors, i.e. thiorphan (3 micromol/l; endopeptidase 24.11 inhibitor), captopril (10 micromol/l; angiotensin converting enzyme inhibitor) and bestatin (10 micromol/l; aminopeptidase inhibitor); all three mammalian tachykinins elicited concentration-related contractions of isolated myometrial preparations. The rank order of agonist potency of the mammalian tachykinins in the presence of the peptidase inhibitors was NKA > SP = NKB, indicating that the contractile effects were mediated by activation of an NK(2) receptor. The NK(2) receptor-selective agonist, [Lys(5), MeLeu(9), Nle(10)]NKA(4-10), produced concentration-related contractile responses, while the respective NK(1) and NK(3) receptor-selective agonists, [Sar(9), Met(O(2))(11)]SP and [N-MePhe(7)]NKB, had no effect either in the absence or presence of the peptidase inhibitors. The NK(2) receptor-selective antagonist, SR48968, produced concentration-related rightward shift in the log concentration curve to [Lys(5), MeLeu(9), Nle(10)]NKA(4-10). This study shows that tachykinins elicit contractile effects on human myometrium obtained from pregnant women near term, and that these effects are mediated by an NK(2) receptor. An excitatory effect of the tachykinins on these preparations could indicate a physiological role for these peptides in enhancing contractility of the uterus in women at term.

Delayed toxicity of two chitinolytic enzyme inhibitors (psammaplin a and pentoxifylline) against eastern subterranean termites (Isoptera: Rhinotermitidae).

PubMed

Hiusen, Timothy J; Kamble-Shripat, T

2013-08-01

By using a no-choice feeding bioassay, delayed toxicity and concentration-dependent mortality of two chitinolytic enzyme inhibitors, pentoxifylline and psammaplin A, were evaluated by determining LT50, LT90, and LT99 (lethal time) against the economically important eastern subterranean termite, Reticulitermes flavipes (Kollar). Pentoxifylline- and psammaplin A-incorporated diets (filter paper) were assayed at 0.01, 0.02, 0.04, 0.08, and 0.21% and 0.0375, 0.075, 0.15, and 0.3% active ingredient (wt:wt), respectively. Acetone-only treated filter paper served as diet for the control treatments. Termite workers were allowed to feed on diet until 100% test population mortality occurred (80-95 d). Both chitinase inhibitors were shown to be toxic to R. flavipes. Concentration-dependent toxicity occurred within the pentoxifylline treatments over the range of 0.01-0.08%, with 0.08% treatments producing an LT50 of 32.2 d. However, mortality in response to psammaplin A treatments lacked concentration-dependent toxicity. Treatment with 0.3% psammaplin A produced an LT50 of 21.3 d. Mortality in response to lower psammaplin A treatments displayed no concentration-dependent trends. This study provides the first report on delayed toxicity of chitinolytic enzyme inhibitors against eastern subterranean termites (order Isoptera) and toxicological data on pentoxifylline and psammaplin A over a range of concentrations.

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

USDA-ARS?s Scientific Manuscript database

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor and Kunitz trypsin concentrations on the stabil...

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.

Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. Themore » AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.« less

Novel design strategy for checkpoint kinase 2 inhibitors using pharmacophore modeling, combinatorial fusion, and virtual screening.

PubMed

Lin, Chun-Yuan; Wang, Yen-Ling

2014-01-01

Checkpoint kinase 2 (Chk2) has a great effect on DNA-damage and plays an important role in response to DNA double-strand breaks and related lesions. In this study, we will concentrate on Chk2 and the purpose is to find the potential inhibitors by the pharmacophore hypotheses (PhModels), combinatorial fusion, and virtual screening techniques. Applying combinatorial fusion into PhModels and virtual screening techniques is a novel design strategy for drug design. We used combinatorial fusion to analyze the prediction results and then obtained the best correlation coefficient of the testing set (r test) with the value 0.816 by combining the Best(train)Best(test) and Fast(train)Fast(test) prediction results. The potential inhibitors were selected from NCI database by screening according to Best(train)Best(test) + Fast(train)Fast(test) prediction results and molecular docking with CDOCKER docking program. Finally, the selected compounds have high interaction energy between a ligand and a receptor. Through these approaches, 23 potential inhibitors for Chk2 are retrieved for further study.

Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix).

PubMed

Klomklao, Sappasith; Benjakul, Soottawat; Visessanguan, Wonnop; Kishimura, Hideki; Simpson, Benjamin K

2007-12-01

Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 degrees C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 degrees C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl2 partially protected trypsin against activity loss at 40 degrees C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.

Identification of a Novel Family of BRAF[superscript V600E] Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Jie; Xie, Peng; Ventocilla, Christian

The BRAF oncoprotein is mutated in about half of malignant melanomas and other cancers, and a kinase activating single valine to glutamate substitution at residue 600 (BRAF{sup V600E}) accounts for over 90% of BRAF-mediated cancers. Several BRAF{sup V600E} inhibitors have been developed, although they harbor some liabilities, thus motivating the development of other BRAF{sup V600E} inhibitor options. We report here the use of an ELISA based high-throughput screen to identify a family of related quinolol/naphthol compounds that preferentially inhibit BRAF{sup V600E} over BRAF{sup WT} and other kinases. We also report the X-ray crystal structure of a BRAF/quinolol complex revealing themore » mode of inhibition, employ structure-based medicinal chemistry efforts to prepare naphthol analogues that inhibit BRAF{sup V600E} in vitro with IC{sub 50} values in the 80-200 nM range under saturating ATP concentrations, and demonstrate that these compounds inhibit MAPK signaling in melanoma cells. Prospects for improving the potency and selectivity of these inhibitors are discussed.« less

Demonstration of 2-hydroxybenzoylglycine as a drug binding inhibitor in newborn infants.

PubMed Central

Suh, B; Wadsworth, S J; Lichtenwalner, D M

1987-01-01

Newborn infants have drug binding defects that share similarities to those of uremic subjects. Since 2-hydroxybenzoylglycine has been chemically defined to be a major drug binding inhibitor in uremia, a search for the presence of a similar compound in the sera of newborn infants was made. An organic substance that has the characteristics of 2-hydroxybenzoylglycine as supported by the retardation factor values on thin-layer chromatograms, retention times of high performance liquid chromatograms, fluorescence emission spectra, and mass spectrum has been demonstrated to be present in the majority of the neonatal sera studied. A strong positive correlation between the levels of the binding inhibitor and the extent of binding defects for nafcillin has been observed. The substance could effectively reduce the total bilirubin concentration when added to the cord sera specimens. It is concluded that 2-hydroxybenzoylglycine plays an important role in drug binding defects observed in the newborn, and the inhibitor may also play a part in the precipitation of bilirubin-induced neurotoxicity in neonates when the substance is abnormally elevated. Images PMID:3654972

Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): a highly potent, selective, and orally bioavailable inhibitor of TGF-β type I receptor kinase as cancer immunotherapeutic/antifibrotic agent.

PubMed

Jin, Cheng Hua; Krishnaiah, Maddeboina; Sreenu, Domalapally; Subrahmanyam, Vura B; Rao, Kota S; Lee, Hwa Jeong; Park, So-Jung; Park, Hyun-Ju; Lee, Kiho; Sheen, Yhun Yhong; Kim, Dae-Kee

2014-05-22

A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized and evaluated to optimize a prototype inhibitor of TGF-β type I receptor kinase (ALK5), 6. Combination of replacement of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety, insertion of a methyleneamino linker, and a o-F substituent in the phenyl ring markedly increased ALK5 inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 μM in a kinase assay and with IC50 values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.

Dynamic monitoring of p53 translocation to mitochondria for the analysis of specific inhibitors using luciferase-fragment complementation.

PubMed

Noda, Natsumi; Awais, Raheela; Sutton, Robert; Awais, Muhammad; Ozawa, Takeaki

2017-12-01

Intracellular protein translocation plays a pivotal role in regulating complex biological processes, including cell death. The tumor suppressor p53 is a transcription factor activated by DNA damage and oxidative stress that also translocates from the cytosol into the mitochondrial matrix to facilitate necrotic cell death. However, specific inhibitors of p53 mitochondrial translocation are largely unknown. To explore the inhibitors of p53, we developed a bioluminescent probe to monitor p53 translocation from cytosol to mitochondria using luciferase fragment complementation assays. The probe is composed of a novel pair of luciferase fragments, the N-terminus of green click beetle luciferase CBG68 (CBGN) and multiple-complement luciferase fragment (McLuc1). The combination of luciferase fragments showed significant luminescence intensity and high signal-to-background ratio. When the p53 connected with McLuc1 translocates from cytosol into mitochondrial matrix, CBGN in mitochondrial matrix enables to complement with McLuc1, resulting in the restoration of the luminescence. The luminescence intensity was significantly increased under hydrogen peroxide-induced oxidative stress following the complementation of CBGN and McLuc1. Pifithrin-μ, a selective inhibitor of p53 mitochondrial translocation, prevented the mitochondrial translocation of the p53 probe in a concentration-dependent manner. Furthermore, the high luminescence intensity made it easier to visualize the p53 translocation at a single cell level under a bioluminescence microscope. This p53 mitochondrial translocation assay is a new tool for high-throughput screening to identify novel p53 inhibitors, which could be developed as drugs to treat diseases in which necrotic cell death is a major contributor. © 2017 Wiley Periodicals, Inc.

Ammonia oxidisers in a non-nitrifying Brazilian savanna soil.

PubMed

Catão, Elisa C P; Thion, Cécile; Krüger, R H; Prosser, James I

2017-11-01

Low nitrification rates in Brazilian savanna (Cerrado) soils have puzzled researchers for decades. Potential mechanisms include biological inhibitors, low pH, low microbial abundance and low soil moisture content, which hinders microbial activity, including ammonia oxidation. Two approaches were used to evaluate these potential mechanisms: (i) manipulation of soil moisture and pH in microcosms containing Cerrado soil and (ii) assessment of nitrification inhibition in slurries containing mixtures of Cerrado soil and an actively nitrifying agricultural soil. Despite high ammonium concentration in Cerrado soil microcosms, little NO3- accumulation was observed with increasing moisture or pH, but in some Cerrado soil slurries, ammonia-oxidising archaea (AOA) amoA transcripts were detected after 14 days. In mixed soil slurries, the final NO3- concentration reflected the initial proportions of agricultural and Cerrado soils in the mixture, providing no evidence of nitrification inhibitors in Cerrado soil. AOA community denaturing gradient gel electrophoresis profiles were similar in the mixed and nitrifying soils. These results suggest that nitrification in Cerrado soils is not constrained by water availability, ammonium availability, low pH or biological inhibitors, and alternative potential explanations for low nitrification levels are discussed. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the treatment of B-cell lymphoma.

PubMed

Ge, Yang; Wang, Changyuan; Song, Shijie; Huang, Jiaxin; Liu, Zhihao; Li, Yongming; Meng, Qiang; Zhang, Jianbin; Yao, Jihong; Liu, Kexin; Ma, Xiaodong; Sun, Xiuli

2018-01-01

The BTK and JAK3 receptor tyrosine kinases are two validated and therapeutically amenable targets in the treatment of B-cell lymphomas. Here we report the identification of several classes of pyrimidine derivatives as potent BTK and JAK3 dual inhibitors. Among these molecules, approximately two thirds displayed strong inhibitory capacity at less than 10 nM concentration, and four compounds (7e, 7g, 7m and 7n) could significantly inhibit the phosphorylation of BTK and JAK3 enzymes at concentrations lower than 1 nM. Additionally, these pyrimidine derivatives also exhibited enhanced activity to block the proliferation of B-cell lymphoma cells compared with the representative BTK inhibitor ibrutinib. In particular, two structure-specific compounds 7b and 7e displayed stronger activity than reference agents in cell-based evaluation, with IC 50 values lower than 10 μM. Further biological studies, including flow cytometric analysis, and a xenograft model for in vivo evaluation, also indicated their efficacy and low toxicity in the treatment of B-cell lymphoma. These findings provide a new insight for the development of novel anti-B-cell lymphoma drugs with multi-target actions. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Biobutanol production from apple pomace: the importance of pretreatment methods on the fermentability of lignocellulosic agro-food wastes.

PubMed

Hijosa-Valsero, María; Paniagua-García, Ana I; Díez-Antolínez, Rebeca

2017-11-01

Apple pomace was studied as a possible raw material for biobutanol production. Five different soft physicochemical pretreatments (autohydrolysis, acids, alkalis, organic solvents and surfactants) were compared in a high-pressure reactor, whose working parameters (temperature, time and reagent concentration) were optimised to maximise the amount of simple sugars released and to minimise inhibitor generation. The pretreated biomass was subsequently subjected to a conventional enzymatic treatment to complete the hydrolysis. A thermal analysis (DSC) of the solid biomass indicated that lignin was mainly degraded during the enzymatic treatment. The hydrolysate obtained with the surfactant polyethylene glycol 6000 (PEG 6000) (1.96% w/w) contained less inhibitors than any other pretreatment, yet providing 42 g/L sugars at relatively mild conditions (100 °C, 5 min), and was readily fermented by Clostridium beijerinckii CECT 508 in 96 h (3.55 g/L acetone, 9.11 g/L butanol, 0.26 g/L ethanol; 0.276 g B /g S yield; 91% sugar consumption). Therefore, it is possible to optimise pretreatment conditions of lignocellulosic apple pomace to reduce inhibitor concentrations in the final hydrolysate and perform successful ABE fermentations without the need of a detoxification stage.

A novel photoelectrochemical biosensor for protein kinase activity assay based on phosphorylated graphite-like carbon nitride.

PubMed

Li, Xue; Zhou, Yunlei; Xu, Yan; Xu, Huijie; Wang, Minghui; Yin, Huanshun; Ai, Shiyun

2016-08-31

Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C3N4) conjugates triggered by Zr(4+) ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC50 value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of sorghum straw hemicellulosic hydrolysate for biotechnological production of xylitol by Candida guilliermondii

PubMed Central

Sene, L.; Arruda, P.V.; Oliveira, S.M.M.; Felipe, M.G.A.

2011-01-01

A preliminary study on xylitol production by Candida guilliermondii in sorghum straw hemicellulosic hydrolysate was performed. Hydrolysate had high xylose content and inhibitors concentrations did not exceed the commonly found values in other hemicellulosic hydrolysates. The highest xylitol yield (0.44 g/g) and productivity (0.19 g/Lh) were verified after 72 hours. PMID:24031733

Bactericidal effect of silver nanoparticles against propagation of Clavibacter michiganensis infection in Lycopersicon esculentum Mill.

PubMed

Rivas-Cáceres, Raymundo Rene; Luis Stephano-Hornedo, Jose; Lugo, Jorge; Vaca, Rocio; Del Aguila, Pedro; Yañez-Ocampo, Gustavo; Mora-Herrera, Martha Elena; Camacho Díaz, Luis Miguel; Cipriano-Salazar, Moisés; Alaba, Peter Adeniyi

2018-02-01

This study explored the use of silver nanoparticle as a bactericidal against the propagation of Clavibacter michiganensis onto tomatoes (Lycopersicon esculentum Mill). In Mexico, tomato production covers about 73% of the total vegetable production but it is affected by outbreak of bacteria canker caused by Clavibacter michiganensis subspecies michiganensis (Cmm). Silver ions possess inhibitor properties, bactericides and high specter antimicrobials. In this study, 6 groups of culture were prepared using 6 different petri dishes where silver nanoparticles of varying concentrations (120, 84, 48, 24, 12 and 0 μg) were added. Furthermore, each group was observed for 20 min, 1, 2, 12 and 24 h. The optimum concentration is 84 μg, which shows an average of 2 Cmm colonies after 20 min. Further increase to 120 μg shows no significant change. However, the average colonies was observed for 48 μg after 1, 2, 12, and 24 h. The obtained results indicate that silver nanoparticles are a promising inhibitor, bactericide and high a specter antimicrobial for treatment or prevention of Cmm. Copyright © 2017. Published by Elsevier Ltd.

Effect of urinary trypsin inhibitor on preterm labor with high granulocyte elastase concentration in cervical secretions.

PubMed

Hayashi, Masako; Oya, Atsuko; Miyake, Hidehiko; Nakai, Akihito; Takeshita, Toshiyuki

2010-04-01

To explore whether intravaginal treatment with urinary trypsin inhibitor (UTI) prevents preterm delivery in patients in preterm labor with increased levels of granulocyte elastase in cervical secretions. The subjects were patients in preterm labor with increased levels of granulocyte elastase in cervical secretions from 16 to 33 weeks gestation. Maternal and neonatal outcomes were compared between patients receiving UTI treatment (UTI group; n=33) and those not receiving UTI treatment (control group; n=40). In patients receiving UTI, the mean gestational age at delivery was greater than that in the control group (37.8 vs. 35.6 weeks, p=0.003), and the rates of premature delivery before 34 and 37 weeks gestation were lower (3% vs. 20%, p=0.028; and 18% vs. 47%, p=0.008, respectively). The percentage of neonates weighing more than 2,500 g was significantly higher in the UTI group, with no neonates weighing less than 1,500 g. The neonatal hospitalization rate was lower in the UTI group (9% vs. 42%, p=0.001). In patients in preterm labor with a high elastase concentration in cervical secretions, treatment with UTI reduced the risk of preterm delivery and improved neonatal outcomes.

Investigation of Diospyros Kaki L.f husk extracts as corrosion inhibitors and bactericide in oil field

PubMed Central

2013-01-01

Background Hydrochloric acid is used in oil-well acidizing commonly for improving the crude oil production of the low-permeable reservoirs, while it is a great challenge for the metal instruments involved in the acidification. Developing natural products as oilfield chemicals is a straight way to find less expensive, green and eco-friendly materials. The great plant resources in Qin-ling and Ba-shan Mountain Area of Shannxi Province enable the investigating of new green oil field chemicals. Diospyros Kaki L.f (persimmon), a famous fruit tree is widely planted in Qin-ling and Ba-shan Mountain Area of Shaanxi Province. It has been found that the crude persimmon extracts are complex mixtures containing vitamins, p-coumaric acid, gallic acid, catechin, flavonoids, carotenoids and condensed tannin and so on, which indicates the extracts of persimmon husk suitable to be used as green and eco-friendly corrosion inhibitors. Findings Extracts of persimmon husk were investigated, by using weight loss and potentiodynamic polarisation techniques, as green and eco-friendly corrosion inhibitors of Q235A steel in 1M HCl. The inhibition efficiency of the extracts varied with extract concentration from 10 to 1,000 mg/L. There are some synergistic effects between the extracts and KI, KSCN and HMTA. Potentiodynamic polarization studies indicate that extracts are mixed-type inhibitors. Besides, the extracts were screened for antibacterial activity against oil field microorganisms, and they showed good to moderate activity against SRB, IB and TGB. Conclusions The inhibition efficiency of the extracts varied with extract concentration from 10 to 1,000 mg/L, and the highest reaches to 65.1% with the con concentration of 1,000 mg/L WE. KI, KSCN and HMTA they can enhance the IE of WE effectively to 97.3% at most, but not effective for KI and KSCN to AE. Tafel polarisation measurements indicate the extracts behave as mixed type inhibitor. Investigation of the antibacterial activity against oil field microorganism showed the extracts can inhibit SRB, IB and TGB with moderate to highly efficiency under 1,000 mg/L, which makes extracts potential to be used as bifunctional oil field chemicals. PMID:23816431

Indolyl aryl sulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors: role of two halogen atoms at the indole ring in developing new analogues with improved antiviral activity.

PubMed

Regina, Giuseppe La; Coluccia, Antonio; Piscitelli, Francesco; Bergamini, Alberto; Sinistro, Anna; Cavazza, Antonella; Maga, Giovanni; Samuele, Alberta; Zanoli, Samantha; Novellino, Ettore; Artico, Marino; Silvestri, Romano

2007-10-04

Indolyl aryl sulfones bearing the 4,5-difluoro (10) or 5-chloro-4-fluoro (16) substitution pattern at the indole ring were potent inhibitors of HIV-1 WT and the NNRTI-resistant strains Y181C and K103N-Y181C. These compounds were highly effective against the 112 and the AB1 strains in lymphocytes and inhibited at nanomolar concentration the multiplication of the IIIBBa-L strain in macrophages. Compound 16 was exceptionally potent against RT WT and RTs carrying the K103N, Y181I, and L100I mutations.

Effect of ibuprofen on menstrual blood prostaglandin levels in dysmenorrheic women.

PubMed

Pulkkinen, M O; Csapo, A I

1979-07-01

In a randomized crossover study 15 dysmenorrheic women were treated during two consecutive menstrual period, once with the potent prostaglandin-synthesis inhibitor: ibuprofen and once with an identical looking placebo. Each patient was medicated for 12 hours during the first day of her menstrual flow and was subsequently fitted with a cervical cup for the collection of menstrual blood during three hours. In these samples the concentrations of prostaglandin (PG)F and PGE were measured by radioimmunoassay. The patients receiving placebo had high PGF levels 135 +/- 27 ng/ml (Mean +/- S.E.) which were significnatly reduced by Ibuprofen to 24 +/- 5 ng/ml (P less than 0.001). The PGE concentrations decreased from 5 +/- 1 ng/ml to 2 +/- 1 ng/ml (P less than 0.05). Ibuprofen also reduced the menstrual pain significantly (P less than 0.001). These results substantiate the earlier conclusion that a causal relationship exists between effective treatment with PG-synthesis inhibitors and decrease in menstrual blood PG levels, intrauterine pressure and dysmenorrheic pain.

A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide.

PubMed

Roh, Changhyun

2012-01-01

Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (-)-catechin gallate and (-)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (-)-catechin gallate and (-)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL(-1), (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

Toward Highly Potent Cancer Agents by Modulating the C-2 Group of the Arylthioindole Class of Tubulin Polymerization Inhibitors

PubMed Central

La Regina, Giuseppe; Bai, Ruoli; Rensen, Whilelmina Maria; Di Cesare, Erica; Coluccia, Antonio; Piscitelli, Francesco; Famiglini, Valeria; Reggio, Alessia; Nalli, Marianna; Pelliccia, Sveva; Pozzo, Eleonora Da; Costa, Barbara; Granata, Ilaria; Porta, Amalia; Maresca, Bruno; Soriani, Alessandra; Iannitto, Maria Luisa; Santoni, Angela; Li, Junjie; Cona, Marlein Miranda; Chen, Feng; Ni, Yicheng; Brancale, Andrea; Dondio, Giulio; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Martini, Claudia; Hamel, Ernest; Lavia, Patrizia; Novellino, Ettore; Silvestri, Romano

2013-01-01

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC50 = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes. PMID:23214452

Improvement of the fermentability of oxalic acid hydrolysates by detoxification using electrodialysis and adsorption.

PubMed

Jeong, So-Yeon; Trinh, Ly Thi Phi; Lee, Hong-Joo; Lee, Jae-Won

2014-01-01

A two-step detoxification process consisting of electrodialysis and adsorption was performed to improve the fermentability of oxalic acid hydrolysates. The constituents of the hydrolysate differed significantly between mixed hardwood and softwood. Acetic acid and furfural concentrations were high in the mixed hardwood, whereas 5-hydroxymethylfurfural (HMF) concentration was relatively low compared with that of the mixed softwood. The removal efficiency of acetic acid reached 100% by electrodialysis (ED) process in both hydrolysates, while those of furfural and HMF showed very low, due to non-ionizable properties. Most of the remaining inhibitors were removed by XAD-4 resin. In the mixed hardwood hydrolysate without removal of the inhibitors, ethanol fermentation was not completed. Meanwhile, both ED-treated hydrolysates successfully produced ethanol with 0.08 and 0.15 g/Lh ethanol productivity, respectively. The maximum ethanol productivity was attained after fermentation with 0.27 and 0.35 g/Lh of detoxified hydrolysates, which were treated by ED, followed by XAD-4 resin. Copyright © 2013 Elsevier Ltd. All rights reserved.

Adsorptive removal of fermentation inhibitors from concentrated acid hydrolyzates of lignocellulosic biomass.

PubMed

Sainio, Tuomo; Turku, Irina; Heinonen, Jari

2011-05-01

Adsorptive purification of concentrated acid hydrolyzate of lignocellulose was investigated. Cation exchange resin (CS16GC), neutral polymer adsorbent (XAD-16), and granulated activated carbon (GAC) were studied to remove furfural, HMF, and acetic acid from a synthetic hydrolyzate containing 20 wt.% H(2)SO(4). Adsorption isotherms were determined experimentally. Loading and regeneration were investigated in a laboratory scale column. GAC has the highest adsorption capacity, but regeneration with water was not feasible. XAD-16 and CS16GC had lower adsorption capacities but also shorter cycle times due to easier regeneration. Productivity increased when regenerating with 50 wt.% EtOH(aq) solution. To compare adsorbents, process performance was quantified by productivity and fraction of inhibitors removed. GAC yields highest performance when high purity is required and ethanol can be used in regeneration. For lower purities, XAD-16 and GAC yield approximately equal performance. When using ethanol must be avoided, CS16GC offers highest productivity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Kinetics of corrosion inhibition of aluminum in acidic media by water-soluble natural polymeric chondroitin-4-sulfate as anionic polyelectrolyte inhibitor.

PubMed

Hassan, Refat M; Ibrahim, Samia M; Takagi, Hideo D; Sayed, Suzan A

2018-07-15

Corrosion inhibition of aluminum (Al) in hydrochloric acid by anionic polyelectrolyte chondroitin-4-sulfate (CS) polysaccharide has been studied using both gasometrical and weight-loss techniques. The results drawn from these two techniques are comparable and exhibit negligible differences. The inhibition efficiency was found to increase with increasing the inhibitor concentration and decreased with increasing temperature. The inhibition action of CS on Al metal surface was found to obey both of Langmuir and Freundlich isotherms. The factors affecting the corrosion rates such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and the temperature were examined. The kinetic parameters were evaluated and a suitable corrosion mechanism consistent with the results obtained is discussed. Copyright © 2018. Published by Elsevier Ltd.

POTENT INHIBITORS OF HUMAN ORGANIC ANION TRANSPORTERS 1 AND 3 FROM CLINICAL DRUG LIBRARIES: DISCOVERY AND MOLECULAR CHARACTERIZATION

PubMed Central

Duan, Peng; Li, Shanshan; Ai, Ni; Hu, Longqin; Welsh, William J.; You, Guofeng

2012-01-01

Transporter-mediated drug-drug interactions in the kidney dramatically influence the pharmacokinetics and other clinical effects of drugs. Human organic anion transporters 1 (hOAT1) and 3 (hOAT3) are the major transporters in the basolateral membrane of kidney proximal tubules, mediating the rate-limiting step in the elimination of a broad spectrum of drugs. In the present study, we screened two clinical drug libraries against hOAT1 and hOAT3. Of the 727 compounds screened, 92 compounds inhibited hOAT1 and 262 compounds inhibited hOAT3. When prioritized based on the peak unbound plasma concentrations of these compounds, three inhibitors for hOAT1 and seven inhibitors for hOAT3 were subsequently identified with high inhibitory potency (>95%). Computational analyses revealed that inhibitors and non-inhibitors can be differentiated from each other on the basis of several physico-chemical features, including: number of hydrogen-bond donors, number of rotatable bonds, and topological polar surface area (TPSA) for hOAT1; and molecular weight, number of hydrogen-bond donors and acceptors, TPSA, partition coefficient (Log P7.4), and polarizability for hOAT3. Pharmacophore modeling identified two common structural features associated with inhibitors for hOAT1 and hOAT3, viz., an anionic hydrogen-bond acceptor atom, and an aromatic center separated by ~5.7 Å. Such model provides mechanistic insights for predicting new OAT inhibitors. PMID:22973893

Vectorization efforts to increase Gram-negative intracellular drug concentration: a case study on HldE-K inhibitors.

PubMed

Atamanyuk, Dmytro; Faivre, Fabien; Oxoby, Mayalen; Ledoussal, Benoit; Drocourt, Elodie; Moreau, François; Gerusz, Vincent

2013-03-14

In this paper, we present different strategies to vectorize HldE kinase inhibitors with the goal to improve their gram-negative intracellular concentration. Syntheses and biological effects of siderophoric, aminoglycosidic, amphoteric, and polycationic vectors are discussed. While siderophoric and amphoteric vectorization efforts proved to be disappointing in this series, aminoglycosidic and polycationic vectors were able for the first time to achieve synergistic effects of our inhibitors with erythromycin. Although these effects proved to be nonspecific, this study provides information about the required stereoelectronic arrangement of the polycationic amines and their basicity requirements to fulfill outer membrane destabilization resulting in better erythromycin synergies.

A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

PubMed

Bordelon, Hali; Russ, Patricia K; Wright, David W; Haselton, Frederick R

2013-01-01

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3) to 5×10(8) copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6), 14×10(6), and 8×10(6) copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

A Magnetic Bead-Based Method for Concentrating DNA from Human Urine for Downstream Detection

PubMed Central

Bordelon, Hali; Russ, Patricia K.; Wright, David W.; Haselton, Frederick R.

2013-01-01

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×103 to 5×108 copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×106, 14×106, and 8×106 copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR. PMID:23861895

Novel Small Molecule Entry Inhibitors of Ebola Virus

PubMed Central

Basu, Arnab; Mills, Debra M.; Mitchell, Daniel; Ndungo, Esther; Williams, John D.; Herbert, Andrew S.; Dye, John M.; Moir, Donald T.; Chandran, Kartik; Patterson, Jean L.; Rong, Lijun; Bowlin, Terry L.

2015-01-01

Background. The current Ebola virus (EBOV) outbreak has highlighted the troubling absence of available antivirals or vaccines to treat infected patients and stop the spread of EBOV. The EBOV glycoprotein (GP) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-EBOV drugs. We report the identification of 2 novel EBOV inhibitors targeting viral entry. Methods. To identify small molecule inhibitors of EBOV entry, we carried out a cell-based high-throughput screening using human immunodeficiency virus–based pseudotyped viruses expressing EBOV-GP. Two compounds were identified, and mechanism-of-action studies were performed using immunoflourescence, AlphaLISA, and enzymatic assays for cathepsin B inhibition. Results. We report the identification of 2 novel entry inhibitors. These inhibitors (1) inhibit EBOV infection (50% inhibitory concentration, approximately 0.28 and approximately 10 µmol/L) at a late stage of entry, (2) induce Niemann-Pick C phenotype, and (3) inhibit GP–Niemann-Pick C1 (NPC1) protein interaction. Conclusions. We have identified 2 novel EBOV inhibitors, MBX2254 and MBX2270, that can serve as starting points for the development of an anti-EBOV therapeutic agent. Our findings also highlight the importance of NPC1-GP interaction in EBOV entry and the attractiveness of NPC1 as an antifiloviral therapeutic target. PMID:26206510

Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Franco, Octávio L; Falcão, Rosana; Fragoso, Rodrigo R; Mello, Luciane V; dos Santos, Roseane C; Grossi-de-Sá, Maria F

2003-01-01

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.

Effects of Anticholinesterases on Catalysis and Induced Conformational Change of the Peripheral Anionic Site of Murine Acetylcholinesterase

PubMed Central

Tong, Fan; Islam, Rafique M.; Carlier, Paul R.; Ma, Ming; Ekström, Fredrik; Bloomquist, Jeffrey R.

2013-01-01

Conventional insecticides targeting acetylcholinesterase (AChE) typically show high mammalian toxicities and because there is resistance to these compounds in many insect species, alternatives to established AChE inhibitors used for pest control are needed. Here we used a fluorescence method to monitor interactions between various AChE inhibitors and the AChE peripheral anionic site, which is a novel target for new insecticides acting on this enzyme. The assay uses thioflavin-T as a probe, which binds to the peripheral anionic site of AChE and yields an increase in fluorescent signal. Three types of AChE inhibitors were studied: catalytic site inhibitors (carbamate insecticides, edrophonium, and benzylpiperidine), peripheral site inhibitors (tubocurarine, ethidium bromide, and propidium iodide), and bivalent inhibitors (donepezil, BW284C51, and a series of bis(n)-tacrines). All were screened on murine AChE to compare and contrast changes of peripheral site conformation in the TFT assay with catalytic inhibition. All the inhibitors reduced thioflavin-T fluorescence in a concentration-dependent manner with potencies (IC50) ranging from 8 nM for bis(6)-tacrine to 159 μM for benzylpiperidine. Potencies in the fluorescence assay were correlated well with their potencies for enzyme inhibition (R2 = 0.884). Efficacies for reducing thioflavin-T fluorescence ranged from 23–36% for catalytic site inhibitors and tubocurarine to near 100% for ethidium bromide and propidium iodide. Maximal efficacies could be reconciled with known mechanisms of interaction of the inhibitors with AChE. When extended to pest species, we anticipate these findings will assist in the discovery and development of novel, selective bivalent insecticides acting on AChE. PMID:24003261

1-Arylsulfonyl-2-(Pyridylmethylsulfinyl) Benzimidazoles as New Proton Pump Inhibitor Prodrugs

PubMed Central

Shin, Jai Moo; Sachs, George; Cho, Young-moon; Garst, Michael

2010-01-01

New arylsulfonyl proton pump inhibitor (PPI) prodrug forms were synthesized. These prodrugs provided longer residence time of an effective PPI plasma concentration, resulting in better gastric acid inhibition. PMID:20032890

The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity.

PubMed Central

Qiao, Dan; Nikitina, Lyudmila A; Buznikov, Gennady A; Lauder, Jean M; Seidler, Frederic J; Slotkin, Theodore A

2003-01-01

Embryonic development in the sea urchin requires trophic actions of the same neurotransmitters that participate in mammalian brain assembly. We evaluated the development of the high-affinity choline transporter, which controls acetylcholine synthesis. A variety of developmental neurotoxicants affect this transporter in mammalian brain. [3H]Hemicholinium-3 binding to the transporter was found in the cell membrane fraction at stages from the unfertilized egg to pluteus, with a binding affinity comparable with that seen in mammalian brain. Over the course of development, the concentration of transporter sites rose more than 3-fold, achieving concentrations comparable with those of cholinergically enriched mammalian brain regions. Dimethylaminoethanol (DMAE), a competitive inhibitor of choline transport, elicited dysmorphology beginning at the mid-blastula stage, with anomalies beginning progressively later as the concentration of DMAE was lowered. Pretreatment, cotreatment, or delayed treatment with acetylcholine or choline prevented the adverse effects of DMAE. Because acetylcholine was protective at a lower threshold, the DMAE-induced defects were most likely mediated by its effects on acetylcholine synthesis. Transient removal of the hyaline layer enabled a charged transport inhibitor, hemicholinium-3, to penetrate sufficiently to elicit similar anomalies, which were again prevented by acetylcholine or choline. These results indicate that the developing sea urchin possesses a high-affinity choline transporter analogous to that found in the mammalian brain, and, as in mammals, the functioning of this transporter plays a key role in the developmental, trophic activity of acetylcholine. The sea urchin model may thus be useful in high-throughput screening of suspected developmental neurotoxicants. PMID:14594623

The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity.

PubMed

Qiao, Dan; Nikitina, Lyudmila A; Buznikov, Gennady A; Lauder, Jean M; Seidler, Frederic J; Slotkin, Theodore A

2003-11-01

Embryonic development in the sea urchin requires trophic actions of the same neurotransmitters that participate in mammalian brain assembly. We evaluated the development of the high-affinity choline transporter, which controls acetylcholine synthesis. A variety of developmental neurotoxicants affect this transporter in mammalian brain. [3H]Hemicholinium-3 binding to the transporter was found in the cell membrane fraction at stages from the unfertilized egg to pluteus, with a binding affinity comparable with that seen in mammalian brain. Over the course of development, the concentration of transporter sites rose more than 3-fold, achieving concentrations comparable with those of cholinergically enriched mammalian brain regions. Dimethylaminoethanol (DMAE), a competitive inhibitor of choline transport, elicited dysmorphology beginning at the mid-blastula stage, with anomalies beginning progressively later as the concentration of DMAE was lowered. Pretreatment, cotreatment, or delayed treatment with acetylcholine or choline prevented the adverse effects of DMAE. Because acetylcholine was protective at a lower threshold, the DMAE-induced defects were most likely mediated by its effects on acetylcholine synthesis. Transient removal of the hyaline layer enabled a charged transport inhibitor, hemicholinium-3, to penetrate sufficiently to elicit similar anomalies, which were again prevented by acetylcholine or choline. These results indicate that the developing sea urchin possesses a high-affinity choline transporter analogous to that found in the mammalian brain, and, as in mammals, the functioning of this transporter plays a key role in the developmental, trophic activity of acetylcholine. The sea urchin model may thus be useful in high-throughput screening of suspected developmental neurotoxicants.

Stable, concentrated solutions of high molecular weight polyaniline and articles therefrom

DOEpatents

Mattes, Benjamin R.; Wang, Hsing-Lin

1999-11-09

Stable, concentrated solutions of high molecular weight polyaniline. In order to process high quality fibers and other articles possessing good mechanical properties, it is known that solution concentrations of the chosen polymer should be in the range from 15-30% (w/w). Moreover, it is desirable to use the highest molecular weight consistent with the solubility properties of the polymer. However, such solutions are inherently unstable, forming gels before processing can be achieved. The present invention describes the addition gel inhibitors (GIs) to the polymer solution, thereby permitting high concentrations (between 15% and 30% (w/w)) of high molecular weight ((M.sub.w)>120,000, and (M.sub.n)>30,000) emeraldine base (EB) polyaniline to be dissolved. Secondary amines have been used for this purpose in concentrations which are small compared to those which might otherwise be used in a cosolvent role therefor. The resulting solutions are useful for generating excellent fibers, films, coatings and other objects, since the solutions are stable for significant time periods, and the GIs are present in too small concentrations to cause polymer deterioration. It is demonstrated that the GIs found to be useful do not act as cosolvents, and that gelation times of the solutions are directly proportional to the concentration of GI. In particular, there is a preferred concentration of GI, which if exceeded causes structural and electrical conductivity degradation of resulting articles. Heating of the solutions significantly improves solubility.

Alterations in brain extracellular dopamine and glycine levels following combined administration of the glycine transporter type-1 inhibitor Org-24461 and risperidone.

PubMed

Nagy, Katalin; Marko, Bernadett; Zsilla, Gabriella; Matyus, Peter; Pallagi, Katalin; Szabo, Geza; Juranyi, Zsolt; Barkoczy, Jozsef; Levay, Gyorgy; Harsing, Laszlo G

2010-12-01

The most dominant hypotheses for the pathogenesis of schizophrenia have focused primarily upon hyperfunctional dopaminergic and hypofunctional glutamatergic neurotransmission in the central nervous system. The therapeutic efficacy of all atypical antipsychotics is explained in part by antagonism of the dopaminergic neurotransmission, mainly by blockade of D(2) dopamine receptors. N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia can be reversed by glycine transporter type-1 (GlyT-1) inhibitors, which regulate glycine concentrations at the vicinity of NMDA receptors. Combined drug administration with D(2) dopamine receptor blockade and activation of hypofunctional NMDA receptors may be needed for a more effective treatment of positive and negative symptoms and the accompanied cognitive deficit in schizophrenia. To investigate this type of combined drug administration, rats were treated with the atypical antipsychotic risperidone together with the GlyT-1 inhibitor Org-24461. Brain microdialysis was applied in the striatum of conscious rats and determinations of extracellular dopamine, DOPAC, HVA, glycine, glutamate, and serine concentrations were carried out using HPLC/electrochemistry. Risperidone increased extracellular concentrations of dopamine but failed to influence those of glycine or glutamate measured in microdialysis samples. Org-24461 injection reduced extracellular dopamine concentrations and elevated extracellular glycine levels but the concentrations of serine and glutamate were not changed. When risperidone and Org-24461 were added in combination, a decrease in extracellular dopamine concentrations was accompanied with sustained elevation of extracellular glycine levels. Interestingly, the extracellular concentrations of glutamate were also enhanced. Our data indicate that coadministration of an antipsychotic with a GlyT-1 inhibitor may normalize hypofunctional NMDA receptor-mediated glutamatergic neurotransmission with reduced dopaminergic side effects characteristic for antipsychotic medication.

The role of various amino acids in enzymatic browning process in potato tubers, and identifying the browning products.

PubMed

Ali, Hussein M; El-Gizawy, Ahmed M; El-Bassiouny, Rawia E I; Saleh, Mahmoud A

2016-02-01

The effects of five structurally variant amino acids, glycine, valine, methionine, phenylalanine and cysteine were examined as inhibitors and/or stimulators of fresh-cut potato browning. The first four amino acids showed conflict effects; high concentrations (⩾ 100mM for glycine and ⩾ 1.0M for the other three amino acids) induced potato browning while lower concentrations reduced the browning process. Alternatively, increasing cysteine concentration consistently reduced the browning process due to reaction with quinone to give colorless adduct. In PPO assay, high concentrations (⩾ 1.11 mM) of the four amino acids developed more color than that of control samples. Visible spectra indicated a continuous condensation of quinone and glycine to give colored adducts absorbed at 610-630 nm which were separated and identified by LC-ESI-MS as catechol-diglycine adduct that undergoes polymerization with other glycine molecules to form peptide side chains. In lower concentrations, the less concentration the less developed color was observed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry.

PubMed

Cerca, Nuno; Martins, Silvia; Cerca, Filipe; Jefferson, Kimberly K; Pier, Gerald B; Oliveira, Rosário; Azeredo, Joana

2005-08-01

To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.

Small-molecule inhibitors of toxT expression in Vibrio cholerae.

PubMed

Anthouard, Rebecca; DiRita, Victor J

2013-08-06

Vibrio cholerae, a Gram-negative bacterium, infects humans and causes cholera, a severe disease characterized by vomiting and diarrhea. These symptoms are primarily caused by cholera toxin (CT), whose production by V. cholerae is tightly regulated by the virulence cascade. In this study, we designed and carried out a high-throughput chemical genetic screen to identify inhibitors of the virulence cascade. We identified three compounds, which we named toxtazin A and toxtazin B and B', representing two novel classes of toxT transcription inhibitors. All three compounds reduce production of both CT and the toxin-coregulated pilus (TCP), an important colonization factor. We present evidence that toxtazin A works at the level of the toxT promoter and that toxtazins B and B' work at the level of the tcpP promoter. Treatment with toxtazin B results in a 100-fold reduction in colonization in an infant mouse model of infection, though toxtazin A did not reduce colonization at the concentrations tested. These results add to the growing body of literature indicating that small-molecule inhibitors of virulence genes could be developed to treat infections, as alternatives to antibiotics become increasingly needed. V. cholerae caused more than 580,000 infections worldwide in 2011 alone (WHO, Wkly. Epidemiol. Rec. 87:289-304, 2012). Cholera is treated with an oral rehydration therapy consisting of water, glucose, and electrolytes. However, as V. cholerae is transmitted via contaminated water, treatment can be difficult for communities whose water source is contaminated. In this study, we address the need for new therapeutic approaches by targeting the production of the main virulence factor, cholera toxin (CT). The high-throughput screen presented here led to the identification of two novel classes of inhibitors of the virulence cascade in V. cholerae, toxtazin A and toxtazins B and B'. We demonstrate that (i) small-molecule inhibitors of virulence gene production can be identified in a high-throughput screen, (ii) targeting virulence gene production is an effective therapeutic strategy, and (iii) small-molecule inhibitors can uncover unknown layers of gene regulation, even in well-studied regulatory cascades.

Effect of food on the bioavailability and tolerability of the JAK2-selective inhibitor fedratinib (SAR302503): Results from two phase I studies in healthy volunteers.

PubMed

Zhang, Meng; Xu, Christine; Ma, Lei; Shamiyeh, Elias; Yin, Jianyun; von Moltke, Lisa L; Smith, William B

2015-07-01

Fedratinib (SAR302503/TG101348) is a Janus kinase 2 (JAK2)-selective inhibitor developed for treatment of patients with myelofibrosis. The effect of food intake on the pharmacokinetics (PKs) and tolerability of single-dose fedratinib was investigated in two Phase I studies (FED12258: 100 mg or 500 mg under fasted or fed [high-fat breakfast] conditions; ALI13451: 500 mg under fasted or fed [low- or high-fat breakfast] conditions) in healthy male subjects. At the 500 mg dose the fed:fasted ratio estimate for area under the plasma concentration-time curve extrapolated to infinity was 0.96 (100 mg; high-fat/fasted), 1.19-1.24 (500 mg; high-fat/fasted), and 1.22 (500 mg; low-fat/fasted). Fedratinib 500 mg attained peak plasma concentration 4 hours after a high-fat breakfast and 2-2.5 hours after a low-fat breakfast or under fasted conditions; terminal half-life was 76-88 hours (fasted) and 73-78 hours (fed). The most frequent adverse events were mild gastrointestinal toxicities, the incidence of which decreased following a high-fat breakfast compared with both fasted and low-fat breakfast conditions (17%, 67%, and 59% of subjects, respectively, in ALI13451). In conclusion, food intake had minimal impact on the PKs of fedratinib, and the tolerability of this drug was improved when taken following a high-fat breakfast. © 2014, The American College of Clinical Pharmacology.

The use of morinda citrifolia as a green corrosion inhibitor for low carbon steel in 3.5% NaCl solution

NASA Astrophysics Data System (ADS)

Kusumastuti, Rahayu; Pramana, Rakhmad Indra; Soedarsono, Johny W.

2017-03-01

The effect and mechanism of green corrosion inhibitor of Morinda Citrifolia (Noni) toward low carbon steel material has been researched. The general background is to develop the cheap and eco-friendly corrosion inhibitor based on components taken from tropical plants that grow +in Indonesia. This research aims to determine the effectiveness of the use of the extracts of noni as green corrosion inhibitor of carbon steel material in aggressive environment. The medium applied for this experiment is 3.5% natrium chloride solution. The variation of the concentration and immersion time duration has been applied as the experimental parameters. All the work was done at room temperature. The corrosion rate was measured by electrochemical polarization method with CMS 600-Gamry instruments and weight loss. The adsorption of inhibitor into the metal surface, which induced bonding formation after immersion was observed by using FTIR method. Inhibition mechanism was observed by polarization curves and fitted by the Langmuir adsorption models. The experimental results show that the higher concentration of inhibitor increasing the inhibition effect. The optimum inhibition is obtained at 3 ppm noni fruit extract, after immersion for about 288 hours. The corrosion rates obtained was 1.385 mpy, with the inhibitor efficiency of 76.92%. The monolayer film is formed coating the surface material as a result of mixed type corrosion inhibitor behavior of Noni. It can be concluded that this green inhibitor is effective to be used for low carbon steel material.

The Divergent Cardiovascular Effects of Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Type 1 Receptor Blockers in Adult Patients With Type 2 Diabetes Mellitus.

PubMed

Strauss, Martin H; Hall, Alistair S

2018-04-01

The renin angiotensin aldosterone system (RAAS) plays a central role in the pathophysiology of hypertension and vascular disease. Angiotensin-converting enzyme inhibitors (ACEi's) suppress angiotensin II (ANG II) concentrations, whereas angiotensin II type 1 (AT 1 ) receptor blockers (ARBs) block the binding of ANG II to AT 1 receptors. ACEi's and ARBs are both effective antihypertensive agents and produce similar risk reductions for stroke, a blood pressure-dependent phenomenon. ACEi's also reduce the risk for myocardial infarction (MI) and all-cause mortality in high-risk hypertensive patients as well as in people with diabetes, vascular disease and congestive heart failure. ARBs, in contrast, do not reduce the risk for MI or death in randomized clinical trials when assessed vs. placebo. Systematic reviews of ARBs that include meta-analyses or metaregression analyses confirm that ARBs lack the cardiovascular-protective effects of ACEi's. Practice guidelines, especially those for high-risk patients, such as those with diabetes mellitus, should reflect the evidence that ACEi's and ARBs have divergent cardiovascular effects: ACEi's reduce mortality, whereas ARBs do not. ACEi's should remain the preferred RAAS inhibitor for patients at high risk. Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

Identification and Characterization of ML352: A Novel, Noncompetitive Inhibitor of the Presynaptic Choline Transporter

PubMed Central

2015-01-01

The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents. PMID:25560927

Therapeutic Management of Familial Hypercholesterolemia: Current and Emerging Drug Therapies.

PubMed

Patel, Roshni S; Scopelliti, Emily M; Savelloni, Julie

2015-12-01

Familial hypercholesterolemia (FH) is a genetic disorder characterized by significantly elevated low-density lipoprotein cholesterol (LDL-C) concentrations that result from mutations of the LDL receptor, apolipoprotein B (apo B-100), and proprotein convertase subtilisin/kexin type 9 (PCSK9). Early and aggressive treatment can prevent premature atherosclerotic cardiovascular disease in these high-risk patients. Given that the cardiovascular consequences of FH are similar to typical hypercholesterolemia, traditional therapies are utilized to decrease LDL-C levels. Patients with FH should receive statins as first-line treatment; high-potency statins at high doses are often required. Despite the use of statins, additional treatments are often necessary to achieve appropriate LDL-C lowering in this patient population. Novel drug therapies that target the pathophysiologic defects of the condition are continuously emerging. Contemporary therapies including mipomersen (Kynamro, Genzyme), an oligonucleotide inhibitor of apo B-100 synthesis; lomitapide (Juxtapid, Aegerion), a microsomal triglyceride transfer protein inhibitor; and alirocumab (Praluent, Sanofi-Aventis/Regeneron) and evolocumab (Repatha, Amgen), PCSK9 inhibitors, are currently approved by the U.S. Food and Drug Administration for use in FH. This review highlights traditional as well as emerging contemporary therapies with supporting clinical data to evaluate current recommendations and discuss the future direction of FH management. © 2015 Pharmacotherapy Publications, Inc.

Evaluation of the Efficacy and Safety of Rivaroxaban Using a Computer Model for Blood Coagulation

PubMed Central

Burghaus, Rolf; Coboeken, Katrin; Gaub, Thomas; Kuepfer, Lars; Sensse, Anke; Siegmund, Hans-Ulrich; Weiss, Wolfgang; Mueck, Wolfgang; Lippert, Joerg

2011-01-01

Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Union and several other countries for the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery and is in advanced clinical development for the treatment of thromboembolic disorders. Its mechanism of action is antithrombin independent and differs from that of other anticoagulants, such as warfarin (a vitamin K antagonist), enoxaparin (an indirect thrombin/Factor Xa inhibitor) and dabigatran (a direct thrombin inhibitor). A blood coagulation computer model has been developed, based on several published models and preclinical and clinical data. Unlike previous models, the current model takes into account both the intrinsic and extrinsic pathways of the coagulation cascade, and possesses some unique features, including a blood flow component and a portfolio of drug action mechanisms. This study aimed to use the model to compare the mechanism of action of rivaroxaban with that of warfarin, and to evaluate the efficacy and safety of different rivaroxaban doses with other anticoagulants included in the model. Rather than reproducing known standard clinical measurements, such as the prothrombin time and activated partial thromboplastin time clotting tests, the anticoagulant benchmarking was based on a simulation of physiologically plausible clotting scenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivity for tissue factor concentration inducing clotting, and a steep concentration–effect relationship, rapidly flattening towards higher inhibitor concentrations, both suggesting a broad therapeutic window. The predicted dosing window is highly accordant with the final dose recommendation based upon extensive clinical studies. PMID:21526168

Gibberellin inhibitors improve embryogenic tissue initiation in conifers.

PubMed

Pullman, Gerald S; Mein, J; Johnson, S; Zhang, Y

2005-02-01

Somatic embryogenesis (SE), the most promising technology to multiply high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and uniformity of future forests in the United States. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (LP; Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation of LP, slash pine (SP; Pinus elliottii), Douglas-fir (DF; Pseudotsuga menziesii), and Norway spruce (NS; Picea abies) were improved through the use of paclobutrazol, a gibberellin synthesis inhibitor. Paclobutrazol was effective at concentrations ranging from 0.25 mg/l to 3.0 mg/l (0.85-10.2 microM) and optimal in LP at 1.0 mg/l. Using control media (no paclobutrazol) and 0.33-1.0 mg/l paclobutrazol, initiation percentages in LP, SP, DF, and NS were improved from 37.7% to 44.2% (across experiments), 19.3% to 28.5%, 16.9% to 23.7%, and 38.8% to 48.5%, respectively. Other gibberellin inhibitors such as flurprimidol, chlormequat-Cl, and daminozide also caused statistically significant increases in LP initiation when added to the medium at concentrations of 0.34, 10.0, and 1.0 microM, respectively. No detrimental effects on subsequent embryo development were observed when 29 new initiations from medium without GA inhibitor and 28 new initiations from medium containing paclobutrazol were tracked through culture capture, liquid culture establishment, cotyledonary embryo development, and germination.

Use of the NP-40 detergent-mediated assay in discovery of inhibitors of beta-hematin crystallization.

PubMed

Sandlin, Rebecca D; Carter, Melissa D; Lee, Patricia J; Auschwitz, Jennifer M; Leed, Susan E; Johnson, Jacob D; Wright, David W

2011-07-01

The protozoan parasite responsible for malaria affects over 500 million people each year. Current antimalarials have experienced decreased efficacy due to the development of drug-resistant strains of Plasmodium spp., resulting in a critical need for the discovery of new antimalarials. Hemozoin, a crystalline by-product of heme detoxification that is necessary for parasite survival, serves as an important drug target. The quinoline antimalarials, including amodiaquine and chloroquine, act by inhibiting the formation of hemozoin. The formation of this crystal does not occur spontaneously, and recent evidence suggests crystallization occurs in the presence of neutral lipid particles located in the acidic digestive vacuole of the parasite. To mimic these conditions, the lipophilic detergent NP-40 has previously been shown to successfully mediate the formation of β-hematin, synthetic hemozoin. Here, an NP-40 detergent-based assay was successfully adapted for use as a high-throughput screen to identify inhibitors of β-hematin formation. The resulting assay exhibited a favorable Z' of 0.82 and maximal drift of less than 4%. The assay was used in a pilot screen of 38,400 diverse compounds at a screening concentration of 19.3 μM, resulting in the identification of 161 previously unreported β-hematin inhibitors. Of these, 48 also exhibited ≥ 90% inhibition of parasitemia in a Plasmodium falciparum whole-cell assay at a screening concentration of 23 μM. Eight of these compounds were identified to have nanomolar 50% inhibitory concentration values near that of chloroquine in this assay.

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kudalkar, Shalley N.; Beloor, Jagadish; Chan, Albert H.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compoundmore » II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0–last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.« less

Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.

PubMed

Hashimoto, Junko; Watanabe, Taku; Seki, Tatsuya; Karasawa, Satoshi; Izumikawa, Miho; Seki, Tomoe; Iemura, Shun-Ichiro; Natsume, Tohru; Nomura, Nobuo; Goshima, Naoki; Miyawaki, Atsushi; Takagi, Motoki; Shin-Ya, Kazuo

2009-09-01

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 microM and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 microM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library.

Identification and characterisation of carnostatine (SAN9812), a potent and selective carnosinase (CN1) inhibitor with in vivo activity.

PubMed

Qiu, Jiedong; Hauske, Sibylle J; Zhang, Shiqi; Rodriguez-Niño, Angelica; Albrecht, Thomas; Pastene, Diego O; van den Born, Jacob; van Goor, Harry; Ruf, Sven; Kohlmann, Markus; Teufel, Michael; Krämer, Bernhard K; Hammes, Hans-Peter; Peters, Verena; Yard, Benito A; Kannt, Aimo

2018-06-20

Carnosinase 1 (CN1) has been postulated to be a susceptibility factor for developing diabetic nephropathy (DN). Although its major substrate, carnosine, is beneficial in rodent models of DN, translation of these findings to humans has been hampered by high CN1 activity in human serum resulting in rapid degradation of carnosine. To overcome this hurdle, we screened a protease-directed small-molecule library for inhibitors of human recombinant CN1. We identified SAN9812 as a potent and highly selective inhibitor of CN1 activity with a K i of 11 nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30 mg/kg SAN9812 led to a sustained reduction in circulating CN1 activity in human CN1 transgenic (TG) mice. Simultaneous administration of carnosine and SAN9812 increased carnosine levels in plasma and kidney by up to 100-fold compared to treatment-naïve CN1-overexpressing mice. To our knowledge, this is the first study reporting on a potent and selective CN1 inhibitor with in vivo activity. SAN9812, also called carnostatine, may be used to increase renal carnosine concentration as a potential therapeutic modality for renal diseases linked to glycoxidative conditions.

Pyridinedicarboxylates, the first mechanism-derived inhibitors for prolyl 4-hydroxylase, selectively suppress cellular hydroxyprolyl biosynthesis. Decrease in interstitial collagen and Clq secretion in cell culture.

PubMed Central

Tschank, G; Raghunath, M; Günzler, V; Hanauske-Abel, H M

1987-01-01

Two pyridinedicarboxylates, predicted [Hanauske-Abel (1983) M.D.-Ph.D. Thesis, Philipps Universität Marburg] and later found to be potent reversible inhibitors of purified prolyl 4-hydroxylase [Majaama, Hanauske-Abel, Günzler & Kivirikko (1984) Eur. J. Biochem. 138, 239-245] were investigated with respect to their effect on hydroxyprolyl biosynthesis in the fibroblast/collagen and the macrophage/Clq systems, and the effect was compared with that of the iron chelator 2,2'-dipyridyl, the compound usually employed to inhibit cellular hydroxyprolyl formation. Only the enzyme-mechanism-derived pyridinedicarboxylates were highly selective inhibitors, and only they lacked overt cytotoxicity. Morphologically, their effect was restricted to the site of cellular hydroxyprolyl biosynthesis, i.e. the cisternae of the rough-surfaced endoplasmic reticulum. They were equally effective in the different cell types studied, and human and guinea-pig fibroblasts showed the same sensitivity. The minimal lipophilicity of the pyridinedicarboxylates necessitated high concentrations to achieve suppression of cellular hydroxyprolyl formation, but lipophilic bio-activatable pro-inhibitors may overcome this disadvantage. For the first time, experimental evidence is presented suggesting that, in cell culture, the biosynthesis of interstitial collagens and Clq can be suppressed selectively, identifying the pyridinedicarboxylates as promising pilot compounds for experiments in vivo. Images Fig. 3. Fig. 4. PMID:2829835

PARP1 expression, activity and ex vivo sensitivity to the PARP inhibitor, talazoparib (BMN 673), in chronic lymphocytic leukaemia

PubMed Central

Herriott, Ashleigh; Tudhope, Susan J.; Junge, Gesa; Rodrigues, Natalie; Patterson, Miranda J.; Woodhouse, Laura; Lunec, John; Hunter, Jill E.; Mulligan, Evan A.; Cole, Michael; Allinson, Lisa M.; Wallis, Jonathan P.; Marshall, Scott; Wang, Evelyn; Curtin, Nicola J.; Willmore, Elaine

2015-01-01

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors. We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 – 190052 pmol PAR/106 cells) compared to that seen in healthy volunteer lymphocytes (2451 – 7519 pmol PAR/106 cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2′-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function. PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib. PMID:26539646

A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

PubMed

Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

2016-05-01

We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. Copyright © 2016 Elsevier Ltd. All rights reserved.

Diethylglyoxal bis(guanylhydrazone): a novel highly potent inhibitor of S-adenosylmethionine decarboxylase with promising properties for potential chemotherapeutic use.

PubMed

Elo, H; Mutikainen, I; Alhonen-Hongisto, L; Laine, R; Jänne, J

1988-07-01

Diethylglyoxal bis(guanylhydrazone) (DEGBG), a novel analog of the antileukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) was synthesized. It was found to be the most powerful inhibitor of yeast S-adenosylmethionine decarboxylase (AdoMetDC) so far studied (Ki approx. 9 nM). This property, together with the finding that the compound is a weaker inhibitor of intestinal diamine oxidase than are MGBG and its glyoxal, ethylglyoxal and ethylmethylglyoxal analogs, makes the compound a promising candidate as a polyamine antimetabolite for chemotherapy studies. DEGBG was also found to potentiate the antiproliferative effect of the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine against mouse L1210 leukemia cells in vitro. DEGBG increased several-fold the intracellular putrescine concentration of cultured L1210 cells, just as MGBG and its ethylglyoxal analog are known to do. The results strongly suggest that DEGBG is worth further studies. Combined with previous studies, they also made possible the construction of some empirical rules concerning the structure-activity relationships of bis(guanylhydrazone) type inhibitors of AdoMetDC. The identity of DEGBG was confirmed by a single-crystal X-ray analysis and by 1H- and 13C-NMR spectroscopy. It consisted of the same isomer as MGBG and several of its analogs are known to consist of.

Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation.

PubMed

Wang, Aoli; Yan, Xiao-E; Wu, Hong; Wang, Wenchao; Hu, Chen; Chen, Cheng; Zhao, Zheng; Zhao, Peng; Li, Xixiang; Wang, Li; Wang, Beilei; Ye, Zi; Wang, Jinhua; Wang, Chu; Zhang, Wei; Gray, Nathanael S; Weisberg, Ellen L; Chen, Liang; Liu, Jing; Yun, Cai-Hong; Liu, Qingsong

2016-10-25

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

Small-molecule inhibitors of FGFR, integrins and FAK selectively decrease L1CAM-stimulated glioblastoma cell motility and proliferation.

PubMed

Anderson, Hannah J; Galileo, Deni S

2016-06-01

The cell adhesion/recognition protein L1CAM (L1; CD171) has previously been shown to act through integrin, focal adhesion kinase (FAK) and fibroblast growth factor receptor (FGFR) signaling pathways to increase the motility and proliferation of glioblastoma cells in an autocrine/paracrine manner. Here, we investigated the effects of clinically relevant small-molecule inhibitors of the integrin, FAK and FGFR signaling pathways on glioblastoma-derived cells to determine their effectiveness and selectivity for diminishing L1-mediated stimulation. The effects of the FGFR inhibitor PD173074, the FAK inhibitors PF431396 and Y15 and the αvβ3/αvβ5 integrin inhibitor cilengitide were assessed in L1-positive and L1-negative variants of the human glioblastoma-derived cell lines T98G and U-118 MG. Their motility and proliferation were quantified using time-lapse microscopy and DNA content/cell cycle analyses, respectively. The application of all four inhibitors resulted in reductions in L1-mediated motility and proliferation rates of L1-positive glioblastoma-derived cells, down to the level of L1-negative cells when used at nanomolar concentrations, whereas no or much smaller reductions in these rates were obtained in L1-negative cells. In addition, we found that single inhibitor treatment resulted in maximum effects (i.e., combinations of FAK or integrin inhibitors with the FGFR inhibitor were rarely more effective). These results suggest that FAK may act as a point of convergence between the integrin and FGFR signaling pathways stimulated by L1 in these cells. We here show for the first time that small-molecule inhibitors of FGFR, integrins and FAK effectively and selectively abolish L1-stimulated migration and proliferation of glioblastoma-derived cells. Our results suggest that these inhibitors have the potential to reduce the aggressiveness of high-grade gliomas expressing L1.

Method for preparing polyaniline fibers

DOEpatents

Mattes, Benjamin R.; Wang, Hsing-Lin

2000-01-01

Stable, concentrated solutions of high molecular weight polyaniline. In order to process high quality fibers and other articles possessing good mechanical properties, it is known that solution concentrations of the chosen polymer should be in the range from 15-30% (w/w). Moreover, it is desirable to use the highest molecular weight consistent with the solubility properties of the polymer. However, such solutions are inherently unstable, forming gels before processing can be achieved. The present invention describes the addition gel inhibitors (GIs) to the polymer solution, thereby permitting high concentrations (>15% (w/w)) of high molecular weight ((M.sub.w)>120,000, and (M.sub.n)>30,000) emeraldine base (EB) polyaniline to be dissolved. Secondary amines have been used for this purpose in concentrations which are small compared to those which might otherwise be used in a cosolvent role therefor. The resulting solutions are useful for generating excellent fibers, films, coatings and other objects, since the solutions are stable for significant time periods, and the GIs are present in too small concentrations to cause polymer deterioration. It is demonstrated that the GIs found to be useful do not act as cosolvents, and that gelation times of the solutions are directly proportional to the concentration of GI. In particular, there is a preferred concentration of GI, which if exceeded causes structural and electrical conductivity degradation of resulting articles. Heating of the solutions significantly improves solubility.

Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes.

PubMed Central

Pan, Y T; Xu, B; Rice, K; Smith, S; Jackson, R; Elbein, A D

1997-01-01

Enterobacter cloacae has been implicated as one of the causative agents in neonatal infection and causes a septicemia thought to be initiated via the gastrointestinal tract. The adhesion of radiolabeled E. cloacae to HT-29 cells was concentration and temperature dependent and was effectively blocked by unlabeled bacteria or by millimolar concentrations of alpha-mannosides and micromolar concentrations of high-mannose oligosaccharides. A variety of well-characterized mannose oligosaccharides were tested as inhibitors of adhesion. The best inhibitor was the Man9(GlcNAc)2-tyrosinamide, which was considerably better than other tyrosinamide-linked oligosaccharides such as Man7(GlcNAc)2, Man6(GlcNAc)2 or Man5(GlcNAc)2. Further evidence that the bacteria preferred Man9(GlcNAc)2 structures was obtained by growing HT-29 cells in the presence of glycoprotein processing inhibitors that block mannosidase I and increase the amount of protein-bound Man9(GlcNAc)2 at the cell surface. Such cells bound 1.5- to 2-fold more bacteria than did control cells. The adhesin involved in binding to high-mannose structures was purified from isolated pili. On sodium dodecyl sulfate-gels, a 35-kDa protein was identified by its specific binding to a mannose-containing biotinylated albumin. The amino acid sequences of several peptides from the 35-kDa subunit showed over 85% identity to FimH, the mannose-specific adhesin of Salmonella typhimurium. Pili were labeled with 125I and examined for the ability to bind to HT-29 cells. Binding showed saturation kinetics and was inhibited by the addition of Man9(GlcNAc)2-tyrosinamide but not by oligosaccharides with fewer mannose residues. Polyclonal antibody against this 35-kDa protein also effectively blocked adhesion of pili or E. cloacae, but no effect was observed with nonspecific antibody. These studies demonstrate that the 35-kDa pilus subunit is a lectin whose specificity is directed toward Man, (GlcNAc)2 oligosaccharides. PMID:9317027

Effect of Molecular Structure on Modulation of Passivation Films on Copper Chemical Mechanical Planarization

NASA Astrophysics Data System (ADS)

Mlynarski, Amy

In order to optimize the chemical mechanical planarization (CMP) process, there is a need to further understand the synergistic relationship between chemical and mechanical parameters to enhance the polishing process. CMP chemistry is very complex, as it contains complexing agents, oxidizing agents, passivating agents, and abrasive particles. This variety of components ensues chaos within the system, which complicates the understanding of the direct impact each component has on the CMP process. In order for there to be efficiency in the polishing process, specifically for copper (Cu) polishing, the chemistry must create a softened passivation layer on the Cu surface that is able to be readily removed by applied mechanical abrasion. Focusing on Cu CMP, the oxidation of Cu to Cu2+ needs to be thoroughly understood in order to probe the formation of creating this ideal passivated layer, which protects recessed Cu regions. The type of film that is formed, the strength of the film, and even the efficiency of film removal will be altered depending on the chemistry of interaction at the Cu surface. This thesis focuses on understanding the working mechanism of the film formation on Cu, depending on the passivating agent added to the system. The different passivating agents used, more specifically benzotriazole (BTA), triazole (TAZ), salicylhydroxamic acid (SHA), and benzimidazole (BIA), have all been known to create a light coat of protection on the recessed metal, providing corrosion resistance. In order to study the differences in these films, many different techniques can be utilized to characterize the films, such as electrochemical scans, referred to as Tafel plots, which will be performed to compare the differences of the films. By altering the temperature within the system, the activation energy for each system can also be determined as another way to characterize the density of the passive film formed. Furthermore, the generation of *OH will be monitored since the formation of *OH is imperative for catalyzing the Cu-amino acid complexes, necessary for obtaining adequate removal rates. The amount of *OH generated from each system would have a direct correlation to the polishing performance for the different systems. Additionally, the effect of changing mechanical parameters or consumables used will alter the polish, more specifically the amount of friction generated during the polishes. This work discovered that when comparing all of the different types of inhibitors, there was a significant difference seen in the type of film formed as well as the stability of the film, strongly dependent on the concentration of the corrosion inhibitor. The calculated activation energy showed a direct correlation to the concentration of the corrosion inhibitor; more specifically, as the concentration of the inhibitor increases, so does the activation energy. By looking at the generated amount of *OH for the complexes, more specifically BTA and SHA, there is a minimal amount of *OH generated within the system compared to that of TAZ and BIA which resemble more like a system containing no inhibitor at all. This would once again show how the structure determines function of the inhibitor in regards to how the complex changes for the different molecules. The removal rates for these systems, both at 100 ppm and 500 ppm, show a strong correlation to the previously discussed activation energies. BTA shows extremely low removal rates, which seems to be diminished at even higher concentration, since the film created is so dense due to the pi-pi electron interactions. Similar trends are seen in the results from TAZ where the removal rates decrease as the concentration of the inhibitor increases. Furthermore, SHA shows significant material removal rates (MRR) at lower concentrations, but the rates are vastly different when increasing the concentration to 500 ppm. This could be because the complex that forms with the surface is a stable ring-like complex, but once enough inhibitor is added, the SHA complex covers the surface entirely, eliminating any chance of Cu-glycine interaction, which would promote removal rate. Unlike the other inhibitors, the removal rates for BIA show that as the concentration of the inhibitor increases, the removal rates significantly increase as well. Since this inhibitor forms a "weaker" complex, comparatively, the more inhibitor added would allow for more of the Cu-glycine interactions to take place.

The phosphatase inhibitor menadione (vitamin K3) protects cells from EGFR inhibition by erlotinib and cetuximab.

PubMed

Perez-Soler, Roman; Zou, Yiyu; Li, Tianhong; Ling, Yi He

2011-11-01

Skin toxicity is the main side effect of epidermal growth factor receptor (EGFR) inhibitors, often leading to dose reduction or discontinuation. We hypothesized that phosphatase inhibition in the skin keratinocytes may prevent receptor dephosphorylation caused by EGFR inhibitors and be used as a new potential strategy for the prevention or treatment of this side effect. Menadione (Vitamin K3) was used as the prototype compound to test our hypothesis. HaCat human skin keratinocyte cells and A431 human squamous carcinoma cells were used. EGFR inhibition was measured by Western blotting and immunofluorescence. Phosphatase inhibition and reactive oxygen species (ROS) generation were measured by standard ELISA and fluorescence assays. Menadione caused significant and reversible EGFR activation in a dose-dependent manner starting at nontoxic concentrations. EGFR activation by menadione was associated with reversible protein tyrosine phosphatase inhibition, which seemed to be mediated by ROS generation as exposure to antioxidants prevented both menadione-induced ROS generation and phosphatase inhibition. Short-term coincubation of cells with nontoxic concentrations of menadione and the EGFR inhibitors erlotinib or cetuximab prevented EGFR dephosphorylation. Seventy-two-hour coincubation of cells with the highest nontoxic concentration of menadione and erlotinib provided for a fourfold cell growth inhibitory protection in HaCat human keratinocyte cells. Menadione at nontoxic concentrations causes EGFR activation and prevents EGFR dephosphorylation by erlotinib and cetuximab. This effect seems to be mediated by ROS generation and secondary phosphatase inhibition. Mild oxidative stress in skin keratinocytes by topical menadione may protect the skin from the toxicity secondary to EGFR inhibitors without causing cytotoxicity. ©2011 AACR

Nephrotoxicity of Epigenetic Inhibitors Used for the Treatment of Cancer

PubMed Central

Scholpa, N.E.; Kolli, R.T.; Moore, M.; Arnold, R.D.; Glenn, T.C.; Cummings, B.S.

2016-01-01

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 hr. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 hr. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury. PMID:27543423

Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.

PubMed

An, S F; Fleming, K A

1991-11-01

A problem associated with use of the polymerase chain reaction to amplify specific DNA fragments from formalin fixed, paraffin wax embedded tissues is the not infrequent failure of amplification. One possible reason for this could be the presence of inhibitor(s), which interfere with the activity of the reaction. It has been shown that such inhibitor(s) exist when amplifying the human beta globin gene (which exists in human genomic DNA as a single copy gene) from routine clinical samples. A variety of methods to remove such inhibitor(s) were investigated. The results indicate that inhibitor(s) are removed by proteinase K digestion, followed by purification with phenol/chloroform, and centrifugation through a Centricon-30 membrane (30,000 molecular weight cut off). Other factors, including the length and concentration of the DNA sequence to be amplified, can also affect amplification.

Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen.

PubMed

Mueller, Niklaus H; Pattabiraman, Nagarajan; Ansarah-Sobrinho, Camilo; Viswanathan, Prasanth; Pierson, Theodore C; Padmanabhan, R

2008-09-01

West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen approximately 32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with K(i) values of 3.2 +/- 0.3 microM and 3.4 +/- 0.6 microM, respectively. These compounds inhibited the dengue virus type 2 protease with K(i) values of 28.6 +/- 5.1 microM and 30.2 +/- 8.6 microM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 +/- 0.4 microM; selectivity index, 100), presumably by inhibition of polyprotein processing.

The economics of inpatient on-demand treatment for haemophilia with high-responding inhibitors: a US retrospective data analysis.

PubMed

Pokras, S M; Petrilla, A A; Weatherall, J; Lee, W C

2012-03-01

Inpatient costs comprise >50% of annual healthcare costs for haemophilia patients with inhibitors but no reports exist on inpatient resource use and costs at a US national level. To quantify inpatient resource use and costs for on-demand treatment of bleeds of US haemophilia patients with inhibitors and compare costs and treatment duration between Factor VIII bypassing agents (BAs). Stays with haemophilia A from 2003-2008 were identified from inpatient billing records. Presence of inhibitors was inferred through use of BA; recombinant activated Factor VII and plasma-derived activated prothrombin complex concentrate. Duration and number of infusions of BA, length of stay, use of opioid-containing analgesics and costs were assessed and compared. Among 1322 stays mean BA treatment duration was 4.6 days with 4.9 infusions, 6.1 nights spent in hospital, and 58% administered opioid-containing analgesics. In unadjusted analyses there were significant differences in the above mentioned outcomes by BA use, reflecting underlying differences between the two patient populations. Average inpatient costs were $82,911. In adjusted analyses, African-American race, greater disease severity, hospital region outside the southern US and older age (cost model only) were significant predictors of longer BA treatment duration and higher costs. The economic burden of inpatient on-demand treatment of haemophilia with inhibitors is substantial and is associated with lengthy stays, high costs and inadequate pain relief. Availability of more effective BAs could reduce the need for re-treatment, reducing treatment costs and other medical costs, while improving health related quality of life. © 2011 Blackwell Publishing Ltd.

Intestinal absorption of forsythoside A in in situ single-pass intestinal perfusion and in vitro Caco-2 cell models

PubMed Central

Zhou, Wei; Di, Liu-qing; Wang, Juan; Shan, Jin-jun; Liu, Shi-jia; Ju, Wen-zheng; Cai, Bao-chang

2012-01-01

Aim: To investigate the mechanisms underlying the intestinal absorption of the major bioactive component forsythoside A (FTA) extracted from Forsythiae fructus. Methods: An in vitro Caco-2 cell model and a single-pass intestinal perfusion in situ model in SD rats were used. Results: In the in vitro Caco-2 cell model, the mean apparent permeability value (Papp-value) was 4.15×10-7 cm/s in the apical-to-basolateral (AP-BL) direction. At the concentrations of 2.6–10.4 μg/mL, the efflux ratio of FTA in the bi-directional transport experiments was approximately 1.00. After the transport, >96% of the apically loaded FTA was retained on the apical side, while >97% of the basolaterally loaded FTA was retained on the basolateral side. The Papp-values of FTA were inversely correlated with the transepithelial electrical resistance. The paracellular permeability enhancers sodium caprate and EDTA, the P-gp inhibitor verapamil and the multidrug resistance related protein (MRP) inhibitors cyclosporine and MK571 could concentration-dependently increase the Papp-values, while the uptake (OATP) transporter inhibitors diclofenac sodium and indomethacin could concentration-dependently decrease the Papp-values. The intake transporter SGLT1 inhibitor mannitol did not cause significant change in the Papp-values. In the in situ intestinal perfusion model, both the absorption rate constant (Ka) and the effective permeability (Peff-values) following perfusion of FTA 2.6, 5.2 and 10.4 μg/mL via the duodenum, jejunum and ileum had no significant difference, although the values were slightly higher for the duodenum as compared to those in the jejunum and ileum. The low, medium and high concentrations of verapamil caused the largest increase in the Peff-values for duodenum, jejunum and ileum, respectively. Sodium caprate, EDTA and cyclosporine resulted in concentration-dependent increase in the Peff-values. Diclofenac sodium and indomethacin caused concentration-dependent decrease in the Peff-values. Mannitol did not cause significant change in the Papp-values for the duodenum, jejunum or ileum. Conclusion: The results suggest that the intestinal absorption of FTA may occur through passive diffusion, and the predominant absorption site may be in the upper part of small intestine. Paracellular transport route is also involved. P-gp, MRPs and OATP may participate in the absorption of FTA in the intestine. The low permeability of FTA contributes to its low oral bioavailability. PMID:22773077

In vitro effects of the cyclooxygenase inhibitor indomethacin and of the phospholipase-C inhibitor U-73122 on carbachol-induced contractions of porcine detrusor muscle.

PubMed

Badawi, Jasmin Katrin; Seja, Tobias; Bross, Stephan

2008-12-01

Prostaglandin synthetase inhibitors belong to one substance class additionally used in the treatment of bladder dysfunctions associated with involuntary bladder contractions. However, the mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) on the detrusor muscle is not clear. In this study, it was examined in vitro whether the NSAID indomethacin exhibited an inhibitory effect on carbachol-induced contractions of the porcine detrusor muscle. Additionally, the inhibitory effect of the phospholipase-C inhibitor U-73122 on carbachol-induced contractions of the porcine detrusor muscle was investigated. Experiments were performed on the muscle strips of the porcine detrusor muscle suspended in a tissue bath. Effects of indomethacin at 10(-6) and 10(-5) M on the maximum carbachol-induced contraction and on the carbachol-response curve were investigated. Additionally, the inhibitory influence of U-73122 at a concentration of 10(-5.5) M on the carbachol-response curve was investigated. Pretreatment with indomethacin at both concentrations did not result in a significant reduction in the maximum contraction compared with the control. In the experiments in which carbachol concentration-response curves were generated, indomethacin exhibited at both concentrations a very small but significant change at carbachol concentrations of 10(-8) and 10(-7.5) M. In the experiments with U-73122, a significant change was found in the concentration-response curve of carbachol at all concentrations of carbachol from 10(-6.5) to 10(-4) M. The mean maximum carbachol-induced contraction was 141.8 +/- 6.8% after incubation with U-73122 and 166.0 +/- 6.4% in the control group (P < 0.05). Indomethacin did not inhibit the carbachol-induced contractions of the porcine detrusor muscle. The cyclooxygenase does not play a significant role in the carbachol-induced bladder contraction of the porcine detrusor muscle. The inhibitory action of the phospholipase-C inhibitor U-73122 on the carbachol-induced contraction was significant, but small. The results point to an inferior role of this pathway.

Synthesis and characterization of a novel eco-friendly corrosion inhibition for mild steel in 1 M hydrochloric acid

NASA Astrophysics Data System (ADS)

Al-Amiery, Ahmed A.; Binti Kassim, Fatin A.; Kadhum, Abdul Amir H.; Mohamad, Abu Bakar

2016-01-01

The acid corrosion inhibition process of mild steel in 1 M HCl by azelaic acid dihydrazide has been investigated using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, open circuit potential (OCP) and electrochemical frequency modulation (EFM). Azelaic acid dihydrazide was synthesized, and its chemical structure was elucidated and confirmed using spectroscopic techniques (infrared, nuclear magnetic resonance and mass spectroscopy). Potentiodynamic polarization studies indicate that azelaic acid dihydrazide is a mixed-type inhibitor. The inhibition efficiency increases with increased inhibitor concentration and reaches its maximum of 93% at 5 × 10-3 M. The adsorption of the inhibitor on a mild steel surface obeys Langmuir’s adsorption isotherm. The effect of temperature on corrosion behavior in the presence of 5 × 10-3 M inhibitor was studied in the temperature range of 30-60 °C. The results indicated that inhibition efficiencies were enhanced with an increase in concentration of inhibitor and decreased with a rise in temperature. To inspect the surface morphology of inhibitor film on the mild steel surface, scanning electron microscopy (SEM) was used before and after immersion in 1.0 M HCl.

Synthesis and characterization of a novel eco-friendly corrosion inhibition for mild steel in 1 M hydrochloric acid

PubMed Central

Al-Amiery, Ahmed A.; Binti Kassim, Fatin A.; Kadhum, Abdul Amir H.; Mohamad, Abu Bakar

2016-01-01

The acid corrosion inhibition process of mild steel in 1 M HCl by azelaic acid dihydrazide has been investigated using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, open circuit potential (OCP) and electrochemical frequency modulation (EFM). Azelaic acid dihydrazide was synthesized, and its chemical structure was elucidated and confirmed using spectroscopic techniques (infrared, nuclear magnetic resonance and mass spectroscopy). Potentiodynamic polarization studies indicate that azelaic acid dihydrazide is a mixed-type inhibitor. The inhibition efficiency increases with increased inhibitor concentration and reaches its maximum of 93% at 5 × 10−3 M. The adsorption of the inhibitor on a mild steel surface obeys Langmuir’s adsorption isotherm. The effect of temperature on corrosion behavior in the presence of 5 × 10−3 M inhibitor was studied in the temperature range of 30–60 °C. The results indicated that inhibition efficiencies were enhanced with an increase in concentration of inhibitor and decreased with a rise in temperature. To inspect the surface morphology of inhibitor film on the mild steel surface, scanning electron microscopy (SEM) was used before and after immersion in 1.0 M HCl. PMID:26795066

Synthesis and characterization of a novel eco-friendly corrosion inhibition for mild steel in 1 M hydrochloric acid.

PubMed

Al-Amiery, Ahmed A; Binti Kassim, Fatin A; Kadhum, Abdul Amir H; Mohamad, Abu Bakar

2016-01-22

The acid corrosion inhibition process of mild steel in 1 M HCl by azelaic acid dihydrazide has been investigated using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, open circuit potential (OCP) and electrochemical frequency modulation (EFM). Azelaic acid dihydrazide was synthesized, and its chemical structure was elucidated and confirmed using spectroscopic techniques (infrared, nuclear magnetic resonance and mass spectroscopy). Potentiodynamic polarization studies indicate that azelaic acid dihydrazide is a mixed-type inhibitor. The inhibition efficiency increases with increased inhibitor concentration and reaches its maximum of 93% at 5 × 10(-3) M. The adsorption of the inhibitor on a mild steel surface obeys Langmuir's adsorption isotherm. The effect of te perature on corrosion behavior in the presence of 5 × 10(-3) M inhibitor was studied in the temperature range of 30-60 °C. The results indicated that inhibition efficiencies were enhanced with an increase in concentration of inhibitor and decreased with a rise in temperature. To inspect the surface morphology of inhibitor film on the mild steel surface, scanning electron microscopy (SEM) was used before and after immersion in 1.0 M HCl.

Multiple paths of electron flow to current in microbial electrolysis cells fed with low and high concentrations of propionate.

PubMed

Hari, Ananda Rao; Katuri, Krishna P; Gorron, Eduardo; Logan, Bruce E; Saikaly, Pascal E

2016-07-01

Microbial electrolysis cells (MECs) provide a viable approach for bioenergy generation from fermentable substrates such as propionate. However, the paths of electron flow during propionate oxidation in the anode of MECs are unknown. Here, the paths of electron flow involved in propionate oxidation in the anode of two-chambered MECs were examined at low (4.5 mM) and high (36 mM) propionate concentrations. Electron mass balances and microbial community analysis revealed that multiple paths of electron flow (via acetate/H2 or acetate/formate) to current could occur simultaneously during propionate oxidation regardless of the concentration tested. Current (57-96 %) was the largest electron sink and methane (0-2.3 %) production was relatively unimportant at both concentrations based on electron balances. At a low propionate concentration, reactors supplemented with 2-bromoethanesulfonate had slightly higher coulombic efficiencies than reactors lacking this methanogenesis inhibitor. However, an opposite trend was observed at high propionate concentration, where reactors supplemented with 2-bromoethanesulfonate had a lower coulombic efficiency and there was a greater percentage of electron loss (23.5 %) to undefined sinks compared to reactors without 2-bromoethanesulfonate (11.2 %). Propionate removal efficiencies were 98 % (low propionate concentration) and 78 % (high propionate concentration). Analysis of 16S rRNA gene pyrosequencing revealed the dominance of sequences most similar to Geobacter sulfurreducens PCA and G. sulfurreducens subsp. ethanolicus. Collectively, these results provide new insights on the paths of electron flow during propionate oxidation in the anode of MECs fed with low and high propionate concentrations.

Kinetic and thermodynamic studies of AISI 4130 steel alloy corrosion in ethylene glycol-water mixture in presence of inhibitors

NASA Astrophysics Data System (ADS)

Khomami, M. Niknejad; Danaee, I.; Attar, A. A.; Peykari, M.

2013-05-01

The electrochemical behavior of steel alloy in ethylene glycol-water mixture was investigated by electrochemical methods. The results obtained showed that corrosion rate was decreased with increasing ethylene glycol concentration. The effect of inorganic inhibitors including NO3 -, NO2 -, Cr2O7 2- and CrO4 2- were studied using electrochemical techniques where the highest inhibition efficiency was obtained for CrO4 2-. In the presence of chromate the inhibitor efficiency increased with its concentration. The inhibiting effect of the chromate was explained on the basis of the competitive adsorption between the inorganic anions and the aggressive Cl- ions, and the adsorption isotherm basically obeys the Langmuir adsorption isotherm. Thermodynamic parameters for steel corrosion and inhibitor adsorption were determined and reveal that the adsorption process is spontaneous. Also, a phenomenon of both physical and chemical adsorption is proposed.

Germ Tube Growth Inhibitor from Cronartium comandrae Aeciospores

PubMed Central

Eppstein, Deborah A.; Tainter, F. H.

1979-01-01

Two compounds showing self-inhibitory action during germination of aeciospores of the comandra blister rust fungus (Cronartium comandrae Pk.) were extracted from these aeciospores by shaking with 0.2 M NH4HCO3 (pH 7.8) for 4 h. One of these, the germination self inhibitor (D. A. Eppstein and F. H. Tainter, Phytopathology 66:1395-1397, 1976), was removed from the ammonium bicarbonate buffer by using chloroform. The water layer which remained contained a substance which, at ca. 10−4 M concentration, had no apparent effect on germ tube emergence but which inhibited normal germ tube growth. Linear germ tube growth ceased or a dendritic or vesicular pattern of growth resulted, depending on the concentration of inhibitor added to extracted germinating spores. The germ tube growth inhibitor appears to be a peptide with a molecular weight of ca. 2,000. Images PMID:16345335

Cholinergic symptoms with low serum cholinesterase from therapeutic cholinesterase inhibitor toxicity.

PubMed

Leikin, Jerrold B; Braund, Victoria; DesLauris, Carol

2014-07-01

Although cholinesterase inhibitors have been frequently used in the treatment of Alzheimer disease, its effects on serum cholinesterase concentrations have been rarely described. We described significant depression of serum cholinesterase levels due to cholinesterase inhibitor toxicity from redundant use of donepezil and rivastigmine in a 78-year-old man. Recovery of serum cholinesterase level was noted upon drug discontinuation and cholinergic symptom resolution. Serum cholinesterase level can be used as a biomarker for central cholinesterase inhibitor toxicity.

Discovery of Potent Benzocycloalkane Derived Diapophytoene Desaturase Inhibitors with an Enhanced Safety Profile for the Treatment of MRSA, VISA, and LRSA Infections.

PubMed

Li, Baoli; Ni, Shuaishuai; Chen, Feifei; Mao, Fei; Wei, Hanwen; Liu, Yifu; Zhu, Jin; Lan, Lefu; Li, Jian

2018-03-09

Blocking the biosynthesis process of staphyloxanthin has emerged as a promising antivirulence strategy. Our previous research revealed that diapophytoene desaturase was an attractive and druggable target against infections caused by pigmented Staphylococcus aureus. Benzocycloalkane-derived compounds were effective inhibitors of diapophytoene desaturase but limited by high hERG (human Ether-a-go-go Related Gene) inhibition activity. Here, we identified a new type of benzo-hepta-containing cycloalkane derivative as diapophytoene desaturase inhibitors. Among the fifty-eight analogues, 48 (hERG inhibition activity, half maximal inhibitory concentration, IC 50 , of 16.1 μM) and 51 (hERG inhibition activity, IC 50 > 40 μM) were distinguished for effectively inhibiting the pigment production of Staphylococcus aureus Newman and three methicillin-resistant Staphylococcus aureus strains, and the four strains were highly sensitize to hydrogen peroxide killing without a bactericidal growth effect. In an in vivo assay, 48 and 51 displayed a comparable effect with linezolid and vancomycin in livers and hearts in mice against Staphylococcus aureus Newman and a more considerable effect against Mu50 and NRS271 with normal administration.

Fragment-based lead generation: identification of seed fragments by a highly efficient fragment screening technology

NASA Astrophysics Data System (ADS)

Neumann, Lars; Ritscher, Allegra; Müller, Gerhard; Hafenbradl, Doris

2009-08-01

For the detection of the precise and unambiguous binding of fragments to a specific binding site on the target protein, we have developed a novel reporter displacement binding assay technology. The application of this technology for the fragment screening as well as the fragment evolution process with a specific modelling based design strategy is demonstrated for inhibitors of the protein kinase p38alpha. In a fragment screening approach seed fragments were identified which were then used to build compounds from the deep-pocket towards the hinge binding area of the protein kinase p38alpha based on a modelling approach. BIRB796 was used as a blueprint for the alignment of the fragments. The fragment evolution of these deep-pocket binding fragments towards the fully optimized inhibitor BIRB796 included the modulation of the residence time as well as the affinity. The goal of our study was to evaluate the robustness and efficiency of our novel fragment screening technology at high fragment concentrations, compare the screening data with biochemical activity data and to demonstrate the evolution of the hit fragments with fast kinetics, into slow kinetic inhibitors in an in silico approach.

Coexisting bacterial populations responsible for multiphasic mineralization kinetics in soil. [Janthinobacterium sp. Rhodococcus sp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidt, S.K.; Gier, M.J.

1990-09-01

Experiments were conducted to study populations of indigenous microorganisms capable of mineralizing 2,4-dinitrophenol (DNP) in two soils. Previous kinetic analyses indicated the presence of two coexisting populations of DNP-mineralizing microorganisms in a forest soil (soil 1). Studies in which eucaryotic and procaryotic inhibitors were added to this soil indicated that both populations were bacterial. Most-probable-number counts with media containing different concentrations of DNP indicated that more bacteria could mineralize low concentrations of DNP than could metabolize high concentrations of it. Enrichments with varying concentrations of DNP and various combinations of inhibitors consistently resulted in the isolation of the same twomore » species of bacteria from soil 1. This soil contained a large number and variety of fungi, but no fungi capable of mineralizing DNP were isolated. The two bacterial isolates were identified as a Janthinobacterium sp. and a Rhodococcus sp. The Janthinobacterium sp. had a low {mu}{sub max} and a low K{sub m} for DNP mineralization, whereas the Rhodococcus sp. had much higher values for both parameters. These differences between the two species of bacteria were similar to differences seen when soil was incubated with different concentrations of DNP. Values for {mu}{sub max} from soil incubations were similar to {mu}{sub max} values obtained in pure culture studies. In contrast, K{sub s} and K{sub m} values showed greater variation between soil and pure culture studies.« less

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 II: production of xylitol and ethanol in the presence of inhibitors.

PubMed

Vajzovic, Azra; Bura, Renata; Kohlmeier, Kevin; Doty, Sharon L

2012-10-01

A systematic study was conducted characterizing the effect of furfural, 5-hydroxymethylfurfural (5-HMF), and acetic acid concentration on the production of xylitol and ethanol by a novel endophytic yeast, Rhodotorula mucilaginosa strain PTD3. The influence of different inhibitor concentrations on the growth and fermentation abilities of PTD3 cultivated in synthetic nutrient media containing 30 g/l xylose or glucose were measured during liquid batch cultures. Concentrations of up to 5 g/l of furfural stimulated production of xylitol to 77 % of theoretical yield (10 % higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in the presence of 5-HMF at concentrations of up to 3 g/l. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99 % of theoretical yield (15 % higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5 g/l of furfural, 5-HMF, and acetic acid, respectively. This yeast would be a potent candidate for the bioconversion of lignocellulosic sugars to biochemicals given that in the presence of low concentrations of inhibitors, its xylitol and ethanol yields are stimulated, and it is capable of metabolizing pretreatment degradation products.

Voriconazole pharmacokinetic variability in cystic fibrosis lung transplant patients.

PubMed

Berge, M; Guillemain, R; Boussaud, V; Pham, M-H; Chevalier, P; Batisse, A; Amrein, C; Dannaoui, E; Loriot, M-A; Lillo-Le Louet, A; Billaud, E M

2009-06-01

Aspergillosis is a high-risk complication in cystic fibrosis (CF) lung transplant patients. Azole antifungal drugs inhibit CYP3A4, resulting in significant metabolic drug-drug interactions. Voriconazole (VRZ) was marketed without therapeutic drug monitoring (TDM) recommendations, consistent with favorable pharmacokinetics, but regular determinations of plasma VRZ concentration were introduced in our center to manage interactions with calcineurin inhibitors and to document the achievement of therapeutic levels. VRZ TDM data analysis for trough concentration (C0) and peak concentration (C2) was carried out, using validated liquid chromatography assay with ultraviolet detection, for 35 CF lung transplant patients (mean age 25 years, mean weight 47 kg, balanced sex ratio) since 2003. Therapeutic range (C0: 1.5 +/- 0.5 - C2 : 4.0 +/- 1.0 mg/L) was expressed relative to pivotal pharmacokinetic trial data. The duration of VRZ treatment ranged from 9 days to 22 months. The recommended standard dose of VRZ (200 mg twice a day, following the loading dose) resulted in significant plasma concentrations (>0.5 mg/L) in 20% of CF lung transplant patients. Therapeutic concentrations were obtained using higher doses (average 570 +/- 160 mg/day, +43%, P<0.01). Despite adaptation, C0 remained <0.5 mg/L (11%), even when the drug was administered intravenously, highlighting the variability of VRZ pharmacokinetics, possibly enhanced by CYP2C19 polymorphism. The risk of inefficacy during periods of underdosage was overcome by treatment with antifungal drug combinations (caspofungin, n=10). The therapeutic index was limited by neurologic effects (14%) and hepatic abnormalities (30%). VRZ concentrations correlated significantly (P<0.01) with aspartate aminotransferase levels but not with bilirubin levels. VRZ acted as a metabolic inhibitor of tacrolimus (C0 to dose ratio 5.8 +/- 2.6, n=31/VRZ versus 1.7 +/- 0.9 alone, P<0.001). Large changes in azole concentration affected the magnitude of the drug-drug interactions and adjustment requirements. TDM is required because VRZ levels are often undetectable in treated CF lung transplant patients, supporting the use of antifungal drug combinations until achievement of VRZ C0 at a steady state between 1 and 2 mg/L. Plasma VRZ concentrations should be determined for the quantitative, individualized management of drug-drug interactions in lung transplant patients, in particular immunosuppressant such as tacrolimus, considering VRZ to be both a target and an inhibitor of CYP3A4.

Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties.

PubMed

De Vita, Daniela; Angeli, Andrea; Pandolfi, Fabiana; Bortolami, Martina; Costi, Roberta; Di Santo, Roberto; Suffredini, Elisabetta; Ceruso, Mariangela; Del Prete, Sonia; Capasso, Clemente; Scipione, Luigi; Supuran, Claudiu T

2017-12-01

We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.

Anaerobic digestion of aircraft deicing fluid wastes: interactions and toxicity of corrosion inhibitors and surfactants.

PubMed

Gruden, Cyndee L; Hernandez, Mark

2002-01-01

Corrosion inhibitors and surfactants are present in aircraft deicing fluids (ADFs) at significant concentrations (> 1% w/w). The purpose of this research was to study the interactions of a common nonionic surfactant with the commercially significant corrosion inhibitors used in modern ADF (4- and 5-methylbenzotriazole [MeBT]), and to determine the effects of their mixture on the conventional anaerobic digestion process. In mesophilic anaerobic microcosms codigesting wastewater solids, propylene glycol, and MeBT, increasing surfactant levels resulted in enhanced MeBT sorption on digester solids. As judged by anaerobic toxicity assays, responses from digesters containing surfactant concentrations below their critical micelle concentration (CMC) suggested that low nonionic surfactant concentrations could facilitate a reduction in the apparent toxicity of MeBT. In microcosms exposed to surfactant concentrations above their CMC, no increase in MeBT solubility was observed, and the anaerobic toxicity response corresponded to control systems not containing surfactant. Direct microscopic measurements of digesting biomass using fluorescent phylogenetic probes (fluorescent in situ hybridization) revealed that members of the domain Bacteria were more sensitive to MeBT in the presence of surfactant than were members of the domain Archaea.

A simple physical mechanism enables homeostasis in primitive cells

NASA Astrophysics Data System (ADS)

Engelhart, Aaron E.; Adamala, Katarzyna P.; Szostak, Jack W.

2016-05-01

The emergence of homeostatic mechanisms that enable maintenance of an intracellular steady state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles, and ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation. This simple physical system enables a primitive homeostatic behaviour: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest that such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.

Detection of antibiotics in goat's milk: effect of detergents on the response of microbial inhibitor tests.

PubMed

Romero, Tamara; Beltrán, María Carmen; Althaus, Rafael Lisandro; Molina, María Pilar

2014-08-01

The aim of the study was to evaluate the interference of acid and alkaline detergents employed in the cleaning of milking equipment of caprine dairy farms on the performance of microbial tests used in antibiotic control (BRT MRL, Delvotest MCS, and Eclipse 100). Eight concentrations of commercial detergents, five acid (0-0.25%) and five alkaline (0-1%) were add to antimicrobial-free goat's milk to evaluate the detergent effect on the response of microbial inhibitor tests. To evaluate the effect of detergents on the detection capability of microbial tests two detergents at 0.5 ml/l (one acid and one basic) and eight concentrations of four β-lactam antibiotics (ampicillin, amoxicillin, cloxacillin and benzylpenicillin) were used. Milk without detergents was used as control. The spiked samples were analysed twelve times by three microbial tests. The results showed that the presence of acid detergents did not affect the response of microbial tests for any of the concentrations tested. However, at concentrations equal to or greater than 2 ml/l alkaline detergents positive results were found in microbial tests (16.7-100%). The detection limits of the screening tests for penicillins were not modified substantially by the presence of detergents. In general, the presence of acid and alkaline detergents in goat's milk did not produce a great interference in the microbial tests, only high concentrations of detergents could cause non-compliant results, but these concentrations are difficult to find in practice if proper cleaning procedures are applied in goat dairy farms.

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat.

PubMed

Algeelani, Sara; Alam, Novera; Hossain, Md Amin; Mikus, Gerd; Greenblatt, David J

2018-08-01

1. Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer's data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat. 2. Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors. 3. The 50% inhibitory concentrations (IC 50 ) of cobicistat and ritonavir were at least 50 µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC 50 0.7 mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC 50 17 µM). 4. Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.

Postauthorization safety surveillance of ADVATE [antihaemophilic factor (recombinant), plasma/albumin-free method] demonstrates efficacy, safety and low-risk for immunogenicity in routine clinical practice.

PubMed

Oldenburg, J; Goudemand, J; Valentino, L; Richards, M; Luu, H; Kriukov, A; Gajek, H; Spotts, G; Ewenstein, B

2010-11-01

  Postauthorization safety surveillance of factor VIII (FVIII) concentrates is essential for assessing rare adverse event incidence. We determined safety and efficacy of ADVATE [antihaemophilic factor (recombinant), plasma/albumin-free method, (rAHF-PFM)] during routine clinical practice. Subjects with differing haemophilia A severities and medical histories were monitored during 12 months of prophylactic and/or on-demand therapy. Among 408 evaluable subjects, 386 (95%) received excellent/good efficacy ratings for all on-demand assessments; the corresponding number for subjects with previous FVIII inhibitors was 36/41 (88%). Among 276 evaluable subjects receiving prophylaxis continuously in the study, 255 (92%) had excellent/good ratings for all prophylactic assessments; the corresponding number for subjects with previous FVIII inhibitors was 41/46 (89%). Efficacy of surgical prophylaxis was excellent/good in 16/16 evaluable procedures. Among previously treated patients (PTPs) with >50 exposure days (EDs) and FVIII≤2%, three (0.75%) developed low-titre inhibitors. Two of these subjects had a positive inhibitor history; thus, the incidence of de novo inhibitor formation in PTPs with FVIII≤2% and no inhibitor history was 1/348 (0.29%; 95% CI, 0.01-1.59%). A PTP with moderate haemophilia developed a low-titre inhibitor. High-titre inhibitors were reported in a PTP with mild disease (following surgery), a previously untreated patient (PUP) with moderate disease (following surgery) and a PUP with severe disease. The favourable benefit/risk profile of rAHF-PFM previously documented in prospective clinical trials has been extended to include a broader range of haemophilia patients, many of whom would have been ineligible for registration studies. © 2010 Blackwell Publishing Ltd.

The -675 4G/5G polymorphism at the Plasminogen Activator Inhibitor 1 (PAI-1) gene modulates plasma Plasminogen Activator Inhibitor 1 concentrations in response to dietary fat consumption.

PubMed

Pérez-Martínez, P; Adarraga-Cansino, M D; Fernández de la Puebla, R A; Blanco-Molina, A; Delgado-Lista, J; Marín, C; Ordovás, J M; López-Miranda, J; Pérez-Jiménez, F

2008-04-01

The objective of the study was to determine whether Plasminogen Activator Inhibitor Type 1 (PAI-1) -675 4G/5G polymorphism is associated with the response of functional plasma PAI-1 concentrations to changes in the amount and quality of dietary fat in healthy subjects. PAI-1 is the major inhibitor of fibrinolysis, and a lower level of fibrinolytic activity could be implicated in an increased risk of IHD. Fifty-nine healthy Spanish volunteers (ten 4G/4G homozygotes, twenty-eight heterozygotes 4G/5G and twenty-one 5G/5G homozygotes) consumed three diets for periods of 4 weeks each: a SFA-rich diet (38 % fat, 20 % SFA), followed by a carbohydrate-rich diet (30 % fat, 55 % carbohydrate) and a MUFA-rich diet (38 % fat, 22 % MUFA) according to a randomized crossover design. At the end of each dietary period plasma lipid and functional plasma PAI-1 concentrations were determined. Subjects carrying the 4G allele (4G/4G and 4G/5G) showed a significant decrease in PAI-1 concentrations after the MUFA diet, compared with the SFA-rich and carbohydrate-rich diets (genotype x diet interaction: P = 0.028). 5G/5G homozygotes had the lowest plasma PAI-1 concentrations compared with 4G/4G and 4G/5G subjects (genotype: P = 0.002), without any changes as a result of the amount and the quality of the dietary fat. In summary, no differences in plasma PAI-1 concentration response were found after changes in dietary fat intake in 5G/5G homozygotes, although these subjects displayed the lowest concentrations of PAI-1. On the other hand, carriers of the 4G allele are more likely to hyper-respond to the presence of MUFA in the diet because of a greater decrease in PAI-1 concentrations.

Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea

PubMed Central

Prinsen, Michael J.; Oliva, Jonathan; Campbell, Mary A.; Arnett, Stacy D.; Tajfirouz, Deena; Ruminski, Peter G.; Yu, Ying; Bond, Brian R.; Ji, Yuhua; Neckermann, Georg; Choy, Robert K. M.; de Hostos, Eugenio; Meyers, Marvin J.

2016-01-01

Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil–treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy. PMID:26907621

High Glucose Concentrations Suppress the Proliferation of Human Periodontal Ligament Stem Cells and Their Differentiation Into Osteoblasts.

PubMed

Kato, Hirohito; Taguchi, Yoichiro; Tominaga, Kazuya; Kimura, Daisuke; Yamawaki, Isao; Noguchi, Masahiro; Yamauchi, Nobuhiro; Tamura, Isao; Tanaka, Akio; Umeda, Makoto

2016-04-01

Diabetes mellitus (DM) is a major risk factor for periodontal disease and affects various cellular functions. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration; however, the effect of hyperglycemia on PDLSCs is unclear. The aim of this study is to investigate whether hyperglycemia affects periodontal tissue regeneration, using human PDLSCs and high-glucose medium as a model of DM. PDLSCs were obtained from healthy adult human mandibular third molars. Cell proliferation, osteoblastic differentiation, and proinflammatory cytokine expression were investigated by culturing PDLSCs in media supplemented with four different glucose concentrations representative of control patients (5.5 mM), patients with postprandial or controlled DM (8.0 mM), and patients with uncontrolled DM (12.0 and 24.0 mM). The molecular effects of hyperglycemia on PDLSC physiology were examined with a focus on the nuclear factor (NF)-(κB signaling pathway. The involvement of NF-κB was investigated with a specific NF-κB inhibitor in PDLSCs under hyperglycemic conditions. High glucose levels inhibited PDLSC proliferation and differentiation into osteoblasts but induced NF-κB activation and subsequent interleukin (IL)-6 and IL-8 expression. Treatment with an NF-κB inhibitor rescued the defects in cell proliferation and osteoblastic differentiation and inhibited the IL-6 expression caused by the high-glucose environment. The results of this study demonstrate that hyperglycemia inhibits human PDLSC proliferation and osteoblastic differentiation.

Treatment of Plants with Gaseous Ethylene and Gaseous Inhibitors of Ethylene Action.

PubMed

Tucker, Mark L; Kim, Joonyup; Wen, Chi-Kuang

2017-01-01

The gaseous nature of ethylene affects not only its role in plant biology but also how you treat plants with the hormone. In many ways, it simplifies the treatment problem. Other hormones have to be made up in solution and applied to some part of the plant hoping the hormone will be taken up into the plant and translocated throughout the plant at the desired concentration. Because all plant cells are connected by an intercellular gas space the ethylene concentration you treat with is relatively quickly reached throughout the plant. In some instances, like mature fruit, treatment with ethylene initiates autocatalytic synthesis of ethylene. However, in most experiments, the exogenous ethylene concentration is saturating, usually >1 μL L -1 , and the synthesis of additional ethylene is inconsequential. Also facilitating ethylene research compared with other hormones is that there are inhibitors of ethylene action 1-MCP (1-methylcyclopropene) and 2,5-NBD (2,5-norbornadiene) that are also gases wherein you can achieve nearly 100% inhibition of ethylene action quickly and with few side effects. Inhibitors for other plant hormones are applied as a solution and their transport and concentration at the desired site is not always known and difficult to measure. Here, our focus is on how to treat plants and plant parts with the ethylene gas and the gaseous inhibitors of ethylene action.

The Na+/H+ exchange inhibitor cariporide is washed out of the myocardium by crystalloid cardioplegia.

PubMed

Bechtel, J F M; Eichler, W; Toerber, K; Weidtmann, B; Hernandez, M; Klotz, K F; Sievers, H H; Bartels, C

2006-08-01

Inhibition of the Na (+)/H (+) exchanger (NHE) is cardioprotective, but dosage and timing of NHE-inhibitors are critical for their efficacy. We studied the effect of a new dosing regime of the NHE-inhibitor cariporide on myocardial function and damage after cardioplegic arrest (CPA) and determined its myocardial and serum concentrations. 3 pigs received a bolus of 180 mg cariporide intravenously (i. v.) and were sacrificed shortly thereafter to allow measurement of the myocardial concentrations of cariporide. Subsequently, 10 pigs were randomized to receive either i. v. cariporide (bolus followed by an infusion of 40 mg/h) or placebo. Cardiopulmonary bypass was initiated, and the heart was arrested for 60 minutes by infusion of St. Thomas Hospital solution. Left ventricular (LV) function was studied using microsonometry. Myocardial damage was assessed by troponin T. Serum concentrations of cariporide were measured throughout the study, and myocardial concentrations were measured before the end of CPA and 180 minutes thereafter. Cariporide was present in all myocardial specimens (median: 1.4 ng/mg) studied previously. In the main study, LV function or myocardial damage did not differ significantly between the groups at any time point. Stable serum cariporide concentrations were achieved (3.4 +/- 0.5 microg/ml). Cariporide was detectable in only one of the myocardial biopsies obtained before the end of CPA, but 180 minutes thereafter, the myocardial cariporide concentration was 2.5 +/- 0.3 ng/mg. We observed no effect of i. v. cariporide on LV function or myocardial damage after cardioplegic arrest. Our data suggest that cariporide is washed out of the myocardium by repeated application of crystalloid cardioplegia. Thus, the mode of delivery also appears to be critical for cardioprotection with NHE-inhibitors.

Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

PubMed

Arnadottir, M; Nilsson-Ehle, P

1994-01-01

The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions.

Edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia injury: heme oxygenase-1 and PI3K/Akt pathway may be involved.

PubMed

Cao, Huifang; Feng, Ying; Ning, Yunye; Zhang, Zinan; Li, Weihao; Li, Qiang

2015-01-01

Hyperoxic acute lung injury (HALI) is a clinical syndrome as a result of prolonged supplement of high concentrations of oxygen. As yet, no specific treatment is available for HALI. The present study aims to investigate the effects of edaravone on hyperoxia-induced oxidative injury and the underlying mechanism. We treated rats and human pulmonary alveolar epithelial cells with hyperoxia and different concentration of edaravone, then examined the effects of edaravone on cell viability, cell injury and two oxidative products. The roles of heme oxygenase-1 (HO-1) and PI3K/Akt pathway were explored using Western blot and corresponding inhibitors. The results showed that edaravone reduced lung biochemical alterations induced by hyperoxia and mortality of rats, dose-dependently alleviated cell mortality, cell injury, and peroxidation of cellular lipid and DNA oxidative damage. It upregulated cellular HO-1 expression and activity, which was reversed by PI3K/Akt pathway inhibition. The administration of zinc protoporphyrin-IX, a HO-1 inhibitor, and LY249002, a PI3K/Akt pathway inhibitor, abolished the protective effects of edaravone in cells. This study indicates that edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1, which is regulated by PI3K/Akt pathway.

Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland.

PubMed

Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Borgatti, Anna Rosa; Pagliarani, Alessandra

2011-02-01

Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC(50)=0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F(0), either in the form of free enzyme or of enzyme-substrate complex (Ki=K'i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg(2+) cofactor, may bind strongly to F(1) subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation. Copyright © 2010 Elsevier Ltd. All rights reserved.

New coumarin derivative as an eco-friendly inhibitor of corrosion of mild steel in Acid medium.

PubMed

Al-Amiery, Ahmed A; Al-Majedy, Yasameen K; Kadhum, Abdul Amir H; Mohamad, Abu Bakar

2014-12-29

The anticorrosion ability of a synthesized coumarin, namely 2-(coumarin-4-yloxy)acetohydrazide (EFCI), for mild steel (MS) in 1 M hydrochloric acid solution has been studied using a weight loss method. The effect of temperature on the corrosion rate was investigated, and some thermodynamic parameters were calculated. The results indicated that inhibition efficiencies were enhanced with an increase in concentration of inhibitor and decreased with a rise in temperature. The IE value reaches 94.7% at the highest used concentration of the new eco-friendly inhibitor. The adsorption of inhibitor on MS surface was found to obey a Langmuir adsorption isotherm. Scanning electron microscopy (SEM) was performed on inhibited and uninhibited mild steel samples to characterize the surface. The Density Function theory (DFT) was employed for quantum-chemical calculations such as EHOMO (highest occupied molecular orbital energy), ELUMO (lowest unoccupied molecular orbital energy) and μ (dipole moment), and the obtained results were found to be consistent with the experimental findings. The synthesized inhibitor was characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic studies.

Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

PubMed

Bilgin, M; Burgazli, K M; Rafiq, A; Mericliler, M; Neuhof, C; Oliva, M L; Parahuleva, M; Soydan, N; Doerr, O; Abdallah, Y; Erdogan, A

2014-01-01

Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

Acute Ethanol Inhibition of γ Oscillations Is Mediated by Akt and GSK3β

PubMed Central

Wang, JianGang; Zhao, JingXi; Liu, ZhiHua; Guo, FangLi; Wang, Yali; Wang, Xiaofang; Zhang, RuiLing; Vreugdenhil, Martin; Lu, Chengbiao

2016-01-01

Hippocampal network oscillations at gamma band frequency (γ, 30–80 Hz) are closely associated with higher brain functions such as learning and memory. Acute ethanol exposure at intoxicating concentrations (≥50 mM) impairs cognitive function. This study aimed to determine the effects and the mechanisms of acute ethanol exposure on γ oscillations in an in vitro model. Ethanol (25–100 mM) suppressed kainate-induced γ oscillations in CA3 area of the rat hippocampal slices, in a concentration-dependent, reversible manner. The ethanol-induced suppression was reduced by the D1R antagonist SCH23390 or the PKA inhibitor H89, was prevented by the Akt inhibitor triciribine or the GSk3β inhibitor SB415286, was enhanced by the NMDA receptor antagonist D-AP5, but was not affected by the MAPK inhibitor U0126 or PI3K inhibitor wortmanin. Our results indicate that the intracellular kinases Akt and GSk3β play a critical role in the ethanol-induced suppression of γ oscillations and reveal new cellular pathways involved in the ethanol-induced cognitive impairment. PMID:27582689

A combined cell based approach to identify P-glycoprotein substrates and inhibitors in a single assay.

PubMed

Balimane, Praveen V; Chong, Saeho

2005-09-14

The objective of this project was to develop a cell based in vitro experimental procedure that can differentiate P-glycoprotein (P-gp) substrates from inhibitors in a single assay. Caco-2 cells grown to confluency on 12-well Transwell were used for this study. The efflux permeability (B to A) of P-gp specific probe (viz., digoxin) in the presence of test compounds (e.g. substrates, inhibitors and non-substrates of P-gp) was monitored, and the influx permeability (A to B) of test compounds was evaluated after complete P-gp blockade. Radiolabelled digoxin was added on the basolateral side with buffer on the apical side. The digoxin concentration appearing on the apical side represents digoxin efflux permeability during the control phase (0-1 h period). After 1 h, a test compound (10 microM) was added on the apical side. The reduced efflux permeability of digoxin suggests that the added test compound is an inhibitor. The influx permeability of test compound is also determined during the 1-2 h study period by measuring the concentration of the test compound in the basolateral side. At the end of 2 h, a potent P-gp inhibitor (GF120918) was added. The increased influx permeability of test compound during the 2-3 h incubation period indicates that the added test compound is a substrate. Samples were taken from both sides at the end of 1-3 h and the concentrations of the test compounds and digoxin were quantitated. Digoxin efflux permeability remained unchanged when incubated with P-gp substrates (e.g., etoposide, rhodamine123, taxol). However, when a P-gp inhibitor was added to the apical side, the digoxin efflux (B to A permeability) was significantly reduced (ketoconazole=51% reduction) as expected. The influx permeability of substrates increased significantly (rhodamine123=70%, taxol=220%, digoxin=290%) after the P-gp inhibitor (GF120918) was introduced, whereas the influx permeability of P-gp inhibitor and non-substrates was not affected by GF120918. Thus, this combined assay provides an efficient cell based in vitro screening tool to simultaneously distinguish compounds that are P-gp substrates from P-gp inhibitors.

Tripeptidyl peptidase II promotes maturation of caspase-1 in Shigella flexneri-induced macrophage apoptosis.

PubMed

Hilbi, H; Puro, R J; Zychlinsky, A

2000-10-01

The invasive enteropathogenic bacterium Shigella flexneri activates apoptosis in macrophages. Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered by Shigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) and clasto-lactacystin beta-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages from Shigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blocked Shigella-induced apoptosis by 95% at a concentration of 1 microM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect on Shigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1beta in Shigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability of Shigella to escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways.

Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 in Shigella flexneri-Induced Macrophage Apoptosis

PubMed Central

Hilbi, Hubert; Puro, Robyn J.; Zychlinsky, Arturo

2000-01-01

The invasive enteropathogenic bacterium Shigella flexneri activates apoptosis in macrophages. Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered by Shigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) and clasto-lactacystin β-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages from Shigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blocked Shigella-induced apoptosis by 95% at a concentration of 1 μM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect on Shigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1β in Shigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability of Shigella to escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways. PMID:10992446

Metallochaperone UreG serves as a new target for design of urease inhibitor: A novel strategy for development of antimicrobials.

PubMed

Yang, Xinming; Koohi-Moghadam, Mohamad; Wang, Runming; Chang, Yuen-Yan; Woo, Patrick C Y; Wang, Junwen; Li, Hongyan; Sun, Hongzhe

2018-01-01

Urease as a potential target of antimicrobial drugs has received considerable attention given its versatile roles in microbial infection. Development of effective urease inhibitors, however, is a significant challenge due to the deeply buried active site and highly specific substrate of a bacterial urease. Conventionally, urease inhibitors are designed by either targeting the active site or mimicking substrate of urease, which is not efficient. Up to now, only one effective inhibitor-acetohydroxamic acid (AHA)-is clinically available, but it has adverse side effects. Herein, we demonstrate that a clinically used drug, colloidal bismuth subcitrate, utilizes an unusual way to inhibit urease activity, i.e., disruption of urease maturation process via functional perturbation of a metallochaperone, UreG. Similar phenomena were also observed in various pathogenic bacteria, suggesting that UreG may serve as a general target for design of new types of urease inhibitors. Using Helicobacter pylori UreG as a showcase, by virtual screening combined with experimental validation, we show that two compounds targeting UreG also efficiently inhibited urease activity with inhibitory concentration (IC)50 values of micromolar level, resulting in attenuated virulence of the pathogen. We further demonstrate the efficacy of the compounds in a mammalian cell infection model. This study opens up a new opportunity for the design of more effective urease inhibitors and clearly indicates that metallochaperones involved in the maturation of important microbial metalloenzymes serve as new targets for devising a new type of antimicrobial drugs.

Potent inhibitors of human organic anion transporters 1 and 3 from clinical drug libraries: discovery and molecular characterization.

PubMed

Duan, Peng; Li, Shanshan; Ai, Ni; Hu, Longqin; Welsh, William J; You, Guofeng

2012-11-05

Transporter-mediated drug-drug interactions in the kidney dramatically influence the pharmacokinetics and other clinical effects of drugs. Human organic anion transporters 1 (hOAT1) and 3 (hOAT3) are the major transporters in the basolateral membrane of kidney proximal tubules, mediating the rate-limiting step in the elimination of a broad spectrum of drugs. In the present study, we screened two clinical drug libraries against hOAT1 and hOAT3. Of the 727 compounds screened, 92 compounds inhibited hOAT1 and 262 compounds inhibited hOAT3. When prioritized based on the peak unbound plasma concentrations of these compounds, three inhibitors for hOAT1 and seven inhibitors for hOAT3 were subsequently identified with high inhibitory potency (>95%). Computational analyses revealed that inhibitors and noninhibitors can be differentiated from each other on the basis of several physicochemical features, including number of hydrogen-bond donors, number of rotatable bonds, and topological polar surface area (TPSA) for hOAT1; and molecular weight, number of hydrogen-bond donors and acceptors, TPSA, partition coefficient (log P(7.4)), and polarizability for hOAT3. Pharmacophore modeling identified two common structural features associated with inhibitors for hOAT1 and hOAT3, viz., an anionic hydrogen-bond acceptor atom, and an aromatic center separated by ∼5.7 Å. Such model provides mechanistic insights for predicting new OAT inhibitors.

Mitotic Arrest and Apoptosis in Breast Cancer Cells Induced by Origanum majorana Extract: Upregulation of TNF-α and Downregulation of Survivin and Mutant p53

PubMed Central

Al Dhaheri, Yusra; Eid, Ali; AbuQamar, Synan; Attoub, Samir; Khasawneh, Mohammad; Aiche, Ghenima; Hisaindee, Soleiman; Iratni, Rabah

2013-01-01

Background In the present study, we investigated the effect of Origanum majorana ethanolic extract on the survival of the highly proliferative and invasive triple-negative p53 mutant breast cancer cell line MDA-MB-231. Results We found that O. majorana extract (OME) was able to inhibit the viability of the MDA-MB-231 cells in a time- and concentration-dependent manner. The effect of OME on cellular viability was further confirmed by the inhibition of colony growth. We showed, depending on the concentration used, that OME elicited different effects on the MDA-MB 231 cells. Concentrations of 150 and 300 µg/mL induced an accumulation of apoptotic–resistant population of cells arrested in mitotis and overexpressing the cyclin-dependent kinase inhibitor, p21 and the inhibitor of apoptosis, survivin. On the other hand, higher concentrations of OME (450 and 600 µg/mL) triggered a massive apoptosis through the extrinsic pathway, including the activation of tumor necrosis factor-α (TNF-α), caspase 8, caspase 3, and cleavage of PARP, downregulation of survivin as well as depletion of the mutant p53 in MDA-MB-231 cells. Furthermore, OME induced an upregulation of γ-H2AX, a marker of double strand DNA breaks and an overall histone H3 and H4 hyperacetylation. Conclusion Our findings provide strong evidence that O. majorana may be a promising chemopreventive and therapeutic candidate against cancer especially for highly invasive triple negative p53 mutant breast cancer; thus validating its complementary and alternative medicinal use. PMID:23451065

Stimulation of EphB2/ephrin-B1 signalling by tumour necrosis factor alpha in human dental pulp stem cells.

PubMed

Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Zhang, Chengfei

2015-04-01

The aim of this study was to investigate whether in vitro stimulation of dental pulp stem cells (DPSCs) by tumour necrosis factor alpha (TNF-α) would induce secretion of EphB2/ephrin-B1 signalling. Dental pulp stem cells isolated from human dental pulp were treated with TNF-α (5-100 ng/ml) over 2-48 h. EphB2/ephrin-B1 mRNA and protein levels were measured by real-time polymerase chain reaction (RT-PCR) and western blot analysis respectively. Additionally, DPSCs were pre-incubated with TNF-α receptor neutralizing antibodies or infected with nuclear factor-kappa B (NF-ĸB) inhibitor, p38 MAPK inhibitor, Jun N-terminal kinase (JNK) inhibitor and MEK inhibitor before TNF-α treatment. Results were analysed by one-way ANOVA. Tumour necrosis factor alpha increased EphB2 mRNA expression in DPSCs at concentrations up to 20 ng/ml and ephrin-B1 at concentrations up to 40 ng/ml (P < 0.05). Its mRNA expression reached maximum at 24 h when treated with TNF-α at 20 ng/ml (P < 0.05). EphB2/ephrin-B1 protein expression levels were high at 16 and 24 h as shown by western blotting. Neutralizing antibodies for TNFR1/2 receptors down-regulated EphB2/ephrin-B1 mRNA expression (P < 0.05) and ephrin-B1 protein expression, but not EphB2 protein expression. JNK-inhibitor inhibited EphB2 mRNA expression only (P < 0.05). EphB2/ephrin-B1 were invoked in DPSCs with TNF-α treatment via the JNK-dependent pathway, but not NF-ĸB, p38 MAPK or MEK signalling. © 2015 John Wiley & Sons Ltd.

Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

PubMed

Tomoda, A; Lachant, N A; Noble, N A; Tanaka, K R

1983-07-01

Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

Screening of Natural Product Derivatives Identifies Two Structurally Related Flavonoids as Potent Quorum Sensing Inhibitors against Gram-Negative Bacteria.

PubMed

Manner, Suvi; Fallarero, Adyary

2018-05-03

Owing to the failure of conventional antibiotics in biofilm control, alternative approaches are urgently needed. Inhibition of quorum sensing (QS) represents an attractive target since it is involved in several processes essential for biofilm formation. In this study, a compound library of natural product derivatives ( n = 3040) was screened for anti-quorum sensing activity using Chromobacterium violaceum as reporter bacteria. Screening assays, based on QS-mediated violacein production and viability, were performed in parallel to identify non-bactericidal QS inhibitors (QSIs). Nine highly active QSIs were identified, while 328 compounds were classified as moderately actives and 2062 compounds as inactives. Re-testing of the highly actives at a lower concentration against C. violaceum , complemented by a literature search, led to the identification of two flavonoid derivatives as the most potent QSIs, and their impact on biofilm maturation in Escherichia coli and Pseudomonas aeruginosa was further investigated. Finally, effects of these leads on swimming and swarming motility of P. aeruginosa were quantified. The identified flavonoids affected all the studied QS-related functions at micromolar concentrations. These compounds can serve as starting points for further optimization and development of more potent QSIs as adjunctive agents used with antibiotics in the treatment of biofilms.

A novel multi-hyphenated analytical method to simultaneously determine xanthine oxidase inhibitors and superoxide anion scavengers in natural products.

PubMed

Qi, Jin; Sun, Li-Qiong; Qian, Steven Y; Yu, Bo-Yang

2017-09-01

Natural products, such as rosmarinic acid and apigenin, can act as xanthine oxidase inhibitors (XOIs) as well as superoxide anion scavengers, and have potential for treatment of diseases associated with high uric acid levels and oxidative stress. However, efficient simultaneous screening of these two bioactivities in natural products has been challenging. We have developed a novel method by assembling a multi-hyphenated high performance liquid chromatography (HPLC) system that combines a photo-diode array, chemiluminescence detector and a HPLC system with a variable wavelength detector, to simultaneously detect components that act as both XOIs and superoxide anion scavengers in natural products. Superoxide anion scavenging activity in the analyte was measured by on-line chemiluminescence chromatography based on pyrogallol-luminol oxidation, while xanthine oxidase inhibitory activity was determined by semi-on-line HPLC analysis. After optimizing multiple elements, including chromatographic conditions (e.g., organic solvent concentration and mobile phase pH), concentrations of xanthine/xanthine oxidase and reaction temperature, our validated analytical method was capable of mixed sample analysis. The final results from our method are presented in an easily understood visual format including comprehensive bioactivity data of natural products. Copyright © 2017. Published by Elsevier B.V.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

PubMed

Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-07-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex

PubMed Central

Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-01-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1. PMID:21447560

DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L -1) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration

DOE PAGES

Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W.; ...

2016-04-01

Distilling and purifying ethanol and other products from second generation lignocellulosic biorefineries adds significant capital and operating costs to biofuel production. The energy usage associated with distillation negatively affects plant gate costs and causes environmental and life-cycle impacts, and the lower titers in fermentation caused by lower sugar concentrations from pretreatment and enzymatic hydrolysis increase energy and water usage and ethanol production costs. In addition, lower ethanol titers increase the volumes required for enzymatic hydrolysis and fermentation vessels increase capital expenditure (CAPEX). Therefore, increasing biofuel titers has been a research focus in renewable biofuel production for several decades. In thismore » work, we achieved approximately 230 g L -1 of monomeric sugars after high solid enzymatic hydrolysis using deacetylation and mechanical refining (DMR) processed corn stover substrates produced at the 100 kg per day scale. The high sugar concentrations and low chemical inhibitor concentrations achieved by the DMR process allowed fermentation to ethanol with titers as high as 86 g L -1, which translates into approximately 10.9% v/v ethanol. To our knowledge, this is the first time that titers greater than 10% v/v ethanol in fermentations derived from corn stover without any sugar concentration or purification steps have been reported. As a result, the potential cost savings from high sugar and ethanol titers achieved by the DMR process are also reported using TEA analysis.« less

DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L -1) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W.

Distilling and purifying ethanol and other products from second generation lignocellulosic biorefineries adds significant capital and operating costs to biofuel production. The energy usage associated with distillation negatively affects plant gate costs and causes environmental and life-cycle impacts, and the lower titers in fermentation caused by lower sugar concentrations from pretreatment and enzymatic hydrolysis increase energy and water usage and ethanol production costs. In addition, lower ethanol titers increase the volumes required for enzymatic hydrolysis and fermentation vessels increase capital expenditure (CAPEX). Therefore, increasing biofuel titers has been a research focus in renewable biofuel production for several decades. In thismore » work, we achieved approximately 230 g L -1 of monomeric sugars after high solid enzymatic hydrolysis using deacetylation and mechanical refining (DMR) processed corn stover substrates produced at the 100 kg per day scale. The high sugar concentrations and low chemical inhibitor concentrations achieved by the DMR process allowed fermentation to ethanol with titers as high as 86 g L -1, which translates into approximately 10.9% v/v ethanol. To our knowledge, this is the first time that titers greater than 10% v/v ethanol in fermentations derived from corn stover without any sugar concentration or purification steps have been reported. As a result, the potential cost savings from high sugar and ethanol titers achieved by the DMR process are also reported using TEA analysis.« less

Indomethacin-induced alterations in corticosteroid and prostaglandin release by isolated adrenocortical cells of the cat.

PubMed Central

Laychock, S G; Rubin, R P

1976-01-01

1 The effects of purported prostaglandin synthesis inhibitors on steroid and prostaglandin (E and F) release from trypsin-dispersed cat adrenocortical cells were investigated. 2 Low indomethacin concentrations potentiated adrenocorticotrophin (ACTH)-evoked prostaglandin and steroid release, whereas higher concentrations depressed both responses to ACTH. The steroidogenic response to exogenous prostaglandin E2 was not markedly altered over a wide range of indomethacin concentrations. 3 Indomethacin enhanced basal steroid release but did not enhance basal prostaglandin E or F release. 4 5,8,11,14-Eicosatetraynoic acid (ETA) elicited a concentration-dependent inhibition of ACTH-induced steroid release, but had little effect on prostaglandin E2-induced steroid release. A high concentration of ETA inhibited prostaglandin E and F release. 5 These data are discussed in relation to the concept that prostaglandins provide a critical link in ACTH-induced corticosteroidogenesis. PMID:181110

Boronic acid-modified lipid nanocapsules: a novel platform for the highly efficient inhibition of hepatitis C viral entry

NASA Astrophysics Data System (ADS)

Khanal, Manakamana; Barras, Alexandre; Vausselin, Thibaut; Fénéant, Lucie; Boukherroub, Rabah; Siriwardena, Aloysius; Dubuisson, Jean; Szunerits, Sabine

2015-01-01

The search for viral entry inhibitors that selectively target viral envelope glycoproteins has attracted increasing interest in recent years. Amongst the handful of molecules reported to show activity as hepatitis C virus (HCV) entry inhibitors are a variety of glycan-binding proteins including the lectins, cyanovirin-N (CV-N) and griffithsin. We recently demonstrated that boronic acid-modified nanoparticles are able to reduce HCV entry through a similar mechanism to that of lectins. A major obstacle to any further development of these nanostructures as viral entry inhibitors is their only moderate maximal inhibition potential. In the present study, we report that lipid nanocapsules (LNCs), surface-functionalized with amphiphilic boronic acid (BA) through their post-insertion into the semi-rigid shell of the LNCs, are indeed far superior as HCV entry inhibitors when compared with previously reported nanostructures. These 2nd generation particles (BA-LNCs) are shown to prevent HCV infection in the micromolar range (IC50 = 5.4 μM of BA moieties), whereas the corresponding BA monomers show no significant effects even at the highest analyzed concentration (20 μM). The new BA-LNCs are the most promising boronolectin-based HCV entry inhibitors reported to date and are thus observed to show great promise in the development of a pseudolectin-based therapeutic agent.The search for viral entry inhibitors that selectively target viral envelope glycoproteins has attracted increasing interest in recent years. Amongst the handful of molecules reported to show activity as hepatitis C virus (HCV) entry inhibitors are a variety of glycan-binding proteins including the lectins, cyanovirin-N (CV-N) and griffithsin. We recently demonstrated that boronic acid-modified nanoparticles are able to reduce HCV entry through a similar mechanism to that of lectins. A major obstacle to any further development of these nanostructures as viral entry inhibitors is their only moderate maximal inhibition potential. In the present study, we report that lipid nanocapsules (LNCs), surface-functionalized with amphiphilic boronic acid (BA) through their post-insertion into the semi-rigid shell of the LNCs, are indeed far superior as HCV entry inhibitors when compared with previously reported nanostructures. These 2nd generation particles (BA-LNCs) are shown to prevent HCV infection in the micromolar range (IC50 = 5.4 μM of BA moieties), whereas the corresponding BA monomers show no significant effects even at the highest analyzed concentration (20 μM). The new BA-LNCs are the most promising boronolectin-based HCV entry inhibitors reported to date and are thus observed to show great promise in the development of a pseudolectin-based therapeutic agent. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03875d

ACE Phenotyping as a Guide Toward Personalized Therapy With ACE Inhibitors.

PubMed

Danilov, Sergei M; Tovsky, Stan I; Schwartz, David E; Dull, Randal O

2017-07-01

Angiotensin-converting enzyme (ACE) inhibitors (ACEI) are widely used in the management of cardiovascular diseases but with significant interindividual variability in the patient's response. To investigate whether interindividual variability in the response to ACE inhibitors is explained by the "ACE phenotype"-for example, variability in plasma ACE concentration, activity, and conformation and/or the degree of ACE inhibition in each individual. The ACE phenotype was determined in plasma of 14 patients with hypertension treated chronically for 4 weeks with 40 mg enalapril (E) or 20 mg E + 16 mg candesartan (EC) and in 20 patients with hypertension treated acutely with a single dose (20 mg) of E with or without pretreatment with hydrochlorothiazide. The ACE phenotyping included (1) plasma ACE concentration; (2) ACE activity (with 2 substrates: Hip-His-Leu and Z-Phe-His-Leu and calculation of their ratio); (3) detection of ACE inhibitors in patient's blood (indicator of patient compliance) and the degree of ACE inhibition (ie, adherence); and (4) ACE conformation. Enalapril reduced systolic and diastolic blood pressure in most patients; however, 20% of patients were considered nonresponders. Chronic treatment results in 40% increase in serum ACE concentrations, with the exception of 1 patient. There was a trend toward better response to ACEI among patients who had a higher plasma ACE concentration. Due to the fact that "20% of patients do not respond to ACEI by blood pressure drop," the initial blood ACE level could not be a predictor of blood pressure reduction in an individual patient. However, ACE phenotyping provides important information about conformational and kinetic changes in ACE of individual patients, and this could be a reason for resistance to ACE inhibitors in some nonresponders.

The consequence of regional gradients of P-gp and CYP3A4 for drug-drug interactions by P-gp inhibitors and the P-gp/CYP3A4 interplay in the human intestine ex vivo.

PubMed

Li, Ming; de Graaf, Inge A M; van de Steeg, Evita; de Jager, Marina H; Groothuis, Geny M M

2017-04-01

Intestinal P-gp and CYP3A4 work coordinately to reduce the intracellular concentration of drugs, and drug-drug interactions (DDIs) based on this interplay are of clinical importance and require pre-clinical investigation. Using precision-cut intestinal slices (PCIS) of human jejunum, ileum and colon, we investigated the P-gp/CYP3A4 interplay and related DDIs with P-gp inhibitors at the different regions of the human intestine with quinidine (Qi), dual substrate of P-gp and CYP3A4, as probe. All the P-gp inhibitors increased the intracellular concentrations of Qi by 2.1-2.6 fold in jejunum, 2.6-3.8 fold in ileum but only 1.2-1.3 fold in colon, in line with the different P-gp expression in these intestinal regions. The selective P-gp inhibitors (CP100356 and PSC833) enhanced 3-hydroxy-quinidine (3OH-Qi) in jejunum and ileum, while dual inhibitors of P-gp and CYP3A4 (verapamil and ketoconazole) decreased the 3OH-Qi production, despite of the increased intracellular Qi concentration, due to inhibition of CYP3A4. The outcome of DDIs based on P-gp/CYP3A4 interplay, shown as remarkable changes in the intracellular concentration of both the parent drug and the metabolite, varied among the intestinal regions, probably due to the different expression of P-gp and CYP3A4, and were different from those found in rat PCIS, which may have important implications for the disposition and toxicity of drugs and their metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro.

PubMed

Lefebvre, Romain A; Van Colen, Inge; Pauwelyn, Vicky; De Maeyer, Joris H

2016-06-15

5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [(3)H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1-1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01-0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid Response to High-Dose, Pulsatile Erlotinib in Afatinib-Refractory Leptomeningeal Carcinomatosis from Adenocarcinoma of the Lung: A Case Report.

PubMed

Fan, Frank S

2016-01-01

Leptomeningeal carcinomatosis occurred in an old female patient who was on a standard dose of afatinib for the treatment of her non-small cell lung cancer harboring an epidermal growth factor receptor gene mutation sensitive to tyrosine kinase inhibitors when extracranial lesions were still under control. Shifting to high-dose, pulsatile erlotinib dramatically saved her from the devastating condition in a very short period of time. Inadequate afatinib concentration in cerebrospinal fluid is reasonably suspected, and there is a call for clinical trials testing high-dose afatinib in leptomeningeal carcinomatosis.

Potentiation of the vascular response to kinins by inhibition of myocardial kininases.

PubMed

Dendorfer, A; Wolfrum, S; Schäfer, U; Stewart, J M; Inamura, N; Dominiak, P

2000-01-01

Inhibitors of angiotensin I-converting enzyme (ACE) are very efficacious in the potentiation of the actions of bradykinin (BK) and are able to provoke a B(2) receptor-mediated vasodilation even after desensitization of this receptor. Because this activity cannot be easily explained only by an inhibition of kinin degradation, direct interactions of ACE inhibitors with the B(2) receptor or its signal transduction have been hypothesized. To clarify the significance of degradation-independent potentiation, we studied the vasodilatory effects of BK and 2 degradation-resistant B(2) receptor agonists in the isolated rat heart, a model in which ACE and aminopeptidase P (APP) contribute equally to the degradation of BK. Coronary vasodilation to BK and to a peptidic (B6014) and a nonpeptidic (FR190997) degradation-resistant B(2) agonist was assessed in the presence or absence of the ACE inhibitor ramiprilat, the APP inhibitor mercaptoethanol, or both. Ramiprilat or mercaptoethanol induced leftward shifts in the BK dose-response curve (EC(50)=3.4 nmol/L) by a factor of 4.6 or 4.9, respectively. Combined inhibition of ACE and APP reduced the EC(50) of BK to 0.18 nmol/L (ie, by a factor of 19) but potentiated the activity of B6014 (EC(50)=1.9 nmol/L) only weakly without altering that of FR190997 (EC(50)=0.34 nmol/L). Desensitization of B(2) receptors was induced by the administration of BK (0.2 micromol/L) or FR190997 (0.1 micromol/L) for 30 minutes; the vascular reactivity to ramiprilat or increasing doses of BK was tested thereafter. After desensitization with BK, but not FR190997, an additional application of ramiprilat provoked a B(2) receptor-mediated vasodilation. High BK concentrations were still effective at the desensitized receptor. The process of desensitization was not altered by ramiprilat. These results show that in this model, all potentiating actions of ACE inhibitors on kinin-induced vasodilation are exclusively related to the reduction in BK breakdown and are equivalently provoked by APP inhibition. The desensitization of B(2) receptors is overcome by increasing BK concentrations, either directly or through the inhibition of ACE. These observations do not suggest any direct interactions of ACE inhibitors with the B(2) receptor or its signal transduction but point to a very high activity of BK degradation in the vicinity of the B(2) receptor in combination with a stimulation-dependent reduction in receptor affinity.

Fructose Rich Diet-Induced High Plasminogen Activator Inhibitor-1 (PAI-1) Production in the Adult Female Rat: Protective Effect of Progesterone

PubMed Central

Castrogiovanni, Daniel; Alzamendi, Ana; Ongaro, Luisina; Giovambattista, Andrés; Gaillard, Rolf C.; Spinedi, Eduardo

2012-01-01

The effect of progesterone (P4) on fructose rich diet (FRD) intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT) dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT) or containing P4 (12 mg/kg). Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND) or containing fructose (10% w/v; CT-FRD and P4-FRD). At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND) rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND) plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1). Lipidemia and adiponectinemia were high (vs. P4-ND) in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production. PMID:23016136

Fructose rich diet-induced high plasminogen activator inhibitor-1 (PAI-1) production in the adult female rat: protective effect of progesterone.

PubMed

Castrogiovanni, Daniel; Alzamendi, Ana; Ongaro, Luisina; Giovambattista, Andrés; Gaillard, Rolf C; Spinedi, Eduardo

2012-08-01

The effect of progesterone (P4) on fructose rich diet (FRD) intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT) dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT) or containing P4 (12 mg/kg). Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND) or containing fructose (10% w/v; CT-FRD and P4-FRD). At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND) rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND) plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1). Lipidemia and adiponectinemia were high (vs. P4-ND) in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production.

Clinical Pharmacokinetics and Pharmacodynamics of Saxagliptin, a Dipeptidyl Peptidase-4 Inhibitor.

PubMed

Boulton, David W

2017-01-01

Saxagliptin is an orally active, highly potent, selective and competitive dipeptidyl peptidase (DPP)-4 inhibitor used in the treatment of type 2 diabetes mellitus at doses of 2.5 or 5 mg once daily. DPP-4 is responsible for degrading the intestinally derived hormones glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP). Inhibition of DPP-4 increases intact plasma GLP-1 and GIP concentrations, augmenting glucose-dependent insulin secretion. Both saxagliptin and its major active metabolite, 5-hydroxy saxagliptin, demonstrate high degrees of selectivity for DPP-4 compared with other DPP enzymes. Saxagliptin is orally absorbed and can be administered with or without food. The half-life of plasma DPP-4 inhibition with saxagliptin 5 mg is ~27 h, which supports a once-daily dosing regimen. Saxagliptin is metabolized by cytochrome P450 (CYP) 3A4/5 and is eliminated by a combination of renal and hepatic clearance. No clinically meaningful differences in saxagliptin or 5-hydroxy saxagliptin pharmacokinetics have been detected in patients with hepatic impairment. No clinically meaningful differences in saxagliptin or 5-hydroxy saxagliptin pharmacokinetics have been detected in patients with mild renal impairment, whereas dose reduction is recommended in patients with moderate or severe renal impairment because of greater systemic exposure [the area under the plasma concentration-time curve (AUC)] to saxagliptin total active moieties. Clinically relevant drug-drug interactions have not been detected; however, limiting the dose to 2.5 mg once daily is recommended in the USA when saxagliptin is coadministered with strong CYP inhibitors, because of increased saxagliptin exposure. In summary, saxagliptin has a predictable pharmacokinetic and pharmacodynamic profile.

Carbonic anhydrase activity in Arabidopsis thaliana thylakoid membrane and fragments enriched with PSI or PSII.

PubMed

Ignatova, Lyudmila K; Rudenko, Natalia N; Mudrik, Vilen A; Fedorchuk, Tat'yana P; Ivanov, Boris N

2011-12-01

The procedure of isolating the thylakoids and the thylakoid membrane fragments enriched with either photosystem I or photosystem II (PSI- and PSII-membranes) from Arabidopsis thaliana leaves was developed. It differed from the one used with pea and spinach in durations of detergent treatment and centrifugation, and in concentrations of detergent and Mg(2+) in the media. Both the thylakoid and the fragments preserved carbonic anhydrase (CA) activities. Using nondenaturing electrophoresis followed by detection of CA activity in the gel stained with bromo thymol blue, one low molecular mass carrier of CA activity was found in the PSI-membranes, and two carriers, a low molecular mass one and a high molecular mass one, were found in the PSII-membranes. The proteins in the PSII-membranes differed in their sensitivity to acetazolamide (AA), a specific CA inhibitor. AA at 5 × 10(-7) M inhibited the CA activity of the high molecular mass protein but stimulated the activity of the low molecular mass carrier in the PSII-membranes. At the same concentration, AA moderately inhibited, by 30%, the CA activity of PSI-membranes. CA activity of the PSII-membranes was almost completely suppressed by the lipophilic CA inhibitor, ethoxyzolamide at 10(-9) M, whereas CA activity of the PSI-membranes was inhibited by this inhibitor even at 5 × 10(-7) M just the same as for AA. The observed distribution of CA activity in the thylakoid membranes from A. thaliana was close to the one found in the membranes of pea, evidencing the general pattern of CA activity in the thylakoid membranes of C3-plants. © Springer Science+Business Media B.V. 2011

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry

PubMed Central

Cerca, Nuno; Martins, Silvia; Cerca, Filipe; Jefferson, Kimberly K.; Pier, Gerald B.; Oliveira, Rosário; Azeredo, Joana

2005-01-01

Objectives To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. Methods Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. Results In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. Conclusions This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis. PMID:15980094

PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries

PubMed Central

Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

2015-01-01

Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K+ concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca2+ release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca2+ waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca2+ concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca2+ entry and promote vasoconstriction. PMID:25888510

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells.

PubMed

Mishima, Masayuki; Tanaka, Kenji; Takeiri, Akira; Harada, Asako; Kubo, Chiyomi; Sone, Sachiko; Nishimura, Yoshikazu; Tachibana, Yukako; Okazaki, Makoto

2008-08-25

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2 microM, 2.4 microM, and 4.8 microM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8 microM, 3.6 microM, and 5.4 microM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.

Comparative sensitivity and inhibitor tolerance of GlobalFiler® PCR Amplification and Investigator® 24plex QS kits for challenging samples.

PubMed

Elwick, Kyleen; Mayes, Carrie; Hughes-Stamm, Sheree

2018-05-01

In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, been buried, decomposed, and/or contain inhibitory substances. This study compares the performance of a relatively new STR kit in the US market (Investigator® 24plex QS kit; Qiagen) with the GlobalFiler® PCR Amplification kit (Thermo Fisher Scientific) when genotyping highly inhibited and low level DNA samples. In this study, DNA samples ranging from 1 ng to 7.8 pg were amplified to define the sensitivity of two systems. In addition, DNA (1 ng and 0.1 ng input amounts) was spiked with various concentrations of five inhibitors common to human remains (humic acid, melanin, hematin, collagen, calcium). Furthermore, bone (N = 5) and tissue samples from decomposed human remains (N = 6) were used as mock casework samples for comparative analysis with both STR kits. The data suggest that the GlobalFiler® kit may be slightly more sensitive than the Investigator® kit. On average STR profiles appeared to be more balanced and average peak heights were higher when using the GlobalFiler® kit. However, the data also show that the Investigator® kit may be more tolerant to common PCR inhibitors. While both STR kits showed a decrease in alleles as the inhibitor concentration increased, more complete profiles were obtained when the Investigator® kit was used. Of the 11 bone and decomposed tissue samples tested, 8 resulted in more complete and balanced STR profiles when amplified with the GlobalFiler® kit. Copyright © 2018 Elsevier B.V. All rights reserved.

Flavonoids Are Inhibitors of Human Organic Anion Transporter 1 (OAT1)–Mediated Transport

PubMed Central

An, Guohua; Wang, Xiaodong

2014-01-01

Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 μM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of <0.3 and 0.47 μM, respectively. In contrast to the tested flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway. PMID:25002746

D-galactose induces necroptotic cell death in neuroblastoma cell lines.

PubMed

Li, Na; He, Yangyan; Wang, Ling; Mo, Chunfen; Zhang, Jie; Zhang, Wei; Li, Junhong; Liao, Zhiyong; Tang, Xiaoqiang; Xiao, Hengyi

2011-12-01

D-Galactose (D-gal) can induce oxidative stress in non-cancer cells and result in cell damage by disturbing glucose metabolism. However, the effect of D-gal on cancer cells is yet to be explored. In this study, we investigated the toxicity of D-gal to malignant cells specifically neuroblastoma cells. As the results, high concentrations of D-gal had significant toxicity to cancer cells, whereas the same concentrations of glucose had no; the viability loss via D-gal treatment was prominent to malignant cells (Neuro2a, SH-SY5Y, PC-3, and HepG2) comparing to non-malignant cells (NIH3T3 and LO(2)). Differing from the apoptosis induced by H(2) O(2), D-gal damaged cells showed the characters of necrotic cell death, such as trypan blue-tangible and early phase LDH leakage. Further experiments displayed that the toxic effect of D-gal can be alleviated by necroptosis inhibitor Necrostatin (Nec-1) and autophagy inhibitor 3-methyladenine (3-MA) but not by caspase inhibitor z-VAD-fmk. D-Gal treatment can transcriptionally up-regulate the genes relevant to necroptosis (Bmf, Bnip3) and autophagy (Atg5, TIGAR) but not the genes related to apoptosis (Caspase3, Bax, and p53). D-Gal did not activate Caspase-3, but prompted puncta-like GFP-LC3 distribution, an indicator for activated autophagy. The involvement of aldose reductase (AR)-mediated polyol pathway was proved because the inhibitor of AR can attenuate the toxicity of D-gal and D-gal treatment elevates the expression of AR. This study demonstrates for the first time that D-gal can induce non-apoptotic but necroptotic cell death in neuroblastoma cells and provides a new clue for developing the strategy against apoptosis-resistant cancers. Copyright © 2011 Wiley Periodicals, Inc.

Short-term exposure of platelets to glucose impairs inhibition of platelet aggregation by cyclooxygenase inhibitors.

PubMed

Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

2011-01-01

Aspirin treatment reduces cardiovascular events and deaths in high-risk non-diabetic patients, but not in patients suffering from diabetes. In these patients, hyperglycemia has been found to cause reduced platelet sensitivity to aspirin. It is supposed that long-term exposure of platelets to glucose leads to non-enzymatic glycosylation and impairs aspirin inhibition of platelet aggregation. On the other hand, short-term exposure of platelets to glucose also attenuates the effect of aspirin on platelets. The aim of the present work was to analyse the effect of short-term exposure of glucose on the inhibition of platelet aggregation by aspirin and other cyclooxygenase (COX) inhibitors. Already a 15 min exposure of platelets to glucose impaired aspirin inhibition of the platelet aggregation induced by collagen, thrombin, adenosine diphosphate (ADP), and arachidonic acid (AA). Aspirin inhibition of platelet aggregation in platelet-rich plasma (PRP) was attenuated by 5.6, 11.2, 16.8, and 22.4 mM of glucose in a concentration-dependent way. The same effect was observed with indomethacin and acetaminophen used as cyclooxygenase inhibitors instead of aspirin. N-methyl-L-arginine, an inhibitor of nitric oxide synthase, prevented the effect of glucose on aspirin, indomethacin and acetaminophen inhibition of platelet aggregation. Other monosaccharides, for example fructose and galactose, impaired aspirin inhibition as did glucose. Lactic acid (0.1, 0.2, 0.4, 0.8 mM), the end product of anaerobic glycolysis in platelets, impaired the inhibition of platelet aggregation with aspirin in a concentration-dependent way but did not affect indomethacin. It is suggested that lactic acid might be a mediator of the effect of glucose on aspirin inhibition in platelets.

Variable Linezolid Exposure in Intensive Care Unit Patients-Possible Role of Drug-Drug Interactions.

PubMed

Töpper, Christoph; Steinbach, Cathérine L; Dorn, Christoph; Kratzer, Alexander; Wicha, Sebastian G; Schleibinger, Michael; Liebchen, Uwe; Kees, Frieder; Salzberger, Bernd; Kees, Martin G

2016-10-01

Standard doses of linezolid may not be suitable for all patient groups. Intensive care unit (ICU) patients in particular may be at risk of inadequate concentrations. This study investigated variability of drug exposure and its potential sources in this population. Plasma concentrations of linezolid were determined by high-performance liquid chromatography in a convenience sample of 20 ICU patients treated with intravenous linezolid 600 mg twice daily. Ultrafiltration applying physiological conditions (pH 7.4/37°C) was used to determine the unbound fraction. Individual pharmacokinetic (PK) parameters were estimated by population PK modeling. As measures of exposure to linezolid, area under the concentration-time curve (AUC) and trough concentrations (Cmin) were calculated and compared with published therapeutic ranges (AUC 200-400 mg*h/L, Cmin 2-10 mg/L). Coadministered inhibitors or inducers of cytochrome P450 and/or P-glycoprotein were noted. Data from 18 patients were included into the PK evaluation. Drug exposure was highly variable (median, range: AUC 185, 48-618 mg*h/L, calculated Cmin 2.92, 0.0062-18.9 mg/L), and only a minority of patients had values within the target ranges (6 and 7, respectively). AUC and Cmin were linearly correlated (R = 0.98), and classification of patients (underexposed/within therapeutic range/overexposed) according to AUC or Cmin was concordant in 15 cases. Coadministration of inhibitors was associated with a trend to higher drug exposure, whereas 3 patients treated with levothyroxine showed exceedingly low drug exposure (AUC ∼60 mg*h/L, Cmin <0.4 mg/L). The median unbound fraction in all 20 patients was 90.9%. Drug exposure after standard doses of linezolid is highly variable and difficult to predict in ICU patients, and therapeutic drug monitoring seems advisable. PK drug-drug interactions might partly be responsible and should be further investigated; protein binding appears to be stable and irrelevant.

Microfluidics platform for single-shot dose-response analysis of chloride channel-modulating compounds.

PubMed

Jin, Byung-Ju; Ko, Eun-A; Namkung, Wan; Verkman, A S

2013-10-07

We previously developed cell-based kinetics assays of chloride channel modulators utilizing genetically encoded yellow fluorescent proteins. Fluorescence platereader-based high-throughput screens yielded small-molecule activators and inhibitors of the cAMP-activated chloride channel CFTR and calcium-activated chloride channels, including TMEM16A. Here, we report a microfluidics platform for single-shot determination of concentration-activity relations in which a 1.5 × 1.5 mm square area of adherent cultured cells is exposed for 5-10 min to a pseudo-logarithmic gradient of test compound generated by iterative, two-component channel mixing. Cell fluorescence is imaged following perfusion with an iodide-containing solution to give iodide influx rate at each location in the image field, thus quantifying modulator effects over a wide range of concentrations in a single measurement. IC50 determined for CFTR and TMEM16A activators and inhibitors by single-shot microfluidics were in agreement with conventional plate reader measurements. The microfluidics approach developed here may accelerate the discovery and characterization of chloride channel-targeted drugs.

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions.

PubMed

Tsuge, Yota; Hori, Yoshimi; Kudou, Motonori; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

2014-10-01

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.

Searching for Novel Inhibitors of the S. aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-benzothiazine Analogues.

PubMed

Felicetti, Tommaso; Cannalire, Rolando; Burali, Maria Sole; Massari, Serena; Manfroni, Giuseppe; Barreca, Maria Letizia; Tabarrini, Oriana; Schindler, Bryan D; Sabatini, Stefano; Kaatz, Glenn W; Cecchetti, Violetta

2017-08-22

Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

EFFECT OF CARBONIC ANHYDRASE INHIBITORS ON THE INORGANIC CARBON UPTAKE BY PHYTOPLANKTON NATURAL ASSEMBLAGES(1).

PubMed

Mercado, Jesús M; Ramírez, Teodoro; Cortés, Dolores; Liger, Esperanza

2009-02-01

The role of carbonic anhydrase (CA) in inorganic carbon acquisition (dissolved inorganic carbon, DIC) was examined in Alboran Sea phytoplankton assemblages. The study area was characterized by a relatively high variability in nutrient concentration and in abundance and taxonomic composition of phytoplankton. Therefore, the relationship between environmental variability and capacity for using HCO3 (-) via external CA (eCA) was examined. Acetazolamide (AZ, an inhibitor of eCA) inhibited the primary productivity (PP) in 50% of the samples, with inhibition percentages ranging from 13% to 60%. The AZ effect was more prominent in the samples that exhibited PP >1 mg C · m(-3)  · h(-1) , indicating that the contribution of eCA to the DIC photosynthetic flux was irrelevant at low PP. The inhibition of primary productivity by AZ was significantly correlated to the abundance of diatoms. However, there was no a relationship between AZ effect and CO2 partial pressure (pCO2 ) or nutrient concentration, indicating that the variability in the PP percentage supported by eCA was mainly due to differences in taxonomic composition of the phytoplankton assemblages. Ethoxyzolamide (EZ, an inhibitor of both external and internal CA) affected 13 of 14 analyzed samples, with PP inhibition percentages varying from 50% to 95%. The effects of AZ and EZ were partially reversed by doubling DIC concentration. These results imply that CA activity (external and/or internal) was involved in inorganic carbon acquisition in most the samples. However, EZ effect was not correlated with pCO2 or taxonomic composition of the phytoplankton. © 2009 Phycological Society of America.

SGLT2 inhibitor lowers serum uric acid through alteration of uric acid transport activity in renal tubule by increased glycosuria

PubMed Central

Chino, Yukihiro; Samukawa, Yoshishige; Sakai, Soichi; Nakai, Yasuhiro; Yamaguchi, Jun-ichi; Nakanishi, Takeo; Tamai, Ikumi

2014-01-01

Sodium glucose cotransporter 2 (SGLT2) inhibitors have been reported to lower the serum uric acid (SUA) level. To elucidate the mechanism responsible for this reduction, SUA and the urinary excretion rate of uric acid (UEUA) were analysed after the oral administration of luseogliflozin, a SGLT2 inhibitor, to healthy subjects. After dosing, SUA decreased, and a negative correlation was observed between the SUA level and the UEUA, suggesting that SUA decreased as a result of the increase in the UEUA. The increase in UEUA was correlated with an increase in urinary d-glucose excretion, but not with the plasma luseogliflozin concentration. Additionally, in vitro transport experiments showed that luseogliflozin had no direct effect on the transporters involved in renal UA reabsorption. To explain that the increase in UEUA is likely due to glycosuria, the study focused on the facilitative glucose transporter 9 isoform 2 (GLUT9ΔN, SLC2A9b), which is expressed at the apical membrane of the kidney tubular cells and transports both UA and d-glucose. It was observed that the efflux of [14C]UA in Xenopus oocytes expressing the GLUT9 isoform 2 was trans-stimulated by 10 mm d-glucose, a high concentration of glucose that existed under SGLT2 inhibition. On the other hand, the uptake of [14C]UA by oocytes was cis-inhibited by 100 mm d-glucose, a concentration assumed to exist in collecting ducts. In conclusion, it was demonstrated that the UEUA could potentially be increased by luseogliflozin-induced glycosuria, with alterations of UA transport activity because of urinary glucose. PMID:25044127

Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose.

PubMed

Olsen, Johan P; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

2016-06-01

The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

The effect of isoenzyme-selective PDE inhibitors on methacholine-induced contraction of guinea-pig and rat ileum.

PubMed Central

Tomkinson, A.; Raeburn, D.

1996-01-01

1. We have examined the effects of the isoenzyme-selective phosphodiesterase (PDE) inhibitors, vinpocetine (type 1), siguazodan (type 3), rolipram (type 4) and zaprinast (type 5) and the non-selective PDE inhibitor enprofylline on methacholine (MCh) contractile concentration-response curves on guinea-pig and rat isolated ileum. 2. In guinea-pig ileum, vinpocetine (10-300 microM), zaprinast (1-300 microM) and enprofylline (100-1000 microM) produced a concentration-dependent depression of the maximum response (Emax) to MCh only without effect on the MCh EC50 values (rank order of potency: zaprinast > vinpocetine > enprofylline). In contrast, siguazodan (10-300 microM) and rolipram (10-300 microM) produced a rightward displacement of the MCh concentration-response curve (increase in EC50: rank order; rolipram > siguazodan), with effects on the MCh maximum seen only at higher concentrations. 3. In the rat ileum, vinpocetine (10-300 microM), zaprinast (0.1-300 microM) and enprofylline (100-1000 microM) caused depression of the MCh maximum contraction (rank order: zaprinast > vinpocetine > enprofylline). Low concentrations of rolipram and siguazodan had no significant effect on the MCh maximum. In the presence of higher concentrations (> 100 microM) of rolipram and siguazodan, a maximum response was not achieved at the highest concentration of MCh tested. As in the guinea-pig ileum, only rolipram (10-300 microM) and siguazodan (10-300 microM) produced a significant, concentration-dependent, rightward displacement of the MCh concentration-response curve (increase in EC50: rank order: rolipram > siguazodan). 4. In the guinea-pig ileum, isoprenaline (0.1 microM) produced a rightward displacement (approximately 3 fold) of the MCh concentration-response curve, accompanied by a significant depression of the maximum response. Increasing the isoprenaline concentration (1 microM) had no further effect on either parameter. Sodium nitroprusside (SNP, > or = 10 microM) produced a concentration-dependent depression of the MCh maximum without an effect on the EC50. 5. In the rat ileum, isoprenaline (1 microM) produced a concentration-dependent rightward displacement (approximately 2.8 fold) of the MCh concentration-response curve with depression of the MCh maximum at higher (> or = 100 microM) concentrations. SNP produced depression of the MCh maximum at a concentration of 10 microM and above. Effects on the MCh EC50 were seen only at 100 and 300 microM. 6. In guinea-pig ileum, isoprenaline (0.1 microM) in combination with rolipram (10 microM) further increased the MCh EC50 and reduced the MCh maximum. The combination of SNP (10 microM) with zaprinast (0.1 microM) produced no further significant effect than SNP alone. 7. In rat ileum, isoprenaline (1 microM) in combination with rolipram (10 microM) further increased the EC50 and reduced the maximum. SNP (10 microM) had no significant effect on either the MCh maximum or EC50. A combination with zaprinast (1 microM) had no further effect. 8. In conclusion, all the PDE inhibitors tested produced a concentration-dependent inhibition of the MCh concentration-response curve, indicating a modulator role for the PDE isoenzymes in gastrointestinal smooth muscle contractility. The PDE inhibitors that elevate cyclic GMP produced a depression of the MCh maximum response only, whilst those that elevate cyclic AMP produced a rightward displacement of the MCh concentration-response curve. This was confirmed by the use of isoprenaline and SNP. This difference in the type of inhibition produced by these PDE isoenzyme inhibitors may reflect a different intracellular site/mechanism by which the cyclic AMP- and cyclic GMP-activated kinases act functionally to antagonize the contractile response. PMID:8864552

Peptide Inhibitors of the Amyloidogenesis of IAPP: Verification of the Hairpin Binding Geometry Hypothesis

PubMed Central

Sivanesam, Kalkena; Shu, Irene; Huggins, Kelly N. L.; Tatarek-Nossol, Marianna; Kapurniotu, Aphrodite; Andersen, Niels H.

2016-01-01

Versions of a previously discovered β-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at sub-stoichiometric concentrations relative to this amyloidogenic peptide. The hairpin binding hypothesis stands confirmed. PMID:27317951

Cetyltrimethyl Ammonium Bromide as Corrosion Inhibitor for Zinc Used in Hydrochloric Acid

NASA Astrophysics Data System (ADS)

Sun, C. X.; Du, J. J.; Ma, Z. W.; Huang, C. S.; Wu, J. Y.

2018-05-01

A compound inhibitor composed of cetyltrimethyl ammonium bromide (CTAB) and bromohexadecyl pyridine was tested as corrosion inhibitor for zinc in hydrochloric acid. The results of static coupon test show that the compound inhibitor can effectively protect zinc from corrosion and the best concentration ratio is CTAB 50 mg/L and bromohexadecyl pyridine 200 mg/L. The polarization results show that the compound inhibitor will cause a negative shift of E0 of zinc in hydrochloric acid. The EIS (electrchemical impedance spectra) results show that the inhibitor leads to a bigger radius and has one time constant. SEM results show that the CTAB and bromohexadecyl pyridine form a uniform and compact membrane on the surface of zinc that can protect zinc from corroding effectively.

High-Throughput Analysis of Ovarian Cycle Disruption by Mixtures of Aromatase Inhibitors

PubMed Central

Golbamaki-Bakhtyari, Nazanin; Kovarich, Simona; Tebby, Cleo; Gabb, Henry A.; Lemazurier, Emmanuel

2017-01-01

Background: Combining computational toxicology with ExpoCast exposure estimates and ToxCast™ assay data gives us access to predictions of human health risks stemming from exposures to chemical mixtures. Objectives: We explored, through mathematical modeling and simulations, the size of potential effects of random mixtures of aromatase inhibitors on the dynamics of women's menstrual cycles. Methods: We simulated random exposures to millions of potential mixtures of 86 aromatase inhibitors. A pharmacokinetic model of intake and disposition of the chemicals predicted their internal concentration as a function of time (up to 2 y). A ToxCast™ aromatase assay provided concentration–inhibition relationships for each chemical. The resulting total aromatase inhibition was input to a mathematical model of the hormonal hypothalamus–pituitary–ovarian control of ovulation in women. Results: Above 10% inhibition of estradiol synthesis by aromatase inhibitors, noticeable (eventually reversible) effects on ovulation were predicted. Exposures to individual chemicals never led to such effects. In our best estimate, ∼10% of the combined exposures simulated had mild to catastrophic impacts on ovulation. A lower bound on that figure, obtained using an optimistic exposure scenario, was 0.3%. Conclusions: These results demonstrate the possibility to predict large-scale mixture effects for endocrine disrupters with a predictive toxicology approach that is suitable for high-throughput ranking and risk assessment. The size of the effects predicted is consistent with an increased risk of infertility in women from everyday exposures to our chemical environment. https://doi.org/10.1289/EHP742 PMID:28886606

Bacterial fatty acid metabolism in modern antibiotic discovery.

PubMed

Yao, Jiangwei; Rock, Charles O

2017-11-01

Bacterial fatty acid synthesis is essential for many pathogens and different from the mammalian counterpart. These features make bacterial fatty acid synthesis a desirable target for antibiotic discovery. The structural divergence of the conserved enzymes and the presence of different isozymes catalyzing the same reactions in the pathway make bacterial fatty acid synthesis a narrow spectrum target rather than the traditional broad spectrum target. Furthermore, bacterial fatty acid synthesis inhibitors are single-targeting, rather than multi-targeting like traditional monotherapeutic, broad-spectrum antibiotics. The single-targeting nature of bacterial fatty acid synthesis inhibitors makes overcoming fast-developing, target-based resistance a necessary consideration for antibiotic development. Target-based resistance can be overcome through multi-targeting inhibitors, a cocktail of single-targeting inhibitors, or by making the single targeting inhibitor sufficiently high affinity through a pathogen selective approach such that target-based mutants are still susceptible to therapeutic concentrations of drug. Many of the pathogens requiring new antibiotic treatment options encode for essential bacterial fatty acid synthesis enzymes. This review will evaluate the most promising targets in bacterial fatty acid metabolism for antibiotic therapeutics development and review the potential and challenges in advancing each of these targets to the clinic and circumventing target-based resistance. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibitors of BRD4 Protein from a Marine-Derived Fungus Alternaria sp. NH-F6

PubMed Central

Ding, Hui; Zhang, Dashan; Zhou, Biao; Ma, Zhongjun

2017-01-01

Bromodomains (BRD) are readers of the epigenetic code that regulate gene transcription through their recognition of acetyl-lysine modified histone tails. Recently, bromodomain-containing proteins such as BRD4 have been demonstrated to be druggable through the discovery of potent inhibitors. These protein–protein interaction inhibitors have the potential to modulate multiple diseases by their profound anti-inflammatory and antiproliferative effects. In order to explore new BRD4 inhibitors as well as lead compounds for the development of new drugs, the secondary metabolites of Alternaria sp. NH-F6, a fungus isolated from deep-sea sediment samples, were analyzed systematically. Five new compounds including two new perylenequinones (1–2), one new alternaric acid (3), 2-(N-vinylacetamide)-4-hydroxymethyl-3-ene-butyrolactone (4), one new cerebroside (5), together with 19 known compounds (6–24) were isolated from the ethyl acetate extracts of this strain. Their structures were elucidated using nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses. Finally, all these compounds were evaluated for their inhibitory activity against BRD4 protein, and compound 2 exhibited a potent inhibition rate of 88.1% at a concentration of 10 µM. This research provides a new BRD4 inhibitor which may possess potential antitumoral, antiviral, or anti-inflammatory pharmaceutical values. PMID:28300771

Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid.

PubMed Central

Barelli, H; Dive, V; Yiotakis, A; Vincent, J P; Checler, F

1992-01-01

A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides. PMID:1332678

Endogenous benzodiazepine-like compounds and diazepam binding inhibitor in serum of patients with liver cirrhosis with and without overt encephalopathy

PubMed Central

Avallone, R; Zeneroli, M; Venturini, I; Corsi, L; Schreier, P; Kleinschnitz, M; Ferrarese, C; Farina, F; Baraldi, C; Pecora, N; Frigo, M; Baraldi, M

1998-01-01

Background/Aim—Despite some controversy, it has been suggested that endogenous benzodiazepine plays a role in the pathogenesis of hepatic encephalopathy. The aim of the present study was to evaluate the concentrations of endogenous benzodiazepines and the peptide, diazepam binding inhibitor, in the blood of patients with liver cirrhosis with and without overt encephalopathy, and to compare these levels with those of consumers of commercial benzodiazepines. 
Subjects—Normal subjects (90), benzodiazepine consumers (14), and cirrhotic patients (113) were studied. 
Methods—Endogenous benzodiazepines were measured by the radioligand binding technique after high performance liquid chromatography (HPLC) purification. The presence of diazepam and N-desmethyldiazepam was assayed by HPLC-electrospray tandem mass spectrometry. Diazepam binding inhibitor was studied in serum by radioimmunoassay. 
Results—Endogenous benzodiazepines were below the limit of detection in 7% of patients with encephalopathy. When detectable, their levels were at least comparable with those of benzodiazepine consumers and correlated with the liver dysfunction but not the stage of encephalopathy. Serum levels of diazepam binding inhibitor tended to decrease when endogenous benzodiazepines levels increased. 
Conclusions—Endogenous benzodiazepines may accumulate in patients with liver cirrhosis during the course of the disease, and the phenomenon appears to be independent of the presence or absence of encephalopathy. 

 Keywords: benzodiazepine consumers; diazepam binding inhibitor; endogenous benzodiazepines; liver cirrhosis; overt hepatic encephalopathy PMID:9691927

The novel imidazopyridine 2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) is a highly selective inhibitor of the inducible nitric-oxide synthase.

PubMed

Strub, Andreas; Ulrich, Wolf-Rüdiger; Hesslinger, Christian; Eltze, Manfrid; Fuchss, Thomas; Strassner, Jochen; Strand, Susanne; Lehner, Martin D; Boer, Rainer

2006-01-01

We have identified imidazopyridine derivatives as a novel class of NO synthase inhibitors with high selectivity for the inducible isoform. 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) showed half-maximal inhibition of crudely purified human inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NO synthases at 86 nM, 17 microM, and 162 microM, respectively. Inhibition of inducible NO synthase was competitive with l-arginine, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. In radioligand and surface plasmon resonance experiments, BYK191023 exhibited an affinity for iNOS, nNOS, and eNOS of 450 nM, 30 microM, and >500 microM, respectively. Inhibition of cellular nitrate/nitrite synthesis in RAW, rat mesangium, and human embryonic kidney 293 cells after iNOS induction showed 40- to 100-fold higher IC(50) values than at the isolated enzyme, in agreement with the much higher l-arginine concentrations in cell culture media and inside intact cells. BYK191023 did not show any toxicity in various rodent and human cell lines up to high micromolar concentrations. The inhibitory potency of BYK191023 was tested in isolated organ models of iNOS (lipopolysaccharide-treated and phenylephrine-precontracted rat aorta; IC(50) = 7 microM), eNOS (arecaidine propargyl ester-induced relaxation of phenylephrine-precontracted rat aorta; IC(50) > 100 microM), and nNOS (field-stimulated relaxation of phenylephrine-precontracted rabbit corpus cavernosum; IC(50) > 100 microM). These data confirm the high selectivity of BYK191023 for iNOS over eNOS and nNOS found at isolated enzymes. In summary, we have identified a new highly selective iNOS inhibitor structurally unrelated to known compounds and l-arginine. BYK191023 is a valuable tool for the investigation of iNOS-mediated effects in vitro and in vivo.

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) III: Endogenous Inhibition of Angiotensin Converting Enzyme (ACE) Provides Protection against Cardiovascular Diseases

PubMed Central

Fagyas, Miklós; Úri, Katalin; Siket, Ivetta M.; Daragó, Andrea; Boczán, Judit; Bányai, Emese; Édes, István; Papp, Zoltán; Tóth, Attila

2014-01-01

ACE inhibitor drugs decrease mortality by up to one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors. Here we investigated the clinical significance of this potential endogenous ACE inhibition. ACE concentration and activity was measured in patient's serum samples (n = 151). ACE concentration was found to be in a wide range (47–288 ng/mL). ACE activity decreased with the increasing concentration of the serum albumin (HSA): ACE activity was 56±1 U/L in the presence of 2.4±0.3 mg/mL HSA, compared to 39±1 U/L in the presence of 12±1 mg/mL HSA (values are mean±SEM). Effects of the differences in ACE concentration were suppressed in human sera: patients with ACE DD genotype exhibited a 64% higher serum ACE concentration (range, 74–288 ng/mL, median, 155.2 ng/mL, n = 52) compared to patients with II genotype (range, 47–194 ng/mL, median, 94.5 ng/mL, n = 28) while the difference in ACE activities was only 32% (range, 27.3–59.8 U/L, median, 43.11 U/L, and range 15.6–55.4 U/L, median, 32.74 U/L, respectively) in the presence of 12±1 mg/mL HSA. No correlations were found between serum ACE concentration (or genotype) and cardiovascular diseases, in accordance with the proposed suppressed physiological ACE activities by HSA (concentration in the sera of these patients: 48.5±0.5 mg/mL) or other endogenous inhibitors. Main implications are that (1) physiological ACE activity can be stabilized at a low level by endogenous ACE inhibitors, such as HSA; (2) angiotensin II elimination may have a significant role in angiotensin II related pathologies. PMID:24690767

Copper and Antibiotics: Discovery, Modes of Action, and Opportunities for Medicinal Applications.

PubMed

Dalecki, Alex G; Crawford, Cameron L; Wolschendorf, Frank

2017-01-01

Copper is a ubiquitous element in the environment as well as living organisms, with its redox capabilities and complexation potential making it indispensable for many cellular functions. However, these same properties can be highly detrimental to prokaryotes and eukaryotes when not properly controlled, damaging many biomolecules including DNA, lipids, and proteins. To restrict free copper concentrations, all bacteria have developed mechanisms of resistance, sequestering and effluxing labile copper to minimize its deleterious effects. This weakness is actively exploited by phagocytes, which utilize a copper burst to destroy pathogens. Though administration of free copper is an unreasonable therapeutic antimicrobial itself, due to insufficient selectivity between host and pathogen, small-molecule ligands may provide an opportunity for therapeutic mimicry of the immune system. By modulating cellular entry, complex stability, resistance evasion, and target selectivity, ligand/metal coordination complexes can synergistically result in high levels of antibacterial activity. Several established therapeutic drugs, such as disulfiram and pyrithione, display remarkable copper-dependent inhibitory activity. These findings have led to development of new drug discovery techniques, using copper ions as the focal point. High-throughput screens for copper-dependent inhibitors against Mycobacterium tuberculosis and Staphylococcus aureus uncovered several new compounds, including a new class of inhibitors, the NNSNs. In this review, we highlight the microbial biology of copper, its antibacterial activities, and mechanisms to discover new inhibitors that synergize with copper. © 2017 Elsevier Ltd. All rights reserved.

Exposure to Acetylcholinesterase Inhibitors Alters the Physiology and Motor Function of Honeybees

PubMed Central

Williamson, Sally M.; Moffat, Christopher; Gomersall, Martha A. E.; Saranzewa, Nastja; Connolly, Christopher N.; Wright, Geraldine A.

2013-01-01

Cholinergic signaling is fundamental to neuromuscular function in most organisms. Sub-lethal doses of neurotoxic pesticides that target cholinergic signaling can alter the behavior of insects in subtle ways; their influence on non-target organisms may not be readily apparent in simple mortality studies. Beneficial arthropods such as honeybees perform sophisticated behavioral sequences during foraging that, if influenced by pesticides, could impair foraging success and reduce colony health. Here, we investigate the behavioral effects on honeybees of exposure to a selection of pesticides that target cholinergic signaling by inhibiting acetylcholinesterase (AChE). To examine how continued exposure to AChE inhibitors affected motor function, we fed adult foraging worker honeybees sub-lethal concentrations of these compounds in sucrose solution for 24 h. Using an assay for locomotion in bees, we scored walking, stopped, grooming, and upside down behavior continuously for 15 min. At a 10 nM concentration, all the AChE inhibitors caused similar effects on behavior, notably increased grooming activity and changes in the frequency of bouts of behavior such as head grooming. Coumaphos caused dose-dependent effects on locomotion as well as grooming behavior, and a 1 μM concentration of coumaphos induced symptoms of malaise such as abdomen grooming and defecation. Biochemical assays confirmed that the four compounds we assayed (coumaphos, aldicarb, chlorpyrifos, and donepezil) or their metabolites acted as AChE inhibitors in bees. Furthermore, we show that transcript expression levels of two honeybee AChE inhibitors were selectively upregulated in the brain and in gut tissues in response to AChE inhibitor exposure. The results of our study imply that the effects of pesticides that rely on this mode of action have subtle yet profound effects on physiological effects on behavior that could lead to reduced survival. PMID:23386834

Potentiation by cholinesterase inhibitors of cholinergic activity in rat isolated stomach and colon.

PubMed

Jarvie, Emma M; Cellek, Selim; Sanger, Gareth J

2008-01-01

Acetylcholinesterase (AChE) inhibitors stimulate gastrointestinal (GI) motility and are potential treatments of conditions associated with inadequate GI motility. The ability of itopride to facilitate neuronally (predominantly cholinergic) mediated contractions of rat isolated stomach, evoked by electrical field stimulation (EFS), has been compared with other cholinesterase inhibitors and with tegaserod, a clinically effective prokinetic and non-selective 5-HT(4) receptor agonist which also facilitates GI cholinergic function. Neostigmine greatly increased EFS-evoked contractions over a narrow concentration range (0.01-1 microM; 754+/-337% facilitation at 1 microM); higher concentrations (1, 3 microM) also increased muscle tension. Donepezil increased EFS-evoked contractions gradually over the full range of concentrations (0.01-10 microM; maximum increase 516+/-20% at 10 microM). Itopride increased the contractions even more gradually, rising to 188+/-84% at 10 microM. The butyrylcholinesterase inhibitor iso-OMPA 0.01-10 microM also increased EFS-evoked contractions, to a maximum of 36+/-5.0% at 10 microM, similar to that caused by tegaserod (35+/-5.2% increase at 1 microM). The effects of tegaserod, but not itopride were inhibited by the 5-HT(4) receptor antagonist SB-204070A 0.3 microM. In rat isolated colon, neostigmine was again the most efficacious, causing a defined maximum increase in EFS-evoked contractions (343+/-82% at 10 microM), without changing muscle tension. Maximum increases caused by donepezil and itopride were, respectively, 57.6+/-20 and 43+/-15% at 10 microM. These data indicate that the abilities of different AChE inhibitors to increase GI cholinergic activity differ markedly. Understanding the reasons is essential if AChE inhibitors are to be optimally developed as GI prokinetics.

Exposure to acetylcholinesterase inhibitors alters the physiology and motor function of honeybees.

PubMed

Williamson, Sally M; Moffat, Christopher; Gomersall, Martha A E; Saranzewa, Nastja; Connolly, Christopher N; Wright, Geraldine A

2013-01-01

Cholinergic signaling is fundamental to neuromuscular function in most organisms. Sub-lethal doses of neurotoxic pesticides that target cholinergic signaling can alter the behavior of insects in subtle ways; their influence on non-target organisms may not be readily apparent in simple mortality studies. Beneficial arthropods such as honeybees perform sophisticated behavioral sequences during foraging that, if influenced by pesticides, could impair foraging success and reduce colony health. Here, we investigate the behavioral effects on honeybees of exposure to a selection of pesticides that target cholinergic signaling by inhibiting acetylcholinesterase (AChE). To examine how continued exposure to AChE inhibitors affected motor function, we fed adult foraging worker honeybees sub-lethal concentrations of these compounds in sucrose solution for 24 h. Using an assay for locomotion in bees, we scored walking, stopped, grooming, and upside down behavior continuously for 15 min. At a 10 nM concentration, all the AChE inhibitors caused similar effects on behavior, notably increased grooming activity and changes in the frequency of bouts of behavior such as head grooming. Coumaphos caused dose-dependent effects on locomotion as well as grooming behavior, and a 1 μM concentration of coumaphos induced symptoms of malaise such as abdomen grooming and defecation. Biochemical assays confirmed that the four compounds we assayed (coumaphos, aldicarb, chlorpyrifos, and donepezil) or their metabolites acted as AChE inhibitors in bees. Furthermore, we show that transcript expression levels of two honeybee AChE inhibitors were selectively upregulated in the brain and in gut tissues in response to AChE inhibitor exposure. The results of our study imply that the effects of pesticides that rely on this mode of action have subtle yet profound effects on physiological effects on behavior that could lead to reduced survival.

The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

PubMed

Lomas, D A; Stone, S R; Llewellyn-Jones, C; Keogan, M T; Wang, Z M; Rubin, H; Carrell, R W; Stockley, R A

1995-10-06

Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

The role of enzyme and substrate concentration in the evaluation of serum angiotensin converting enzyme (ACE) inhibition by enalaprilat in vitro.

PubMed

Weisser, K; Schloos, J

1991-10-09

The relationship between serum angiotensin converting enzyme (ACE) activity and concentration of the ACE inhibitor enalaprilat was determined in vitro in the presence of different concentrations (S = 4-200 mM) of the substrate Hip-Gly-Gly. From Henderson plots, a competitive tight-binding relationship between enalaprilat and serum ACE was found yielding a value of approximately 5 nM for serum ACE concentration (Et) and an inhibition constant (Ki) for enalaprilat of approximately 0.1 nM. A plot of reaction velocity (Vi) versus total inhibitor concentration (It) exhibited a non-parallel shift of the inhibition curve to the right with increasing S. This was reflected by apparent Hill coefficients greater than 1 when the commonly used inhibitory sigmoid concentration-effect model (Emax model) was applied to the data. Slopes greater than 1 were obviously due to discrepancies between the free inhibitor concentration (If) present in the assay and It plotted on the abscissa and could, therefore, be indicators of tight-binding conditions. Thus, the sigmoid Emax model leads to an overestimation of Ki. Therefore, a modification of the inhibitory sigmoid Emax model (called "Emax tight model") was applied, which accounts for the depletion of If by binding, refers to It and allows estimation of the parameters Et and IC50f (free concentration of inhibitor when 50% inhibition occurs) using non-linear regression analysis. This model could describe the non-symmetrical shape of the inhibition curves and the results for Ki and Et correlated very well with those derived from the Henderson plots. The latter findings confirm that the degree of ACE inhibition measured in vitro is, in fact, dependent on the concentration of substrate and enzyme present in the assay. This is of importance not only for the correct evaluation of Ki but also for the interpretation of the time course of serum ACE inhibition measured ex vivo. The non-linear model has some advantages over the linear Henderson equation: it is directly applicable without conversion of the data and avoids the stochastic dependency of the variables, allowing non-linear regression of all data points contributing with the same weight.

Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.

PubMed

Jenkins, David Paul; Yu, Weifeng; Brown, Brandon M; Løjkner, Lars Damgaard; Wulff, Heike

2013-01-01

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

Responses of the terrestrial ammonia-oxidizing archaeon Ca. Nitrososphaera viennensis and the ammonia-oxidizing bacterium Nitrosospira multiformis to nitrification inhibitors.

PubMed

Shen, Tianlin; Stieglmeier, Michaela; Dai, Jiulan; Urich, Tim; Schleper, Christa

2013-07-01

Nitrification inhibitors have been used for decades to improve nitrogen fertilizer utilization in farmland. However, their effect on ammonia-oxidizing Archaea (AOA) in soil is little explored. Here, we compared the impact of diverse inhibitors on nitrification activity of the soil archaeon Ca. Nitrososphaera viennensis EN76 and compared it to that of the ammonia-oxidizing bacterium (AOB) Nitrosospira multiformis. Allylthiourea, amidinothiourea, and dicyandiamide (DCD) inhibited ammonia oxidation in cultures of both N. multiformis and N. viennensis, but the effect on N. viennensis was markedly lower. In particular, the effective concentration 50 (EC50) of allylthiourea was 1000 times higher for the AOA culture. Among the tested nitrification inhibitors, DCD was the least potent against N. viennensis. Nitrapyrin had at the maximal soluble concentration only a very weak inhibitory effect on the AOB N. multiformis, but showed a moderate effect on the AOA. The antibiotic sulfathiazole inhibited the bacterium, but barely affected the archaeon. Only the NO-scavenger carboxy-PTIO had a strong inhibitory effect on the archaeon, but had little effect on the bacterium in the concentrations tested. Our results reflect the fundamental metabolic and cellular differences of AOA and AOB and will be useful for future applications of inhibitors aimed at distinguishing activities of AOA and AOB in soil environments. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Corrosion inhibitor for aqueous ammonia absorption system

DOEpatents

Phillips, Benjamin A.; Whitlow, Eugene P.

1998-09-22

A method of inhibiting corrosion and the formation of hydrogen and thus improving absorption in an ammonia/water absorption refrigeration, air conditioning or heat pump system by maintaining the hydroxyl ion concentration of the aqueous ammonia working fluid within a selected range under anaerobic conditions at temperatures up to 425.degree. F. This hydroxyl ion concentration is maintained by introducing to the aqueous ammonia working fluid an inhibitor in an amount effective to produce a hydroxyl ion concentration corresponding to a normality of the inhibitor relative to the water content ranging from about 0.015 N to about 0.2 N at 25.degree. C. Also, working fluids for inhibiting the corrosion of carbon steel and resulting hydrogen formation and improving absorption in an ammonia/water absorption system under anaerobic conditions at up to 425.degree. F. The working fluids may be aqueous solutions of ammonia and a strong base or aqueous solutions of ammonia, a strong base, and a specified buffer.

Corrosion inhibitor for aqueous ammonia absorption system

DOEpatents

Phillips, B.A.; Whitlow, E.P.

1998-09-22

A method is described for inhibiting corrosion and the formation of hydrogen and thus improving absorption in an ammonia/water absorption refrigeration, air conditioning or heat pump system by maintaining the hydroxyl ion concentration of the aqueous ammonia working fluid within a selected range under anaerobic conditions at temperatures up to 425 F. This hydroxyl ion concentration is maintained by introducing to the aqueous ammonia working fluid an inhibitor in an amount effective to produce a hydroxyl ion concentration corresponding to a normality of the inhibitor relative to the water content ranging from about 0.015 N to about 0.2 N at 25 C. Also, working fluids for inhibiting the corrosion of carbon steel and resulting hydrogen formation and improving absorption in an ammonia/water absorption system under anaerobic conditions at up to 425 F. The working fluids may be aqueous solutions of ammonia and a strong base or aqueous solutions of ammonia, a strong base, and a specified buffer. 5 figs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stauderman, K.A.; Gandhi, V.C.; Jones, D.J.

Fluoxetine, a selective 5-Ht uptake inhibitor, inhibited 15 mM K{sup +}-induced ({sup 3}H)5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K{sup +} used to depolarize the synaptosomes and the concentration of external Ca{sup 2+}. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of ({sup 3}H)5-HT releasemore » induced by the Ca{sup 2+}-ionophore A 23187 or Ca{sup 2+}-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K{sup +}-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca{sup 2+} channels and Ca{sup 2+} entry.« less

Is platelet function as measured by Thrombelastograph monitoring in whole blood affected by platelet inhibitors?

PubMed

Bailey, Lori A; Sistino, Joseph J; Uber, Walter E

2005-03-01

Platelet inhibitors, especially the glycoprotein (GP) IIb/IIIa receptor antagonists, have demonstrated their effectiveness in reducing the acute ischemic complications of percutaneous coronary intervention (PCI) and in improving clinical outcomes in patients with acute coronary crisis. Three common platelet inhibitors observed in emergent cardiopulmonary bypass (CPB) for failed PCI are abciximab, eptifibatide, and tirofiban. An in vitro model was constructed in two parts to determine whether platelet aggregation inhibition induced by platelet inhibitors would be demonstrated by the Thrombelastograph (TEG) monitor when compared with baseline samples with no platelet inhibitor. In part A, 20 mL of fresh whole blood was divided into four groups: group I = baseline, group A = abcix-imab microg/mL, group E = eptifibatide ng/mL, and group T = tirofiban ng/mL. Platelet inhibitor concentrations in whole blood were derived starting with reported serum concentrations with escalation to achieve 80% platelet inhibition using the Medtronic hemoSTATUS and/or Lumi-aggregometer. A concentration range determined by our in vitro tests were chosen for each drug using concentrations achieving less than, equal to, or greater than 80% platelet inhibition. In part B, TEG analysis was then performed using baseline and concentrations for each drug derived in part A. Parameters measured were clot formation reaction time (R), coagulation time (K), maximum amplitude (MA) and alpha angle (A). Groups E1000 and E2000 extended R over control by 37% and 23%, respectively (p = 0.01 and 0.03). Groups E1000 and E2000 increased K times by 45% and 58% (p = .02 and .04). T160 samples prolonged K by 20% (p = 0.01). The angle or clot strength (A) was decreased in groups T160 and E1000 by 23% (+ 7.06 SD) and 18% (+ 11.23 SD), respectively (p = 0.001 and 0.01). The MA decrease was statistically significant in the T160, E1000 and E2000 by 9%, 6% and 13% respectively (p = 0.01). Samples treated with abciximab were comparable to control values for all parameters measured. Although statistical significance could be demonstrated with some parameters, sensitivity was only observed at increased doses and was not seen with all agents tested. In our in vitro model, the TEG monitor was unable to demonstrate clinically significant differences in platelet function and may not be reflective of platelet function in samples which have been treated with these GP IIb/IIIa inhibitors.

Mechanism of the Antimicrobial Action of Pyrithione: Effects on Membrane Transport, ATP Levels, and Protein Synthesis

PubMed Central

Chandler, Carol J.; Segel, Irwin H.

1978-01-01

Pyrithione is a general inhibitor of membrane transport processes in fungi. A brief preincubation of Penicillium mycelia with pyrithione resulted in a marked decrease in the activities of a variety of independently regulated transport systems, including those for inorganic sulfate, inorganic phosphate, methylamine (actually, the NH4+ permease), choline-O-sulfate, glucose, l-methionine (a specific system), and several hydrophobic l-α-amino acids (the general amino acid permease). The degree of inhibition at any fixed pyrithione concentration and exposure time increased as the pH of the incubation medium was decreased. This result strongly suggests that the active species is the un-ionized molecule and that pyrithione acts by collapsing a transmembrane ΔpH driving force. The degree of transport inhibition caused by a given concentration of pyrithione increased with increasing time of exposure to the inhibitor. However, exposure time and pyrithione concentration were not reciprocally related. At “low” pyrithione concentrations, transport inhibition plateaued at some finite value. This observation suggests that the fungi can detoxify low levels of the inhibitor. The concentration of pyrithione required for a given degree of growth inhibition increased as the experimental mycelial density increased. This phenomenon was consistent with the suggestion that the fungi are capable of inactivating pyrithione. PMID:28693

The CYP2C8 inhibitor gemfibrozil does not affect the pharmacokinetics of zafirlukast.

PubMed

Karonen, Tiina; Neuvonen, Pertti J; Backman, Janne T

2011-02-01

Gemfibrozil, a strong inhibitor of cytochrome P450 (CYP) 2C8 in vivo, was recently found to markedly increase the plasma concentrations of montelukast in humans. Like montelukast, zafirlukast is a substrate of CYP2C9 and CYP3A4 and a potent inhibitor of CYP2C8 in vitro. To investigate the contribution of CYP2C8 to the metabolism of zafirlukast in vivo, we studied the effect of gemfibrozil on the pharmacokinetics of zafirlukast. Ten healthy subjects in a randomized cross-over study took gemfibrozil 600 mg or placebo twice daily for 5 days, and on day 3, a single oral dose of 20 mg zafirlukast. The plasma concentrations of zafirlukast were measured for 72 h postdose. The mean total area under the plasma concentration-time curve of zafirlukast during the gemfibrozil phase was 102% (geometric mean ratio; 95% confidence interval 89-116%) of that during the placebo phase. Furthermore, there were no statistically significant differences in the peak plasma concentration, time of peak concentration, or elimination half-life of zafirlukast between the phases. Gemfibrozil has no effect on the pharmacokinetics of zafirlukast, indicating that CYP2C8 does not play a significant role in the elimination of zafirlukast.

Chromenylchalcones with inhibitory effects on monoamine oxidase B.

PubMed

Jo, Geunhyeong; Ahn, Seunghyun; Kim, Bong-Gyu; Park, Hye Ri; Kim, Young Hwa; Choo, Hyun Ah; Koh, Dongsoo; Chong, Youhoon; Ahn, Joong-Hoon; Lim, Yoongho

2013-12-15

Structure-activity relationship (SAR) calculations were used to find monoamine oxidase-B (MAO-B) inhibitors by identifying pharmacophores exhibiting high inhibitory activities. Several such chromenylchalcones were designed and synthesized accordingly. Their inhibitory effects on MAO-B were determined using an HPLC-based method and an MAO-B enzyme assay kit. (E)-3-(6-Methoxy-2H-chromen-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one exhibited a half-maximal inhibitory concentration of 320 nM. Its molecular-level binding mode with the three-dimensional structure of MAO-B was elucidated using an in silico docking study. The chromenylchalcone scaffold, which is derived from natural products including isoflavonoids and chalcones, had not been previously reported as an MAO-B inhibitor. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat.

PubMed

Killi, Uday K; Wsol, Vladimir; Soukup, Ondrej; Kuca, Kamil; Winder, Michael; Tobin, Gunnar

2014-02-01

Obidoxime, a weak acetylcholine-esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration-dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90-290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80-450%). Physostigmine concentration-dependently increased methacholine-evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor-evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime. © 2013 Wiley Publishing Asia Pty Ltd.

Curcumin and its cyclohexanone analogue inhibited human Equilibrative nucleoside transporter 1 (ENT1) in pancreatic cancer cells.

PubMed

Revalde, Jezrael L; Li, Yan; Wijeratne, Tharaka S; Bugde, Piyush; Hawkins, Bill C; Rosengren, Rhonda J; Paxton, James W

2017-05-15

Our group investigated combining the phytochemical curcumin and gemcitabine in a liposome, to improve gemcitabine's activity against pancreatic tumours. While optimising the curcumin: gemcitabine ratio for co-encapsulation, we found that increasing curcumin concentrations relative to gemcitabine resulted in antagonistic interactions. As curcumin is a promiscuous transporter inhibitor; we suspected that increased resistance occurred via inhibition of Equilibrative nucleoside transporter 1 (ENT1)-mediated gemcitabine uptake. To test our hypothesis, we determined whether curcumin and a related analogue, 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone (or A13), inhibited ENT1-mediated accumulation of [ 3 H]uridine and [ 3 H]gemcitabine into pancreatic cancer cells. We then confirmed the inhibition of gemcitabine accumulation by investigating whether curcumin/A13 could increase gemcitabine resistance in growth inhibition assays. We found that curcumin and A13 concentration-dependently inhibited the ENT1-mediated accumulation of both uridine and gemcitabine in MIA PaCa-2 and PANC-1 cells. We also found that non-toxic concentrations of curcumin and A13 significantly increased the resistance of both cell lines to gemcitabine. Increased resistance only occurred when curcumin/A13 was co-incubated with gemcitabine, and not with sequential exposure (i.e., curcumin first, followed by gemcitabine, or vice versa). We also found that the curcumin analogue (3E,5E)-3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (or EF24) did not inhibit gemcitabine accumulation, making it more suitable in combinations than curcumin/A13. From these results, we concluded that curcumin and A13 are inhibitors of the ENT1 transporter, but only at high concentrations (2-20µM). Curcumin is unlikely to inhibit gemcitabine uptake in tumours but may interfere with the oral absorption of ENT1 substrates due to high gut concentrations readily achievable from over-the-counter tablets/capsules. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficiency and corrosion rate analysis of organic inhibitor utilization from bawang dayak leaves (EleutherineamericanaMerr.) on API 5L steel

NASA Astrophysics Data System (ADS)

Sari, Shaimah Rinda; Sari, Eli Novita; Rizky, Yoel; Sulistijono, Triana, Yunita

2018-05-01

This research studied the inhibition of corrosion by bawang dayak leaves extract (EleutherineamericanaMerr.) on API 5L steel in brine water environment (3.5% NaCl). The inhibitor was extracted using maceration process from bawang dayak leaves that was cultivated in Paser District, East Kalimantan. The test of antioxidant activity showed that bawang dayak leaves extract is a very powerful antioxidant with IC50 value of 27.30204. The results from FTIR test show the presence of electronegative atoms and double bonds of the alkenes groups that provide the potential of the extract as a corrosion inhibitor. Efficiency of inhibition reached up to 93.158% for the addition of inhibitor with 300 ppm concentration and 20 days of immersion time. This inhibitory behavior is also supported by polarization measurements where the lowest corrosion rate of 0.00128 mm/year is obtained at the same concentration and immersion time.

The effect of MRN complex and ATM kinase inhibitors on Zebrafish embryonic development

NASA Astrophysics Data System (ADS)

Kumaran, Malina; Fazry, Shazrul

2018-04-01

Zebrafish is an ideal animal model to study developmental biology due to its transparent embryos and rapid development stages of embryogenesis. Here we investigate the role of DNA damage proteins, specifically Mre11/Rad50/NBN (MRN) complex and ataxia-telangiectasia mutated (ATM) kinase during embryogenesis by inhibiting its function using specific MRN complex (Mirin) and ATM Kinase inhibitors (Ku60019 and Ku55933). Zebrafish embryos at midblastula transition (MBT) stage are treated with Mirin, Ku60019 and Ku55933. The embryonic development of the embryos was monitored at 24 hours-post fertilisation (hpf), 48 hpf and 72 hpf. We observed that at the lowest concentrations (3 µM of Mirin, 1.5 nM of Ku60019 and 3 nM of Ku55933), the inhibitors treated embryos have 100% survivability. However, with increasing inhibitor concentration, the survivability drops. Control or mock treatment of all embryos shows 100 % survivability rate. This study suggests that DNA damage repair proteins may be crucial for normal zebrafish embryo development and survival.

Difluoromethane, a new and improved inhibitor of methanotrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, L.G.; Sasson, C.; Oremland, R.S.

1998-11-01

Difluoromethane (HFC-32; DFM) is compared to acetylene and methyl fluoride as an inhibitor of methanotrophy in cultures and soils. DFM was found to be a reversible inhibitor of CH{sub 4} oxidation by Methylococcus capsulatus (Bath). Consumption of CH{sub 4} in soil was blocked by additions of low levels of DFM (0.03 kPa), and this inhibition was reversed by DFM removal. Although a small quantity of DFM was consumed during these incubations, its remaining concentration was sufficiently elevated to sustain inhibition. Methanogenesis in anaerobic soil slurries, including acetoclastic methanogenesis, was unaffected by levels of DFM which inhibit methanotrophy. Low levels ofmore » DFM (0.03 kPa) also inhibited nitrification and N{sub 2}O production by soils. DFM is proposed as an improved inhibitor of CH{sub 4} oxidation over acetylene and/or methyl fluoride on the basis of its reversibility, its efficacy at low concentrations, its lack of inhibition of methanogenesis, and its low cost.« less

Phytoextraction of Cadmium and Zinc By Sedum plumbizincicola Using Different Nitrogen Fertilizers, a Nitrification Inhibitor and a Urease Inhibitor.

PubMed

Arnamwong, Suteera; Wu, Longhua; Hu, Pengjie; Yuan, Cheng; Thiravetyan, Paitip; Luo, Yongming; Christie, Peter

2015-01-01

Cadmium (Cd) and zinc (Zn) phytoavailability and their phytoextraction by Sedum plumbizincicola using different nitrogen fertilizers, nitrification inhibitor (dicyandiamide, DCD) and urease inhibitor (N-(n-Butyl) thiophosphoric triamide, NBPT) were investigated in pot experiments where the soil was contaminated with 0.99 mg kg(-1) of Cd and 241 mg kg(-1) Zn. The soil solution pH varied between 7.30 and 8.25 during plant growth which was little affected by the type of N fertilizer. The (NH4)2SO4+DCD treatment produced higher NH4+-N concentrations in soil solution than the (NH4)2SO4 and NaNO3 treatment which indicated that DCD addition inhibited the nitrification process. Shoot Cd and Zn concentrations across all treatments showed ranges of 52.9-88.3 and 2691-4276 mg kg(-1), respectively. The (NH4)2SO4+DCD treatment produced slightly higher but not significant Cd and Zn concentrations in the xylem sap than the NaNO3 treatment. Plant shoots grown with NaNO3 had higher Cd concentrations than (NH4)2SO4+DCD treatment at 24.0 and 15.4 mg kg(-1), respectively. N fertilizer application had no significant effect on shoot dry biomass. Total Cd uptake in the urea+DCD treatment was higher than in the control, urea+NBPT, urea+NBPT+DCD, or urea treatments, by about 17.5, 23.3, 10.7, and 25.1%, respectively.

Neutral endopeptidase regulates neurogenic inflammatory responses induced by stimulation of human oral keratinocytes with bacterial lipopolysaccharide and nicotine.

PubMed

Nakata, Motoki; Awano, Shuji; Kinoshita, Naomasa; Yoshida, Akihiro; Ansai, Toshihiro

2013-10-01

Neutral endopeptidase (NEP) is present on various epithelial cells and inactivates numerous physiologically active peptides. Neutral endopeptidase may regulate proinflammatory signals in oral mucosal epithelium. However, the function of NEP in oral mucosal epithelium is unknown. The present study investigated the action of NEP upon proinflammatory signals on human oral keratinocytes and the influence of endothelin-converting enzyme (ECE)-1, an enzyme similar to NEP, on the functions of NEP. Oral keratinocytes were cultured in medium containing inflammatory inducers [lipopolysaccharide (LPS) and nicotine], NEP inhibitors, and ECE-1/NEP inhibitors, either alone or in combination. The concentrations of substance P (SP) and interleukin-1β (IL-1β) were measured in the supernatant. Additionally, the concentrations of SP and IL-1β were measured in the supernatant of cells incubated with LPS or nicotine after transfection with NEP small interfering RNA (siRNA). The concentrations of SP and IL-1β were significantly increased in cells incubated with NEP inhibitors and, to a lesser extent, in cells incubated with ECE-1/NEP inhibitors, compared with controls (cells incubated with LPS or nicotine alone). The concentrations of SP and IL-1β in cells transfected with NEP siRNA were significantly augmented compared with controls. In conclusion, the present study demonstrated that NEP down-regulated the levels of SP and IL-1β produced from human oral keratinocytes, although ECE-1 may be partly related to the down-regulation. © 2013 Eur J Oral Sci.

Pharmacokinetic Interactions between Nelfinavir and 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Atorvastatin and Simvastatin

PubMed Central

Hsyu, Poe-Hirr; Schultz-Smith, Melissa D.; Lillibridge, James H.; Lewis, Ronald H.; Kerr, Bradley M.

2001-01-01

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are effective agents in lowering cholesterol and triglycerides and are being used by human immunodeficiency virus-positive patients to treat the lipid elevation that may be associated with antiretroviral therapy. Many HMG-CoA reductase inhibitors and protease inhibitors are metabolized by the same cytochrome P450 enzyme 3A4 (CYP3A4). In addition, many protease inhibitors are potent inhibitors of CYP3A4. Therefore, coadministration of these two classes of drugs may cause significant drug interactions. This open-label, multiple-dose study was performed to determine the interactions between nelfinavir, a protease inhibitor, and two HMG-CoA reductase inhibitors, atorvastatin and simvastatin, in healthy volunteers. Thirty-two healthy subjects received either atorvastatin calcium (10 mg once a day) or simvastatin (20 mg once a day) for the first 14 days of the study. Nelfinavir (1,250 mg twice a day) was added on days 15 to 28. Pharmacokinetic assessment was performed on days 14 and 28. The study drugs were well tolerated. Nelfinavir increased the steady-state area under the plasma concentration-time curve during one dosing period (AUCτ) of atorvastatin 74% and the maximum concentration (Cmax) of atorvastatin 122% and increased the AUCτ of simvastatin 505% and the Cmax of simvastatin 517%. Neither atorvastatin nor simvastatin appeared to alter the pharmacokinetics of nelfinavir. It is recommended that coadministration of simvastatin with nelfinavir should be avoided, whereas atorvastatin should be used with nelfinavir with caution. PMID:11709322

Heterogeneous Binding and Central Nervous System Distribution of the Multitargeted Kinase Inhibitor Ponatinib Restrict Orthotopic Efficacy in a Patient-Derived Xenograft Model of Glioblastoma.

PubMed

Laramy, Janice K; Kim, Minjee; Gupta, Shiv K; Parrish, Karen E; Zhang, Shuangling; Bakken, Katrina K; Carlson, Brett L; Mladek, Ann C; Ma, Daniel J; Sarkaria, Jann N; Elmquist, William F

2017-11-01

This study investigated how differences in drug distribution and free fraction at different tumor and tissue sites influence the efficacy of the multikinase inhibitor ponatinib in a patient-derived xenograft model of glioblastoma (GBM). Efficacy studies in GBM6 flank (heterotopic) and intracranial (orthotopic) models showed that ponatinib is effective in the flank but not in the intracranial model, despite a relatively high brain-to-plasma ratio. In vitro binding studies indicated that flank tumor had a higher free (unbound) drug fraction than normal brain. The total and free drug concentrations, along with the tissue-to-plasma ratio (Kp) and its unbound derivative (Kp,uu), were consistently higher in the flank tumor than the normal brain at 1 and 6 hours after a single dose in GBM6 flank xenografts. In the orthotopic xenografts, the intracranial tumor core displayed higher Kp and Kp,uu values compared with the brain-around-tumor (BAT). The free fractions and the total drug concentrations, hence free drug concentrations, were consistently higher in the core than in the BAT at 1 and 6 hours postdose. The delivery disadvantages in the brain and BAT were further evidenced by the low total drug concentrations in these areas that did not consistently exceed the in vitro cytotoxic concentration (IC 50 ). Taken together, the regional differences in free drug exposure across the intracranial tumor may be responsible for compromising efficacy of ponatinib in orthotopic GBM6. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Materials Evaluation. Part II. Development of Corrosion Inhibitors.

DTIC Science & Technology

1979-09-01

concentration upon the pitting behavior of Al 7075-T6 in an inhibited solution 46 24 Polarization behavior of 4340 steel 47 25 Polarization behavior of copper ...However, this combination itself was not effective in inhibiting the corrosion of high- strength aluminum alloys, copper , and other alloys used in...79 JUNE MONTHLY COMPOSITE COMPLETE ANALYSIS (Results expressed in milligrams per liter) Calcium Ca 61.60 Magnesium Mg 7.00 Sulfates S04 55.00 Chlorides

Inhibition of PI3K-AKT-mTOR pathway sensitizes endometrial cancer cell lines to PARP inhibitors.

PubMed

Philip, Charles-André; Laskov, Ido; Beauchamp, Marie-Claude; Marques, Maud; Amin, Oreekha; Bitharas, Joanna; Kessous, Roy; Kogan, Liron; Baloch, Tahira; Gotlieb, Walter H; Yasmeen, Amber

2017-09-08

Phosphatase and Tensin homolog (PTEN) is a tumor suppressor gene. Loss of its function is the most frequent genetic alteration in endometrioid endometrial cancers (70-80%) and high grade tumors (90%). We assessed the sensitivity of endometrial cancer cell lines to PARP inhibitors (olaparib and BMN-673) and a PI3K inhibitor (BKM-120), alone or in combination, in the context of their PTEN mutation status. We also highlighted a direct pathway linking PTEN to DNA repair. Using endometrial cancer cellular models with known PTEN status, we evaluated their homologous recombination (HR) functionality by RAD51 foci formation assay. The 50% Inhibitory concentration (IC50) of PI3K and PARP inhibitors in these cells was assessed, and western blotting was performed to determine the expression of proteins involved in the PI3K/mTOR pathway. Moreover, we explored the interaction between RAD51 and PI3K/mTOR by immunofluorescence. Next, the combination effect of PI3K and PARP inhibitors on cell proliferation was evaluated by a clonogenic assay. Cells with mutated PTEN showed over-activation of the PI3K/mTOR pathway. These cells were more sensitive to PARP inhibition compared to PTEN wild-type cells. In addition, PI3K inhibitor treatment reduced RAD51 foci formation in PTEN mutated cells, and sensitized these cells to PARP inhibitor. Targeting both PARP and PI3K might lead to improved personalized therapeutic approaches in endometrial cancer patients with PTEN mutations. Understanding the complex interaction of PTEN mutations with DNA repair in endometrial cancer will help to better select patients that are likely to respond to some of the new and costly targeted therapies.

Cyclic AMP-specific phosphodiesterase-4 as a target for the development of antidepressant drugs.

PubMed

Zhang, Han-Ting

2009-01-01

Phosphodiesterase-4 (PDE4), one of eleven PDE enzyme families, specifically catalyzes hydrolysis of cyclic AMP (cAMP); it has four subtypes (PDE4A-D) with at least 25 splice variants. PDE4 plays a critical role in the control of intracellular cAMP concentrations. PDE4 inhibitors produce antidepressant actions in both animals and humans via enhancement of cAMP signaling in the brain. However, their clinical utility has been hampered by side effects, in particular nausea and emesis. While there is still a long way to go before PDE4 inhibitors with high therapeutic indices are available for treatment of depressive disorders, important advances have been made in the development of PDE4 inhibitors as antidepressants. First, limited, but significant studies point to PDE4D as the major PDE4 subtype responsible for antidepressant-like effects of PDE4 inhibitors, although the role of PDE4A cannot be excluded. Second, PDE4D may contribute to emesis, the major side effect of PDE4 inhibitors. For this reason, identification of roles of PDE4D splice variants in mediating antidepressant activity is particularly important. Recent studies using small interfering RNAs (siRNAs) have demonstrated the feasibility to identify cellular functions of individual PDE4 variants. Third, mixed inhibitors of PDE4 and PDE7 or PDE4 and serotonin reuptake have been developed and may be potential antidepressants with minimized side effects. Finally, relatively selective inhibitors of one or two PDE4 subtypes have been synthesized using structure- and scaffold-based design. This review also discusses the relationship between PDE4 and antidepressant activity based on structures, brain distributions, and pharmacological properties of PDE4 and its isoforms.

Identification and mechanism of action analysis of the new PARP-1 inhibitor 2″-hydroxygenkwanol A.

PubMed

Dal Piaz, Fabrizio; Ferro, Piera; Vassallo, Antonio; Vasaturo, Michele; Forte, Giovanni; Chini, Maria Giovanna; Bifulco, Giuseppe; Tosco, Alessandra; De Tommasi, Nunziatina

2015-09-01

Poly(ADP-ribose) polymerase 1 (PARP-1) activity has been implicated in the pathogenesis of numerous diseases as cancer, inflammation, diabetes and neurodegenerative disorders, therefore the research for new PARP-1 inhibitors is still an active area. To identify new potential PARP-1 inhibitors, we performed a screening of a small-molecule library consisting of polyphenols isolated from plants used in the traditional medicine, by Surface Plasmon Resonance (SPR). Biochemical and cellular assays were performed to confirm SPR results and select the promising candidate(s). Finally, limited proteolysis and ligand docking analyses allowed defining the protein region involved in the interaction with the putative inhibitor(s). The dimeric spiro-flavonoid 2″-hydroxygenkwanol A, member of a relatively recently discovered class of flavonoids containing a spirane C-atom, has been identified as possible PARP-1 inhibitor. This compound showed a high affinity for the polymerase (KD: 0.32±0.05μM); moreover PARP-1 activity in the presence of 2″-hydroxygenkwanol A was significantly affected both when using the recombinant protein and when measuring the cellular effects. Finally, our study suggests this compound to efficiently interact with the protein catalytic domain, into the nicotine binding pocket. 2″-hydroxygenkwanol A efficiently binds and inhibits PARP-1 at submicromolar concentrations, thus representing a promising lead for the design of a new class of PARP-1 modulators, useful as therapeutic agents and/or biochemical tools. Our study has identified an additional class of plant molecules, the spiro-biflavonoids, with known beneficial pharmacological properties but with an unknown mechanism of action, as a possible novel class of PARP-1 activity inhibitors. Copyright © 2015 Elsevier B.V. All rights reserved.

Nitrous Oxide Emissions Affected by Biochar and Nitrogen Stabilizers

NASA Astrophysics Data System (ADS)

Gao, S.; Cai, Z.; Xu, M.

2016-12-01

Both biochar and N fertilizer stabilizers (N transformation inhibitors) are potential strategies to reduce nitrous oxide (N2O) emissions from fertilization, but the mechanisms and/or N transformation processes affecting the N dynamics are not fully understood. This research investigated N2O emissions and N transformations in soil amended with biochar and N transformation inhibitors. The soil was a sandy loam soil and adjusted to 10% soil water content and incubated at 25oC. Biochar amendment at 1% (w/w), Agrotain® Ultra (urease inhibitor), Agrotain® Plus (urease and nitrification inhibitor), and N-Serve® 24 (nitrification inhibitor) as well as another potential nitrification inhibitor, potassium thiosulfate (KTS), at 0.25-1:1 K2O/N ratios (w/w) were tested. Emissions of N2O, soil mineral N species change, and soil pH were determined for 35 days after fertilizers were applied. Biochar, Agrotain® Ultra or Plus, or N-Serve® 24 all effectively reduced N2O emissions by more than 60% as compared to no amendment control. The KTS, however, was only effective in reducing N2O emissions at a high ratio (1:1 K2O/N, w/w). There was a strong correlation between N2O emission and the concentration of nitrite (NO2-) in soil but not other mineral species. All the amendments showed that their effects on N transformation and N2O emissions were completed within a few weeks after application. Laboratory analysis indicated that biochar affected the N dynamics most likely via adsorption of ammonium (NH4+) and the inhibitors by affecting N transformation rate. This research has gained further understanding on how biochar and N stabilizers affect N2O emissions and the knowledge can assist in developing mitigation strategies.

Effects of phosphoramidon on endothelin-1 and big endothelin-1 production in human aortic endothelial cells.

PubMed

Matsumura, Y; Tsukahara, Y; Kojima, T; Murata, S; Murakami, A; Takada, K; Takaoka, M; Morimoto, S

1995-03-01

Using cultured human aortic endothelial cells, we examined the effects of phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, on the release of endogenous endothelin-1 (ET-1) and big endothelin-1 (big ET-1), and on the generation of ET-1 from exogenously applied big ET-1. Phosphoramidon, at concentrations of 10(-6) to 2 x 10(-4) M, caused a biphasic alteration of the ET-1 release, i.e., at lower concentrations of the drug, there were slight but unexpected increases of the release, whereas higher concentrations led to a decrease which is due to the drug-induced inhibition of ECE. The former effect appears to be based on the inhibition of ET-1 degradation by neutral endopeptidase 24.11 (NEP), since kelatorphan, a specific NEP inhibitor, produced a similar increasing effect on ET-1 release. Phosphoramidon enhanced the big ET-1 release from the cells in a concentration-dependent manner. When high concentrations of phosphoramidon were added, there was a dramatic increase in the release of big ET-1, which cannot be explained only by the drug-induced inhibition of ECE. This increase in big ET-1 release appeared to be partly due to a transient stimulation of the expression of prepro ET-1 mRNA. The amount of ET-1 generated from exogenously applied big ET-1 was markedly decreased by phosphoramidon in a concentration-dependent manner. In a similar fashion, phosphoramidon markedly inhibited ECE activity of the membrane fraction of cultured cells. Thus, ET-1 generation from exogenously applied big ET-1 reflects the functional phosphoramidon-sensitive ECE activities in human aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide Inhibitors of the amyloidogenesis of IAPP: verification of the hairpin-binding geometry hypothesis.

PubMed

Sivanesam, Kalkena; Shu, Irene; Huggins, Kelly N L; Tatarek-Nossol, Marianna; Kapurniotu, Aphrodite; Andersen, Niels H

2016-08-01

Versions of a previously discovered β-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at substoichiometric concentrations relative to this amyloidogenic peptide. The hairpin-binding hypothesis stands confirmed. © 2016 Federation of European Biochemical Societies.

Identification of novel serum peptide biomarkers for high-altitude adaptation: a comparative approach

NASA Astrophysics Data System (ADS)

Yang, Juan; Li, Wenhua; Liu, Siyuan; Yuan, Dongya; Guo, Yijiao; Jia, Cheng; Song, Tusheng; Huang, Chen

2016-05-01

We aimed to identify serum biomarkers for screening individuals who could adapt to high-altitude hypoxia at sea level. HHA (high-altitude hypoxia acclimated; n = 48) and HHI (high-altitude hypoxia illness; n = 48) groups were distinguished at high altitude, routine blood tests were performed for both groups at high altitude and at sea level. Serum biomarkers were identified by comparing serum peptidome profiling between HHI and HHA groups collected at sea level. Routine blood tests revealed the concentration of hemoglobin and red blood cells were significantly higher in HHI than in HHA at high altitude. Serum peptidome profiling showed that ten significantly differentially expressed peaks between HHA and HHI at sea level. Three potential serum peptide peaks (m/z values: 1061.91, 1088.33, 4057.63) were further sequence identified as regions of the inter-α trypsin inhibitor heavy chain H4 fragment (ITIH4 347-356), regions of the inter-α trypsin inhibitor heavy chain H1 fragment (ITIH1 205-214), and isoform 1 of fibrinogen α chain precursor (FGA 588-624). Expression of their full proteins was also tested by ELISA in HHA and HHI samples collected at sea level. Our study provided a novel approach for identifying potential biomarkers for screening people at sea level who can adapt to high altitudes.

Novel method to assess antiretroviral target trough concentrations using in vitro susceptibility data.

PubMed

Acosta, Edward P; Limoli, Kay L; Trinh, Lan; Parkin, Neil T; King, Jennifer R; Weidler, Jodi M; Ofotokun, Ighovwerha; Petropoulos, Christos J

2012-11-01

Durable suppression of HIV-1 replication requires the establishment of antiretroviral drug concentrations that exceed the susceptibility of the virus strain(s) infecting the patient. Minimum plasma drug concentrations (C(trough)) are correlated with response, but determination of target C(trough) values is hindered by a paucity of in vivo concentration-response data. In the absence of these data, in vitro susceptibility measurements, adjusted for serum protein binding, can provide estimations of suppressive in vivo drug concentrations. We derived serum protein binding correction factors (PBCF) for protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and an integrase inhibitor by measuring the effect of a range of human serum concentrations on in vitro drug susceptibility measured with the PhenoSense HIV assay. PBCFs corresponding to 100% HS were extrapolated using linear regression and ranged from 1.4 for nevirapine to 77 for nelfinavir. Using the mean 95% inhibitory concentration (IC(95)) for ≥1,200 drug-susceptible viruses, we calculated protein-bound IC(95) (PBIC(95)) values. PBIC(95) values were concordant with the minimum effective C(trough) values that were established in well-designed pharmacodynamic studies (e.g., indinavir, saquinavir, and amprenavir). In other cases, the PBIC(95) values were notably lower (e.g., darunavir, efavirenz, and nevirapine) or higher (nelfinavir and etravirine) than existing target recommendations. The establishment of PBIC(95) values as described here provides a convenient and standardized approach for estimation of the minimum drug exposure that is required to maintain viral suppression and prevent the emergence of drug-resistant variants, particularly when in vivo concentration-response relationships are lacking.

Novel Method To Assess Antiretroviral Target Trough Concentrations Using In Vitro Susceptibility Data

PubMed Central

Limoli, Kay L.; Trinh, Lan; Parkin, Neil T.; King, Jennifer R.; Weidler, Jodi M.; Ofotokun, Ighovwerha; Petropoulos, Christos J.

2012-01-01

Durable suppression of HIV-1 replication requires the establishment of antiretroviral drug concentrations that exceed the susceptibility of the virus strain(s) infecting the patient. Minimum plasma drug concentrations (Ctrough) are correlated with response, but determination of target Ctrough values is hindered by a paucity of in vivo concentration-response data. In the absence of these data, in vitro susceptibility measurements, adjusted for serum protein binding, can provide estimations of suppressive in vivo drug concentrations. We derived serum protein binding correction factors (PBCF) for protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and an integrase inhibitor by measuring the effect of a range of human serum concentrations on in vitro drug susceptibility measured with the PhenoSense HIV assay. PBCFs corresponding to 100% HS were extrapolated using linear regression and ranged from 1.4 for nevirapine to 77 for nelfinavir. Using the mean 95% inhibitory concentration (IC95) for ≥1,200 drug-susceptible viruses, we calculated protein-bound IC95 (PBIC95) values. PBIC95 values were concordant with the minimum effective Ctrough values that were established in well-designed pharmacodynamic studies (e.g., indinavir, saquinavir, and amprenavir). In other cases, the PBIC95 values were notably lower (e.g., darunavir, efavirenz, and nevirapine) or higher (nelfinavir and etravirine) than existing target recommendations. The establishment of PBIC95 values as described here provides a convenient and standardized approach for estimation of the minimum drug exposure that is required to maintain viral suppression and prevent the emergence of drug-resistant variants, particularly when in vivo concentration-response relationships are lacking. PMID:22964257

The cytochrome P4503A4 inhibitor clarithromycin increases the plasma concentrations and effects of repaglinide.

PubMed

Niemi, M; Neuvonen, P J; Kivistö, K T

2001-07-01

Our objective was to study the effects of the macrolide antibiotic clarithromycin on the pharmacokinetics and pharmacodynamics of repaglinide, a novel short-acting antidiabetic drug. In a randomized, double-blind, 2-phase crossover study, 9 healthy volunteers were treated for 4 days with 250 mg oral clarithromycin or placebo twice daily. On day 5 they received a single dose of 250 mg clarithromycin or placebo, and 1 hour later a single dose of 0.25 mg repaglinide was given orally. Plasma repaglinide, serum insulin, and blood glucose concentrations were measured up to 7 hours. Clarithromycin increased the mean total area under the concentration-time curve of repaglinide by 40% (P <.0001) and the peak plasma concentration by 67% (P <.005) compared with placebo. The mean elimination half-life of repaglinide was prolonged from 1.4 to 1.7 hours (P <.05) by clarithromycin. Clarithromycin increased the mean incremental area under the concentration-time curve from 0 to 3 hours of serum insulin by 51% (P <.05) and the maximum increase in the serum insulin concentration by 61% (P <.01) compared with placebo. No statistically significant differences were found in the blood glucose concentrations between the placebo and clarithromycin phases. Even low doses of the cytochrome P4503A4 (CYP3A4) inhibitor clarithromycin increase the plasma concentrations and effects of repaglinide. Concomitant use of clarithromycin or other potent inhibitors of CYP3A4 with repaglinide may enhance its blood glucose-lowering effect and increase the risk of hypoglycemia.

[Determination of minimal concentrations of biocorrosion inhibitors by a bioluminescence method in relation to bacteria, participating in biocorrosion].

PubMed

Efremenko, E N; Azizov, R E; Makhlis, T A; Abbasov, V M; Varfolomeev, S D

2005-01-01

By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Katon, Khazar, VFIKS-82, Nitro-1, Kaspii-2, and Kaspii-4) suppressing most common microbial corrosion agents: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only dividing cells but also their dormant living counterparts, are two- to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.

Calcite crystal growth rate inhibition by polycarboxylic acids

USGS Publications Warehouse

Reddy, M.M.; Hoch, A.R.

2001-01-01

Calcite crystal growth rates measured in the presence of several polycarboxyclic acids show that tetrahydrofurantetracarboxylic acid (THFTCA) and cyclopentanetetracarboxylic acid (CPTCA) are effective growth rate inhibitors at low solution concentrations (0.01 to 1 mg/L). In contrast, linear polycarbocylic acids (citric acid and tricarballylic acid) had no inhibiting effect on calcite growth rates at concentrations up to 10 mg/L. Calcite crystal growth rate inhibition by cyclic polycarboxyclic acids appears to involve blockage of crystal growth sites on the mineral surface by several carboxylate groups. Growth morphology varied for growth in the absence and in the presence of both THFTCA and CPTCA. More effective growth rate reduction by CPTCA relative to THFTCA suggests that inhibitor carboxylate stereochemical orientation controls calcite surface interaction with carboxylate inhibitors. ?? 20O1 Academic Press.

Chitosan: A macromolecule as green corrosion inhibitor for mild steel in sulfamic acid useful for sugar industry.

PubMed

Gupta, Neeraj Kumar; Joshi, P G; Srivastava, Vandana; Quraishi, M A

2018-01-01

The present investigation aims at investigation of low cost nontoxic carbohydrate biopolymer chitosan as corrosion inhibitor alone and in combination with KI for mild steel in 1M sulfamic acid medium using gravimetric, electrochemical and surface analysis techniques. It is found that chitosan alone exhibits inhibition efficiency of 73.8% at 200ppm concentration. However, in combination with KI (5ppm), it gave more than 90% inhibition efficiency. The significant increase in the inhibition performance of chitosan has been explained by the synergistic mechanism. The results of Potentiodynamic polarization study shows that chitosan and its blend with KI decreases both anodic and cathodic reactions occurring at mild steel surface in 1M sulfamic acid medium by blocking active sites of the metal and acts as mixed type inhibitor. EIS study reveals that the polarization resistance increases with increase in the concentration of inhibitors which increases charge transfer resistance across the metal/solution interface. The adsorption of chitosan followed the Langmuir adsorption isotherm. The formation of inhibitor film on metal surface was supported by scanning electron microscopy (SEM) and atomic force microscopy (AFM) surface studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

PubMed

Singh, Samsher; Kalia, Nitin P; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R; Kumar, Ashok; Bharate, Sandip B; Khan, Inshad A

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P -glycoprotein ( P -gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P -gp with half maximal inhibitory concentration (IC 50 ) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P -gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria

PubMed Central

Singh, Samsher; Kalia, Nitin P.; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R.; Kumar, Ashok; Bharate, Sandip B.; Khan, Inshad A.

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time–kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four–eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer. PMID:29046665

Discovery of 4-Methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-((1-nicotinoylpiperidin-4-yl)oxy)benzamide (CHMFL-ABL/KIT-155) as a Novel Highly Potent Type II ABL/KIT Dual Kinase Inhibitor with a Distinct Hinge Binding.

PubMed

Wang, Qiang; Liu, Feiyang; Wang, Beilei; Zou, Fengming; Qi, Ziping; Chen, Cheng; Yu, Kailin; Hu, Chen; Qi, Shuang; Wang, Wenchao; Hu, Zhenquan; Liu, Juan; Wang, Wei; Wang, Li; Liang, Qianmao; Zhang, Shanchun; Ren, Tao; Liu, Qingsong; Liu, Jing

2017-01-12

The discovery of a novel potent type II ABL/c-KIT dual kinase inhibitor compound 34 (CHMFL-ABL/KIT-155), which utilized a hydrogen bond formed by NH on the kinase backbone and carbonyl oxygen of 34 as a unique hinge binding, is described. 34 potently inhibited purified ABL (IC 50 : 46 nM) and c-KIT kinase (IC 50 : 75 nM) in the biochemical assays and displayed high selectivity (S Score (1) = 0.03) at the concentration of 1 μM among 468 kinases/mutants in KINOMEscan assay. It exhibited strong antiproliferative activities against BCR-ABL/c-KIT driven CML/GISTs cancer cell lines through blockage of the BCR-ABL/c-KIT mediated signaling pathways, arresting cell cycle progression and induction of apoptosis. 34 possessed a good oral PK property and effectively suppressed the tumor progression in the K562 (CML) and GIST-T1 (GISTs) cells mediated xenograft mouse model. The distinct hinge-binding mode of 34 provided a novel pharmacophore for expanding the chemical structure diversity for the type II kinase inhibitors discovery.

Effect of acarbose on postprandial blood glucose concentrations in healthy cats fed low and high carbohydrate diets.

PubMed

Singh, Ranee; Rand, Jacquie S; Coradini, Marcia; Morton, John M

2015-10-01

Feeding a low carbohydrate diet is recommended for diabetic cats; however, some cats may require diets containing moderate-to-high carbohydrate and may benefit from the use of therapeutic agents to improve glycemic control. The aim of the study was to determine the effect of the α-glucosidase inhibitor acarbose on postprandial plasma glucose concentration when combined with commercially available feline diets high and low in carbohydrate. Twelve healthy, adult, non-obese, neutered cats were enrolled. Plasma glucose concentrations were assessed over 24 h after feeding high and low carbohydrate diets, with and without acarbose, during single and multiple meal tests, in a crossover study. Commercially available feline diets were used, which were high and low in carbohydrate (providing 51% and 7% of metabolizable energy, respectively). In cats fed the high carbohydrate diet as a single meal, mean 24 h glucose concentrations were lower when acarbose was administered. Mean glucose concentrations were lower in the first 12 h when acarbose was given once daily, whereas no significant difference was observed in mean results from 12-24 h. Acarbose had little effect in cats eating multiple meals. Compared with consumption of the high carbohydrate diet with acarbose, lower mean 24 h and peak glucose concentrations were achieved by feeding the low carbohydrate diet alone. In healthy cats meal-fed diets of similar composition to the diets used in this study, acarbose has minimal effect when a low carbohydrate diet is fed but reduces postprandial glucose concentrations over 24 h when a high carbohydrate diet is fed. However, mean glucose concentrations over 24 h are still higher when a high carbohydrate diet with acarbose is fed relative to the low carbohydrate diet without acarbose. Future studies in diabetic cats are warranted to confirm these findings. © ISFM and AAFP 2014.

HU-331, a novel cannabinoid-based anticancer topoisomerase II inhibitor.

PubMed

Kogan, Natalya M; Schlesinger, Michael; Priel, Esther; Rabinowitz, Ruth; Berenshtein, Eduard; Chevion, Mordechai; Mechoulam, Raphael

2007-01-01

Anthracyclines, a large group of quinonoid compounds, are used to treat some forms of cancer. Although highly effective in cancer therapy, the mechanism of action of these compounds is not specific; they act on cancer and other cells by numerous mechanisms. A new anticancer quinone (HU-331) was synthesized from cannabidiol. It shows significant high efficacy against human cancer cell lines in vitro and against in vivo tumor grafts in nude mice. In this study, we investigated its mode of action and present evidence on its unique mechanism. HU-331 does not cause cancer cell cycle arrest, cell apoptosis, or caspase activation. HU-331-caused cell death of human cancer cell lines is not mediated by reactive oxygen intermediates/species, as exposure to HU-331 failed to elicit the generation of reactive oxygen species. HU-331 inhibits DNA topoisomerase II even at nanomolar concentrations but has only a slight nonsignificant effect on DNA topoisomerase I action. The cannabinoid quinone HU-331 is a highly specific inhibitor of topoisomerase II, compared with most known anticancer quinones. It might represent a new potent anticancer drug.

The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.

PubMed

Halestrap, A P

1975-04-01

1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor.

Cost of care of haemophilia with inhibitors.

PubMed

Di Minno, M N D; Di Minno, G; Di Capua, M; Cerbone, A M; Coppola, A

2010-01-01

In Western countries, the treatment of patients with inhibitors is presently the most challenging and serious issue in haemophilia management, direct costs of clotting factor concentrates accounting for >98% of the highest economic burden absorbed for the healthcare of patients in this setting. Being designed to address questions of resource allocation and effectiveness, decision models are the golden standard to reliably assess the overall economic implications of haemophilia with inhibitors in terms of mortality, bleeding-related morbidity, and severity of arthropathy. However, presently, most data analyses stem from retrospective short-term evaluations, that only allow for the analysis of direct health costs. In the setting of chronic diseases, the cost-utility analysis, that takes into account the beneficial effects of a given treatment/healthcare intervention in terms of health-related quality of life, is likely to be the most appropriate approach. To calculate net benefits, the quality adjusted life year, that significantly reflects such health gain, has to be compared with specific economic impacts. Differences in data sources, in medical practice and/or in healthcare systems and costs, imply that most current pharmacoeconomic analyses are confined to a narrow healthcare payer perspective. Long-term/lifetime prospective or observational studies, devoted to a careful definition of when to start a treatment; of regimens (dose and type of product) to employ, and of inhibitor population (children/adults, low-responding/high responding inhibitors) to study, are thus urgently needed to allow for newer insights, based on reliable data sources into resource allocation, effectiveness and cost-utility analysis in the treatment of haemophiliacs with inhibitors.

Fragment-based discovery of novel and selective mPGES-1 inhibitors Part 1: identification of sulfonamido-1,2,3-triazole-4,5-dicarboxylic acid.

PubMed

Lee, Kijae; Pham, Van Chung; Choi, Min Ji; Kim, Kyung Ju; Lee, Kyung-Tae; Han, Seong-Gu; Yu, Yeon Gyu; Lee, Jae Yeol

2013-01-01

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase that catalyzes the conversion of prostaglandin PGH(2) to PGE(2) and represents a novel target for therapeutic treatment of inflammatory disorders. It is essential to identify mPGES-1 inhibitor with novel scaffold as new hit or lead compound for the purpose of the next-generation anti-inflammatory drugs. Herein we report the discovery of sulfonamido-1,2,3-triazole-4,5-dicarboxylic derivatives as a novel class of mPGES-1 inhibitors identified through fragment-based virtual screening and in vitro assays on the inhibitory activity of the actual compounds. 1-[2-(N-Phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f) inhibits human mPGES-1 (IC(50) of 1.1 μM) with high selectivity (ca.1000-fold) over both COX-1 and COX-2 in a cell-free assay. In addition, the activity of compound 6f was again tested at 10 μM concentration in presence of 0.1% Triton X-100 and found to be reduced to 1/4 of its original activity without this detergent. Compared to the complete loss of activity of nuisance inhibitor with the detergent, therefore, compound 6f would be regarded as a partial nuisance inhibitor of mPGES-1 with a novel scaffold for the optimal design of more potent mPGES-1 inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effect of peptidases on the ability of exogenous and endogenous neurokinins to produce neurokinin 1 receptor internalization in the rat spinal cord.

PubMed

Marvizon, Juan Carlos G; Wang, Xueren; Lao, Li-Jun; Song, Bingbing

2003-12-01

The ability of peptidases to restrict neurokinin 1 receptor (NK1R) activation by exogenously applied or endogenously released neurokinins was investigated by measuring NK1R internalization in rat spinal cord slices. Concentration-response curves for substance P and neurokinin A were obtained in the presence and absence of 10 microm thiorphan, an inhibitor of neutral endopeptidase (EC 3.4.24.11), plus 10 microm captopril, an inhibitor of dipeptidyl carboxypeptidase (EC 3.4.15.1). These inhibitors significantly decreased the EC50 of substance P to produce NK1R internalization from 32 to 9 nm, and the EC50 of neurokinin A from 170 to 60 nm. Substance P was significantly more potent than neurokinin A, both with and without these peptidase inhibitors. In the presence of peptidase inhibitors, neurokinin B was 10 times less potent than neurokinin A and 64 times less potent than substance P (EC50=573 nm). Several aminopeptidase inhibitors (actinonin, amastatin, bacitracin, bestatin and puromycin) failed to further increase the effect of thiorphan plus captopril on the NK1R internalization produced by 10 nm substance P. Electrical stimulation of the dorsal root produced NK1R internalization by releasing endogenous neurokinins. Thiorphan plus captopril increased NK1R internalization produced by 1 Hz stimulation, but not by 30 Hz stimulation. Therefore, NEN and DCP restrict NK1R activation by endogenous neurokinins when they are gradually released by low-frequency firing of primary afferents, but become saturated or inhibited when primary afferents fire at a high frequency.

Intestinal transport of HDND-7, a novel hesperetin derivative, in in vitro MDCK cell and in situ single-pass intestinal perfusion models.

PubMed

Chen, Ruonan; Li, Lan; Shen, Chenlin; Huang, Cheng; Ma, Taotao; Meng, Xiaoming; Qian, Zhengyue; Li, Yangyang; Li, Jun

2017-08-01

1. Hesperetin (HDND) possesses extensive bioactivities, however, its poor solubility and low bioavailability limit its application. HDND-7, a derivative of HDND, has better solubility and high bioavailability. In this study, we investigated the intestinal absorption mechanisms of HDND-7. 2. MDCK cells were used to examine the transport mechanisms of HDND-7 in vitro, and a rat in situ intestinal perfusion model was used to characterize the absorption of HDND-7. The concentration of HDND-7 was determined by HPLC. 3. In MDCK cells, HDND-7 was effectively absorbed in a concentration-dependent manner in both directions. Moreover, HDND-7 showed pH-dependent and TEER-independent transport in both directions. The transport of HDND-7 was significantly reduced at 4 °C or in the presence of NaN3. Furthermore, the efflux of HDND-7 was apparently reduced in the presence of MRP2 inhibitors MK-571 or probenecid. However, P-gp inhibitor verapamil had no effect on the transport of HDND-7. The in situ intestinal perfusion study indicated HDND-7 was well-absorbed in four intestinal segments. Furthermore, MRP2 inhibitors may slightly increase the absorption of HDND-7 in jejunum. 4. In summary, all results indicated that HDND-7 might be absorbed mainly by passive diffusion via transcellular pathway, MRP2 but P-gp may participate in the efflux of HDND-7.

Pharmacology of irinotecan.

PubMed

Kuhn, J G

1998-08-01

Irinotecan (CPT-11 [Camptosar]), a semisynthetic derivative of the plant alkaloid camptothecin, is bioactivated by carboxylesterases (EC3.1.1-) to the topoisomerase I inhibitor SN-38, a minor metabolite. Bioactivation of intravenously administered irinotecan by carboxylesterases occurs predominantly in the liver. Two human carboxylesterase isoforms responsible for SN-38 formation have been characterized. At relevant hepatic irinotecan concentrations up to 12 micrograms/mL, a low-Km isoform is responsible for irinotecan bioactivation. High concentrations of drugs commonly coadministered with irinotecan do not inhibit carboxylesterase activity. Intestinal carboxylesterases can also generate SN-38, followed by subsequent oral absorption. A second major polar metabolite of irinotecan, aminopentanecarboxylic acid (APC), is the product of CYP3A4-mediated oxidation of the terminal piperidine ring. APC is 100-fold less active than SN-38 as a topoisomerase I inhibitor and is a relatively weak inhibitor of acetylcholinesterase. SN-38 is eliminated mainly through conjugation by hepatic uridine glucuronosyltransferase (UGT*1.1), the same isoezyme responsible for glucuronidation of bilirubin. Grade 4 irinotecan-related toxicity (ie, neutropenia, diarrhea) has recently been reported in two patients with deficient UGT*1.1 activity. SN-38 glucuronide (SN-38G), which has only 1/100th the antitumor activity of SN-38, is actively secreted into the bile by a canalicular multispecific organic anion transporter. Deconjugation of SN-38G to SN-38 by beta-glucuronidase produced by the intestinal flora may contribute to enterohepatic recirculation of SN-38 and delayed intestinal toxicity.

Application of response surface methodology method in designing corrosion inhibitor

NASA Astrophysics Data System (ADS)

Asmara, Y. P.; Athirah; Siregar, J. P.; Kurniawan, T.; Bachtiar, D.

2017-10-01

In oil and gas pipelines and offshore structure, inhibitors have been considered to be the first choice to reduce corrosion rate. There are many corrosion inhibitor compositions available in the market. To produce the best corrosion inhibitor requires many experimental data which is not efficient. These experiments used response surface methodology (RSM) to select corrosion inhibitor compositions. The experiments investigated effects of corrosion inhibition on corrosion rate of low carbon steel in 3% NaCl solution with different concentrations of selected main inhibitor compositions which are ethyl acetate (EA), ethylene glycol (EG) and sodium benzoate (SB). Corrosion rate were calculated using linear polarization resistance (LPR). All of the experiments were set in natural conditions at pH 7. MINITAB® version 15 was used for data analysis. It is shown that a quadratic model is a representative model can predict best corrosion inhibitor composition comprehensibly.

PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine monophosphate, is a potent and pan-genotype inhibitor of hepatitis C virus replication.

PubMed

Lam, Angela M; Murakami, Eisuke; Espiritu, Christine; Steuer, Holly M Micolochick; Niu, Congrong; Keilman, Meg; Bao, Haiying; Zennou, Veronique; Bourne, Nigel; Julander, Justin G; Morrey, John D; Smee, Donald F; Frick, David N; Heck, Julie A; Wang, Peiyuan; Nagarathnam, Dhanapalan; Ross, Bruce S; Sofia, Michael J; Otto, Michael J; Furman, Phillip A

2010-08-01

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075+/-0.050 microM (mean+/-standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 microM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.

How Roots Perceive and Respond to Gravity.

ERIC Educational Resources Information Center

Moore, Randy

1984-01-01

Discusses graviperception and gravitropism by plant roots. Indicates that graviperception occurs via sedimentation of amyloplasts in columella cells of the root cap and that the minimal graviresponsiveness of lateral roots may be due to the intensity of their caps to establish a concentration gradient of inhibitor(s) sufficient to affect…

Oral Treatment of Rodents with Insecticides for Control of Sand Flies (Diptera: Psychodidae) and the Fluorescent Tracer Technique (FTT) as a Tool to Evaluate Potential Sand Fly Control Methods

DTIC Science & Technology

2011-03-01

chitin synthesis inhibitors diflubenzuron and novaluron evaluated in these studies were...effective against sand fly larvae and palatable to hamsters. In contrast to the chitin synthesis inhibitors and juvenile hormone analogs...concentrations (mg/kg) Effects Chitin synthesis inhibitor Diflubenzuron 8.97, 89.7, 897 Mortality at larva-to-pupa molt Novaluron 9.88, 98.8,

Studies on the neuroprotective action of kynurenine mono-oxygenase inhibitors in post-ischemic brain damage.

PubMed

Moroni, Flavio; Carpenedo, Raffaella; Cozzi, Andrea; Meli, Elena; Chiarugi, Alberto; Pellegrini-Giampietro, Domenico E

2003-01-01

Kynurenine 3-mono-oxygenase (KMO) inhibitors facilitate kynurenic acid (KYNA) neosynthesis and reduce the formation of 3OH-kynurenine (3-HK) and quinolinic acid (QUIN). They also attenuate post-ischemic brain damage and decrease glutamate (Glu) content in brain extracellular spaces. To investigate KMO mechanism(s) of neuroprotection, we performed experiments in gerbils subjected to bilateral carotid occlusion and in organotypic rat hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD). In gerbils, direct application of KYNA (100 nM, through reverse microdialysis in the hippocampus) completely prevented the increase in Glu output induced by transient (5 min) occlusion of the carotids. In rat hippocampal slices exposed for 30 min to OGD, KMO inhibitors (m-nitrobenzoyl)-alanine (mNBA, 30-100 microM) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-8048, 1-10 microM) reduced post-ischemic neuronal death and increased KYNA concentrations in the incubation medium. KYNA may antagonize glycineb or alpha7 nicotinic acetylcholine receptors but the concentrations in the incubation medium never reached values that could efficiently antagonize receptor function. On the contrary, 3-HK (1-10 microM) added to slices exposed to OGD in the presence of KMO inhibitors completely prevented the neuroprotective effects of the inhibitors. Our findings suggest that KMO inhibitors reduce OGD-induced pyramidal cell death by decreasing 3-HK (and possibly QUIN) synthesis.

Self-adaption of methane-producing communities to pH disturbance at different acetate concentrations by shifting pathways and population interaction.

PubMed

Hao, Liping; Lü, Fan; Li, Lei; Wu, Qing; Shao, Liming; He, Pinjing

2013-07-01

To investigate the competition among acetate-utilizing microorganisms at different acetate levels, bioconversion processes of 50, 100, 150 and 200 mM acetate in the presence and absence of methanogenic inhibitor CH3F were monitored in thermophilic methanogenic system. The successive response of methane-producing community during the deteriorative and recovery phases caused by pH disturbance was analyzed. High acetate concentration (>50mM) inhibited the activity of acetoclastic methanogenesis (AM). The increasing pH (>7.5) enhanced this inhibition. The syntrophic acetate oxidizing (SAO) bacteria and hydrogenotrophic methanogens including Methanomicrobiales and Methanobacteirales were more tolerant to the stress from high acetate concentration and high pH. Resumption from alkali condition to normal pH stimulated the growth of acetate oxidizing syntrophs. The reaction rate of SAO-HM was lower than that of AM. These results point to the possibility to regenerate the deteriorated anaerobic digesters by addition of acclimatized inocula rich in acetate-oxidizing syntrophs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Silicon uptake and transport is an active process in Cucumis sativus.

PubMed

Liang, Yongchao; Si, Jin; Römheld, Volker

2005-09-01

Cucumis sativus is a species known to accumulate high levels of silicon (Si) in the tops, though the mechanism for its high Si uptake is little understood. In a series of hydroponic experiments, we examined uptake and xylem loading of Si in C. sativus along with Vicia faba at three levels of Si (0.085, 0.17 and 1.70 mm). Measured Si uptake in C. sativus was more than twice as high as calculated from the rate of transpiration assuming no discrimination between silicic acid and water in uptake. Measured Si uptake in V. faba, however, was significantly lower than the calculated uptake. Concentration of Si in xylem exudates was several-fold higher in C. sativus, but was significantly lower in V. faba compared with the Si concentration in external solutions, regardless of Si levels. Silicon uptake was strongly inhibited by low temperature and 2,4-dinitrophenol, a metabolic inhibitor, in C. sativus but not in V. faba. It can be concluded that Si uptake and transport in C. sativus is active and independent of external Si concentrations, in contrast to the process in V. faba.

Cholesteryl ester transfer protein inhibition as a strategy to reduce cardiovascular risk

PubMed Central

Barter, Philip J.; Rye, Kerry-Anne

2012-01-01

Human and rabbit plasma contain a cholesteryl ester transfer protein (CETP) that promotes net mass transfers of cholesteryl esters from high density lipoproteins (HDL) to other plasma lipoprotein fractions. As predicted, inhibition of CETP in both humans and rabbits increases the concentration of cholesterol in the potentially protective HDL fraction, while decreasing it in potentially proatherogenic non-HDL fractions. Inhibition of CETP in rabbits also inhibits the development of diet-induced atherosclerosis. However, use of the CETP inhibitor torcetrapib in humans did not reduce atheroma in three imaging trials and caused an excess of deaths and cardiovascular events in a large clinical outcome trial. The precise explanation for the harm caused by torcetrapib is unknown but may relate to documented, potentially harmful effects unrelated to inhibition of CETP. More recently, a trial using the weak CETP inhibitor dalcetrapib, which raises HDL levels less effectively than torcetrapib and does not lower non-HDL lipoprotein levels, was terminated early for reasons of futility. There was no evidence that dalcetrapib caused harm in that trial. Despite these setbacks, the hypothesis that CETP inhibitors will be antiatherogenic in humans is still being tested in studies with anacetrapib and evacetrapib, two CETP inhibitors that are much more potent than dalcetrapib and that do not share the off-target adverse effects of torcetrapib. PMID:22550134

Nonprenylated Xanthones from Gentiana lutea, Frasera caroliniensis, and Centaurium erythraea as Novel Inhibitors of Vascular Smooth Muscle Cell Proliferation.

PubMed

Waltenberger, Birgit; Liu, Rongxia; Atanasov, Atanas G; Schwaiger, Stefan; Heiss, Elke H; Dirsch, Verena M; Stuppner, Hermann

2015-11-13

Aberrant proliferation of vascular smooth muscle cells (VSMC) plays a major role in restenosis, the pathological renarrowing of the blood vessel lumen after surgical treatment of stenosis. Since available anti-proliferative pharmaceuticals produce unfavorable side effects, there is high demand for the identification of novel VSMC proliferation inhibitors. A natural product screening approach using a resazurin conversion assay enabled the identification of gentisin (1) from Gentiana lutea as a novel inhibitor of VSMC proliferation with an IC50 value of 7.84 µM. Aiming to identify further anti-proliferative compounds, 13 additional nonprenylated xanthones, isolated from different plant species, were also tested. While some compounds showed no or moderate activity at 30 µM, 1-hydroxy-2,3,4,5-tetramethoxyxanthone (4), swerchirin (6), and methylswertianin (7) showed IC50 values between 10.2 and 12.5 µM. The anti-proliferative effect of 1, 4, 6, and 7 was confirmed by the quantification of DNA synthesis (BrdU incorporation) in VSMC. Cell death quantification (determined by LDH release in the culture medium) revealed that the compounds are not cytotoxic in the investigated concentration range. In conclusion, nonprenylated xanthones are identified as novel, non-toxic VSMC proliferation inhibitors, which might contribute to the development of new therapeutic applications to combat restenosis.

Urea transporter proteins as targets for small-molecule diuretics.

PubMed

Esteva-Font, Cristina; Anderson, Marc O; Verkman, Alan S

2015-02-01

Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

PubMed

Nishiura, T; Abe, K

1999-01-01

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

A Potent and Selective Quinoxalinone-Based STK33 Inhibitor Does Not Show Synthetic Lethality in KRAS-Dependent Cells

PubMed Central

2012-01-01

The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers. To test this hypothesis, we initiated a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). Several hits were identified, and one of these, a quinoxalinone derivative, was optimized. Extensive SAR studies were performed and led to the chemical probe ML281 that showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors that were reported in the course of these studies. Even at the highest concentration tested (10 μM), ML281 had no effect on the viability of KRAS-dependent cancer cells. These results are consistent with other recent reports using small-molecule STK33 inhibitors. Small molecules having different chemical structures and kinase-selectivity profiles are needed to fully understand the role of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable addition to small-molecule probes of STK33. PMID:23256033

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani.

PubMed

Bhaumik, D; Datta, A K

1988-04-01

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.

Chemical biology tools to study pantetheinases of the vanin family.

PubMed

Schalkwijk, Joost; Jansen, Patrick

2014-08-01

VNNs (vanins) are pantetheinases that hydrolyse pantetheine to pantothenic acid and cysteamine. Studies with Vnn1-knockout mice have indicated a role of VNN-1 in inflammation and stress responses. VNN-1 is highly expressed in liver and is under transcriptional control of PPAR (peroxisome-proliferator-activated receptor)-α and nutritional status, suggesting a role in energy metabolism. Recently, the specific substrates and inhibitors of VNNs were obtained as tools to study VNN biology and to investigate whether VNNs are potential drug targets. Oral administration of RR6, a pantothenone with nanomolar anti-VNN potency, completely inhibited plasma VNN activity in rats and showed favourable pharmacokinetics. Prolonged RR6 administration caused alterations of hepatic and plasma lipid concentrations upon fasting. VNN inhibitors were found to protect pantothenamides (pantetheine analogues with antibiotic activity) against breakdown by plasma VNN, thereby preserving their antibiotic activity. Combination of pantothenamides with a VNN inhibitor showed a strong activity against Staphylococcus aureus and Staphylococcus pneumoniae when assayed in the presence of 10% serum. Recent studies have reported plasma stable pantothenamides that were active against the malaria parasite Plasmodium falciparum. We conclude that VNN inhibitors and pantothenate derivatives that target enzymes in the CoA (coenzyme A) biosynthetic pathway may have potential use as novel drugs in infection, inflammation and metabolism.

The influence of angiotensin converting enzyme inhibitors on lipid peroxidation in sera and aorta of rabbits in diet-induced hypercholesterolemia.

PubMed

Wojakowski, W; Gminski, J; Siemianowicz, K; Goss, M; Machalski, M

2000-11-01

In hypercholesterolemia increased lipid and lipoprotein peroxidation occurs. The renin-angiotensin system plays an important role in atherogenesis. Angiotensin II induces smooth muscle cells proliferation and stimulates oxidation of LDL particles and foam cell accumulation. Inhibition of ang II production leads to decrease in lipid peroxide production. The aim of this study was to assess the lipid peroxidation expressed as concentration of thiobarbituric acid reactive species (TBARS) in sera and aorta homogenates after administration of two doses of angiotensin-converting enzyme (ACE) inhibitors (captopril, enalapril and quinapril) in diet-induced hypercholesterolemia in rabbits. Sixty-four New Zealand rabbits were used. Animals were fed with standard fodder, special diet (1% cholesterol content) or special diet + tested ACEI. Two doses of ACE inhibitors were used: i), equivalent to applied to humans, ii), dose 10 times higher. The animals were divided into 8 groups: control, standard fodder; B, special diet; C1, C2, special diet + captopril in doses 2.5 and 25 mg/kg/24 h, respectively; E1, E2, special diet + enalapril in doses 0.75 and 7.5 mg/kg/24 h, respectively; Q1 and Q2, special diet + quinapril in doses 0.75 and 7.5 mg/kg per day, respectively. In cholesterol-fed rabbits and in groups receiving lower doses of tested ACE inhibitors, the serum TBARS concentration at 6 months was significantly higher in comparison to the control. The higher doses of enalapril, quinapril and captopril, prevented the cholesterol-induced rise in TBARS concentration. Lower dose of captopril attenuated the rise in TBARS concentration, it was significantly lower in comparison to group B, but higher than in the control group. In animals from groups B, E1, C1, Q1 TBARS concentration in aortae was significantly higher as compared to control group. Both doses of captopril and higher doses of enalapril and quinapril inhibited the rise of lipid peroxides concentration induced by cholesterol-rich diet.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygenmore » and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high-content screen for the identification of compounds that target cells in dormant tumor spheroid regions. • Metabolite profiling by NMR spectroscopy on 3D tumor spheroids. • Identification of respiratory chain inhibitors to specifically induce cell death in inner tumor spheroid regions. • Respiratory chain inhibitors enhance cytostatic based therapy in vitro.« less

Dyslipidemia, Diet and Physical Exercise in Children on Treatment With Antiretroviral Medication in El Salvador: A Cross-sectional Study.

PubMed

Sonego, Michela; Sagrado, Maria José; Escobar, Gustavo; Lazzerini, Marzia; Rivas, Estefanie; Martín-Cañavate, Rocio; Pérez de López, Elsy; Ayala, Sandra; Castaneda, Luis; Aparicio, Pilar; Custodio, Estefanía

2016-10-01

Dyslipidemias are common in HIV-infected children, especially if treated with protease inhibitors, but there are few data on how to treat dyslipidemias in this population. We estimated the dyslipidemia prevalence and its association with treatment, diet and physical exercise in children on antiretroviral treatment at the El Salvador reference center for pediatric HIV care (CENID). Information was gathered regarding socio-demographic characteristics, treatment, diet and physical activity of 173 children aged 5-18 years and receiving antiretroviral therapy. Triglycerides, total cholesterol, low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), viral load and CD4 T-lymphocytes were measured. Abnormal concentrations were defined as triglycerides ≥130 mg/dL in 10- to 18-year olds and ≥100 mg/dL in <10-year olds; total cholesterol ≥200 mg/dL; LDL-C ≥130 mg/dL and HDL-C ≤35 mg/dL. We adjusted 4 different multivariate models to assess the independent association of each type of dyslipidemia with protease inhibitors, diet and physical exercise. Of the 173 children, 83 (48%) had hypertriglyceridemia and 25 (14.5%) hypercholesterolemia. High LDL-C concentrations were observed in 17 children (9.8%) and low HDL-C in 38 (22%). Treatment with protease inhibitors was significantly associated with hypertriglyceridemia [prevalence ratio (PR) 2.8; 95% confidence interval (CI): 2.0-3.8] and hypercholesterolemia (PR 9.0; 95% CI: 3.6-22.2). Higher adherence to a "high fat/sugar diet" was associated with hypercholesterolemia (PR 1.6; 95% CI: 1.1-2.3) and high LDL-C (PR 1.7; 95% CI: 1.0-2.9). Compared with those exercising <3 times/week, children exercising ≥7 times were less likely to have low HDL-C (PR = 0.4; 95% CI: 0.2-0.7). These results suggest that a healthy diet and exercise habits can contribute to controlling some aspects of the lipid profile in this population.

Small Quaternary Inhibitors K298 and K524: Cholinesterases Inhibition, Absorption, Brain Distribution, and Toxicity.

PubMed

Karasova, Jana Zdarova; Hroch, Milos; Musilek, Kamil; Kuca, Kamil

2016-02-01

Inhibitors of acetylcholinesterase (AChE) may be used in the treatment of various cholinergic deficits, among them being myasthenia gravis (MG). This paper describes the first in vivo data for promising small quaternary inhibitors (K298 and K524): acute toxicity study, cholinesterase inhibition, absorption, and blood-brain barrier penetration. The newly prepared AChE inhibitors (bis-quinolinium and quinolinium compounds) possess a positive charge in the molecule which ensures that anti-AChE action is restricted to peripheral effect. HPLC-MS was used for determination of real plasma and brain concentration in the pharmacokinetic part of the study, and standard non-compartmental analysis was performed. The maximum plasma concentrations were attained at 30 min (K298; 928.76 ± 115.20 ng/ml) and 39 min (K524; 812.40 ± 54.96 ng/ml) after i.m. Both compounds are in fact able to target the central nervous system. It seems that the difference in the CNS distribution profile depends on an active efflux system. The K524 brain concentration was actively decreased to below an effective level; in contrast, K298 progressively accumulated in brain tissue. Peripheral AChE inhibitors are still first-line treatment in the mild forms of MG. Commonly prescribed carbamates have many severe side effects related to AChE carbamylation. The search for new treatment strategies is still important. Unlike carbamates, these new compounds target AChE via apparent π-π or π-cationic interaction aside at the AChE catalytic site.

[Tranexamic acid as first-line emergency treatment for episodes of bradykinin-mediated angioedema induced by ACE inhibitors].

PubMed

Beauchêne, C; Martins-Héricher, J; Denis, D; Martin, L; Maillard, H

2018-05-04

Episodes of acquired bradykinin-mediated angioedema due to angiotensin-converting enzyme (ACE) inhibitors may result in fatal outcomes. There is no consensus regarding emergency pharmacological management of these episodes. Treatment options include icatibant and C1INH concentrate. Tranexamic acid is administered for moderate episodes. Its efficacy in the treatment of ACE inhibitor-induced episodes of angioedema is not established. The aim of this retrospective study is to assess the benefits of emergency tranexamic acid administration in the management of ACE inhibitor-induced episodes of angioedema. Retrospective analysis of the medical files of patients who consulted between 2010 and 2016 in two French tertiary care hospitals for a bradykinic angioedema attributed to an ACE treatment. All of them had received tranexamic acid as a first line treatment. Thirty three patients who had experienced severe episode of angioedema were included. Twenty seven patients showed significant improvement when treated with tranexamic acid alone. The six remaining patients were treated with icatibant (5/33) or C1INH concentrate (1/33), due to partial improvement after tranexamic acid therapy. None of the patients were intubated, no fatalities were recorded and no side effects were reported. Tranexamic acid is an easily accessible and affordable therapy that may provide effective treatment for ACE inhibitor-induced episodes of angioedema. It may help while waiting for a more specific treatment (icatibant and C1INH concentrate) that is at times unavailable in emergency departments. Copyright © 2018 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier SAS. All rights reserved.

Benzoate-induced stress enhances xylitol yield in aerobic fed-batch culture of Candida mogii TISTR 5892.

PubMed

Wannawilai, Siwaporn; Sirisansaneeyakul, Sarote; Chisti, Yusuf

2015-01-20

Production of the natural sweetener xylitol from xylose via the yeast Candida mogii TISTR 5892 was compared with and without the growth inhibitor sodium benzoate in the culture medium. Sodium benzoate proved to be an uncompetitive inhibitor in relatively poorly oxygenated shake flask aerobic cultures. In a better controlled aerobic environment of a bioreactor, the role of sodium benzoate could equally well be described as competitive, uncompetitive or noncompetitive inhibitor of growth. In intermittent fed-batch fermentations under highly aerobic conditions, the presence of sodium benzoate at 0.15gL(-1) clearly enhanced the xylitol titer relative to the control culture without the sodium benzoate. The final xylitol concentration and the average xylitol yield on xylose were nearly 50gL(-1) and 0.57gg(-1), respectively, in the presence of sodium benzoate. Both these values were substantially higher than reported for the same fermentation under microaerobic conditions. Therefore, a fed-batch aerobic fermentation in the presence of sodium benzoate is promising for xylitol production using C. mogii. Copyright © 2014 Elsevier B.V. All rights reserved.

A real-time fluorescence assay for protease activity and inhibitor screening based on the aggregation-caused quenching of a perylene probe.

PubMed

Wang, Yan; Zhang, Zhifang; Zhang, Ya; Yu, Cong

2018-06-01

We have established a real-time and label-free fluorescence turn-on strategy for protease activity detection and inhibitor screening via peptide-induced aggregation-caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly-l-glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective. Copyright © 2018 John Wiley & Sons, Ltd.

A mid-infrared flow-through sensor for label-free monitoring of enzyme inhibition.

PubMed

Armenta, S; Tomischko, W; Lendl, B

2008-12-01

Label-free monitoring of acetylcholinesterase (AChE) activity was achieved with a mid-infrared flow-through sensor. The flow-through sensor comprised agarose beads, carrying covalently immobilized AChE, which were placed in a temperature-controlled (37 degrees C) CaF(2) flow cell with an optical path of 60 mum. The sensor was incorporated into a computer-controlled sequential injection (SI) system for automated liquid handling. Different mixtures of enzyme substrate acetylcholine (ACh) and inhibitor (tacrine) were prepared and fed into the flow-through sensor. The flow was stopped as soon as the prepared mixtures reached the sensor. Enzymatic hydrolysis of ACh by AChE was directly monitored as it took place in the flow-through sensor. The inhibition effect of tacrine was calculated from the reaction-induced spectral changes, revealing an important decrease in the activity of AChE, approaching zero when the inhibitor concentration is high enough. The developed mid-infrared flow-through sensor is flexible and can be used to study the inhibitor activity of different target molecules as well as different enzymes.

N-ω-chloroacetyl-l-ornithine, a new competitive inhibitor of ornithine decarboxylase, induces selective growth inhibition and cytotoxicity on human cancer cells versus normal cells.

PubMed

Medina-Enríquez, Miriam Marlene; Alcántara-Farfán, Verónica; Aguilar-Faisal, Leopoldo; Trujillo-Ferrara, José Guadalupe; Rodríguez-Páez, Lorena; Vargas-Ramírez, Alba Laura

2015-06-01

Many cancer cells have high expression of ornithine decarboxylase (ODC) and there is a concerted effort to seek new inhibitors of this enzyme. The aim of the study was to initially characterize the inhibition properties, then to evaluate the cytotoxicity/antiproliferative cell based activity of N-ω-chloroacetyl-l-ornithine (NCAO) on three human cancer cell lines. Results showed NCAO to be a reversible competitive ODC inhibitor (Ki = 59 µM) with cytotoxic and antiproliferative effects, which were concentration- and time-dependent. The EC50,72h of NCAO was 15.8, 17.5 and 10.1 µM for HeLa, MCF-7 and HepG2 cells, respectively. NCAO at 500 µM completely inhibited growth of all cancer cells at 48 h treatment, with almost no effect on normal cells. Putrescine reversed NCAO effects on MCF-7 and HeLa cells, indicating that this antiproliferative activity is due to ODC inhibition.

High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

PubMed

Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

2017-07-01

Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

Preclinical rationale for combination of crizotinib with mitomycin C for the treatment of advanced colorectal cancer.

PubMed

Lev, Avital; Deihimi, Safoora; Shagisultanova, Elena; Xiu, Joanne; Lulla, Amriti R; Dicker, David T; El-Deiry, Wafik S

2017-09-02

Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the United States. We analyzed 26 MSI-High and 558 non-MSI-High CRC tumors. BRCA2 mutations were highly enriched (50%) in MSI-High CRC. Immunohistochemistry showed that BRCA2-mutated MSI-High CRC had high c-MET (64%) expression compared with BRCA-WT (17%). We hypothesized a mechanistic link between BRCA2-deficiency and c-MET overexpression and synergistic interaction between drugs that treat BRCA-deficient tumors (mitomycin C (MMC) or PARP inhibitors) and c-MET inhibitors (crizotinib). We tested CRC cell lines for sensitivity to MMC plus crizotinib or other drug combinations including PARP-inhibitors. Combined treatment of tumor cells with crizotinib and MMC led to increased apoptosis as compared with each drug alone. Additionally, combination treatment with increasing concentrations of both drugs demonstrated a synergistic anti-cancer effect (CI = 0.006-0.74). However, we found no evidence for c-MET upregulation upon effective BRCA2 knockdown in tumor cells -/+DNA damage. Although we found no mechanistic link between BRCA2 deficiency and c-MET overexpression, c-MET is frequently overexpressed in CRC and BRCA2 is mutated especially in MSI-H CRC. The combination of crizotinib with MMC appeared synergistic regardless of MSI or BRCA2 status. Using an in-vivo CRC xenograft model we found reduced tumor growth with combined crizotinib and MMC therapy (p = 0.0088). Our preclinical results support clinical testing of the combination of MMC and crizotinib in advanced CRC. Targeting cell survival mediated by c-MET in combination with targeting DNA repair may be a reasonable strategy for therapy development in CRC or other cancers.

Drug-drug interactions with sodium-glucose cotransporters type 2 (SGLT2) inhibitors, new oral glucose-lowering agents for the management of type 2 diabetes mellitus.

PubMed

Scheen, André J

2014-04-01

Inhibitors of sodium-glucose cotransporters type 2 (SGLT2) reduce hyperglycaemia by decreasing renal glucose threshold and thereby increasing urinary glucose excretion. They are proposed as a novel approach for the management of type 2 diabetes mellitus. They have proven their efficacy in reducing glycated haemoglobin, without inducing hypoglycaemia, as monotherapy or in combination with various other glucose-lowering agents, with the add-on value of promoting some weight loss and lowering arterial blood pressure. As they may be used concomitantly with many other drugs, we review the potential drug-drug interactions (DDIs) regarding the three leaders in the class (dapagliglozin, canagliflozin and empagliflozin). Most of the available studies were performed in healthy volunteers and have assessed the pharmacokinetic interferences with a single administration of the SGLT2 inhibitor. The exposure [assessed by peak plasma concentrations (Cmax) and area under the concentration-time curve (AUC)] to each SGLT2 inhibitor tested was not significantly influenced by the concomitant administration of other glucose-lowering agents or cardiovascular agents commonly used in patients with type 2 diabetes. Reciprocally, these medications did not influence the pharmacokinetic parameters of dapagliflozin, canagliflozin or empagliflozin. Some modest changes were not considered as clinically relevant. However, drugs that could specifically interfere with the metabolic pathways of SGLT2 inhibitors [rifampicin, inhibitors or inducers of uridine diphosphate-glucuronosyltransferase (UGT)] may result in significant changes in the exposure of SGLT2 inhibitors, as shown for dapagliflozin and canagliflozin. Potential DDIs in patients with type 2 diabetes receiving chronic treatment with an SGLT2 inhibitor deserve further attention, especially in individuals treated with several medications or in more fragile patients with hepatic and/or renal impairment.

Effect of fluid flow, pH and tobacco extracts concentration as organic inhibitors to corrosion characteristics of AISI 1045 steel in 3.5% NaCl environment containing CO2 gas

NASA Astrophysics Data System (ADS)

Kurniawan, Budi Agung; Pratiwi, Vania Mitha; Ahmadi, Nafi'ul Fikri

2018-04-01

Corrosion become major problem in most industries. In the oil and gas company, corrosion occurs because of reaction between steel and chemical species inside crude oil. Crude oil or nature gas provide corrosive species, such as CO2, O2, H2S and so on. Fluid containing CO2 gas causes CO2 corrosion which attack steel as well as other corrosion phenomena. This CO2 corrosion commonly called as sweet environment and produce FeCO3 as corrosion products. Fluid flow factor in pipelines during the oil and gas transportation might increase the rate of corrosion itself. Inhibitor commonly use used as corrosion protection because its simplicity in usage. Nowadays, organic inhibitor become main issue in corrosion protection because of biodegradable, low cost, and environmental friendly. This research tried to use tobacco leaf extract as organic inhibitor to control corrosion in CO2 environment. The electrolyte solution used was 3.5% NaCl at pH 4 and pH 7. Weight loss test results showed that the lowest corrosion rate was reach at 132.5 ppm inhibitor, pH 7 and rotational speed of 150 rpm with corrosion rate of 0.091 mm/y. While at pH 4, the lowest corrosion rate was found at rotational speed of 150 rpm with inhibitor concentration of 265 ppm and corrosion rate of 0.327 mm/y. FTIR results indicate the presence of nicotine functional groups on the steel surface. However, based on corrosion rate, it is believed that corrosion occurs, and FeCO3 was soluble in electrolyte. Tobacco leaf extract inhibitors worked by a physisorption mechanism, where tobacco inhibitors formed thin layer on the steel surface.

Phytochemical investigation on leaf extract of Cordia salicifolia Cham.

PubMed

Menghini, Luigi; Epifano, Francesco; Leporini, Lidia; Pagiotti, Rita; Tirillini, Bruno

2008-03-01

The dichloromethane extract of leaves of Cordia salicifolia Cham. (Family Boraginaceae) was fractionated by SiO(2) column chromatography and analyzed by gas chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy. The apolar extract is characterized by a very high content of (+)-spathulenol (0.53%). The major component of the extract exhibited a very weak activity as an inhibitor of growth of Helicobacter pylori in vitro (minimum inhibitory concentration = 200 microg/mL).

Turning tumor-promoting copper into an anti-cancer weapon via high-throughput chemistry.

PubMed

Wang, F; Jiao, P; Qi, M; Frezza, M; Dou, Q P; Yan, B

2010-01-01

Copper is an essential element for multiple biological processes. Its concentration is elevated to a very high level in cancer tissues for promoting cancer development through processes such as angiogenesis. Organic chelators of copper can passively reduce cellular copper and serve the role as inhibitors of angiogenesis. However, they can also actively attack cellular targets such as proteasome, which plays a critical role in cancer development and survival. The discovery of such molecules initially relied on a step by step synthesis followed by biological assays. Today high-throughput chemistry and high-throughput screening have significantly expedited the copper-binding molecules discovery to turn "cancer-promoting" copper into anti-cancer agents.

The Inhibitor Ko143 Is Not Specific for ABCG2.

PubMed

Weidner, Lora D; Zoghbi, Sami S; Lu, Shuiyu; Shukla, Suneet; Ambudkar, Suresh V; Pike, Victor W; Mulder, Jan; Gottesman, Michael M; Innis, Robert B; Hall, Matthew D

2015-09-01

Imaging ATP-binding cassette (ABC) transporter activity in vivo with positron emission tomography requires both a substrate and a transporter inhibitor. However, for ABCG2, there is no inhibitor proven to be specific to that transporter alone at the blood-brain barrier. Ko143 [[(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4- b]indole-3-propanoic acid 1,1-dimethylethyl ester], a nontoxic analog of fungal toxin fumitremorgin C, is a potent inhibitor of ABCG2, although its specificity in mouse and human systems is unclear. This study examined the selectivity of Ko143 using human embryonic kidney cell lines transfected with ABCG2, ABCB1, or ABCC1 in several in vitro assays. The stability of Ko143 in rat plasma was measured using high performance liquid chromatography. Our results show that, in addition to being a potent inhibitor of ABCG2, at higher concentrations (≥1 μM) Ko143 also has an effect on the transport activity of both ABCB1 and ABCC1. Furthermore, Ko143 was found to be unstable in rat plasma. These findings indicate that Ko143 lacks specificity for ABCG2 and this should be taken into consideration when using Ko143 for both in vitro and in vivo experiments. U.S. Government work not protected by U.S. copyright.

Protease inhibitors from several classes work synergistically against Callosobruchus maculatus.

PubMed

Amirhusin, Bahagiawati; Shade, Richard E; Koiwa, Hisashi; Hasegawa, Paul M; Bressan, Ray A; Murdock, Larry L; Zhu-Salzman, Keyan

2007-07-01

Targeting multiple digestive proteases may be more effective in insect pest control than inhibition of a single enzyme class. We therefore explored possible interactions of three antimetabolic protease inhibitors fed to cowpea bruchids in artificial diets, using a recombinant soybean cysteine protease inhibitor scN, an aspartic protease inhibitor pepstatin A, and soybean Kunitz trypsin inhibitor KI. scN and pepstatin, inhibiting major digestive cysteine and aspartic proteases, respectively, significantly prolonged the developmental time of cowpea bruchids individually. When combined, the anti-insect effect was synergistic, i.e., the toxicity of the mixture was markedly greater than that of scN or pepstatin alone. KI alone did not impact insect development even at relatively high concentrations, but its anti-insect properties became apparent when acting jointly with scN or scN plus pepstatin. Incubating KI with bruchid midgut extract showed that it was partially degraded. This instability may explain its lack of anti-insect activity. However, this proteolytic degradation was inhibited by scN and/or pepstatin. Protection of KI from proteolysis in the insect digestive tract thus could be the basis for the synergistic effect. These observations support the concept that cowpea bruchid gut proteases play a dual role; digesting protein for nutrient needs and protecting insects by inactivating dietary proteins that may otherwise be toxic. Our results also suggest that transgenic resistance strategies that involve multigene products are likely to have enhanced efficacy and durability.

A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA)-chi 2-fluorescein.

PubMed

Wiley, J S; Brocklebank, A M; Snook, M B; Jamieson, G P; Sawyer, W H; Craik, J D; Cass, C E; Robins, M J; McAdam, D P; Paterson, A R

1991-02-01

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Investigation of interaction studies of cefpirome with ACE-inhibitors in various buffers

NASA Astrophysics Data System (ADS)

Nawaz, Muhammad; Arayne, Muhammad Saeed; Sultana, Najma; Abbas, Hira Fatima

2015-02-01

This work describes a RP-HPLC method for the determination and interaction studies of cefpirome with ACE-inhibitors (captopril, enalapril and lisinopril) in various buffers. The separation and interaction of cefpirome with ACE-inhibitors was achieved on a Purospher Star, C18 (5 μm, 250 × 4.6 mm) column. Mobile phase consisted of methanol: water (80:20, v/v, pH 3.3); however, for the separation of lisinopril, it was modified to methanol-water (40:60, v/v, pH 3.3) and pumped at a flow rate of 1 mL min-1. In all cases, UV detection was performed at 225 nm. Interactions were carried out in physiological pH i.e., pH 1 (simulated gastric juice), 4 (simulated full stomach), 7.4 (blood pH) and 9 (simulated GI), drug contents were analyzed by reverse phase high performance liquid chromatography. Method was found linear in the concentration range of 1.0-50.0 μg mL-1 with correlation coefficient (r2) of 0.999. Precision (RSD%) was less than 2.0%, indicating good precision of the method and accuracy was 98.0-100.0%. Furthermore, cefpirome-ACE-inhibitors' complexes were also synthesized and results were elucidated on the basis of FT-IR, and 1H NMR. The interaction results show that these interactions are pH dependent and for the co-administration of cefpirome and ACE-inhibitors, a proper interval should be given.

Qualitative and quantitative two-dimensional thin-layer chromatography/high performance liquid chromatography/diode-array/electrospray-ionization-time-of-flight mass spectrometry of cholinesterase inhibitors.

PubMed

Mroczek, Tomasz

2016-09-10

Recently launched thin-layer chromatography-mass spectrometry (TLC-MS) interface enabling extraction of compounds directly from TLC plates into MS ion source was unusually extended into two-dimensional thin-layer chromatography/high performance liquid chromatography (2D, TLC/HPLC) system by its a direct connection to a rapid resolution 50×2.1mm, I.D. C18 column compartment followed by detection by diode array (DAD) and electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS). In this way, even not separated bands of complicated mixtures of natural compounds could be analysed structurally, only within 1-2min after development of TLC plates. In comparison to typically applied TLC-MS interface, no ion suppression for acidic mobile phases was observed. Also, substantial increase in ESI-TOF-MS sensitivities and quality of spectra, were noticed. It has been utilised in combination with TLC- based bioautographic approaches of acetylcholinesterase (AChE) inhibitors, However, it can be also applied in any other procedures related to bioactivity (e.g. 2,2-Diphenyl-1-picryl-hydrazyl-DPPH screen test for radicals). This system has been also used for determination of half maximal inhibitory concentration (IC50 values) of the active inhibitor-galanthamine, as an example. Moreover, AChE inhibitory potencies of some of purified plant extracts, never studied before, have been quantitatively measured. This is first report of usage such the 2D TLC/HPLC/MS system both for qualitative and quantitative evaluation of cholinesterase inhibitors in biological matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

Corrosion of radioactive waste tanks containing washed sludge and precipitates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bickford, D.F.; Congdon, J.W.; Oblath, S.B.

1988-05-01

At the US Department of Energy (DOE) Savannah River Plant, the corrosion of carbon steel storage tanks containing alkaline, high-level radioactive waste is controlled by specification of limits on waste composition and temperature. Laboratory tests, conducted to determine minimum corrosion inhibitor levels, indicated pitting of carbon steel near the waterline for proposed storage conditions. In situ electrochemical measurements of full-scale radioactive process demonstrations were conducted to assess the validity of laboratory tests. The in situ results are compared to those of laboratory tests, with particular regard given to simulated solution composition. Transition metal hydroxide sludge contains strong passivating species formore » carbon steel. Washed precipitate contains organic species that lower solution pH and tend to reduce passivating films, requiring higher inhibitor concentrations than the 0.01 M nitrite required for reactor fuel reprocessing wastes.« less