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Sample records for aldosterone synthase inhibitors

  1. Progress towards clinically useful aldosterone synthase inhibitors.

    PubMed

    Cerny, Matthew A

    2013-01-01

    Owing to the high degree of similarity between aldosterone synthase (CYP11B2) and cortisol synthase (CYP11B1), the design of selective inhibitors of one or the other of these two enzymes was, at one time, thought to be impossible. Through development of novel enzyme screening assays and significant medicinal chemistry efforts, highly potent inhibitors of CYP11B2 have been identified with selectivities approaching 1000-fold between the two enzymes. Many of these molecules also possess selectivity against other steroidogenic cytochromes P450 (e.g. CYP17A1 and CYP19A1) as well as hepatic drug metabolizing P450s. Though not as well developed or explored, inhibitors of CYP11B1, with selectivities approaching 50-fold, have also been identified. The therapeutic benefits of affecting the renin-angiotensin-aldosterone system have been well established with the therapeutically useful angiotensin-converting enzymes inhibitors, angiotensin receptor blockers, and mineralocorticoid receptor antagonists. Data regarding the additional benefits of an aldosterone synthase inhibitor (ASi) are beginning to emerge from animal models and human clinical trials. Despite great promise and much progress, additional challenges still exist in the path towards development of a therapeutically useful ASi.

  2. A novel aldosterone synthase inhibitor ameliorates mortality in pressure-overload mice with heart failure.

    PubMed

    Furuzono, Shinji; Meguro, Masaki; Miyauchi, Satoru; Inoue, Shinichi; Homma, Tsuyoshi; Yamada, Keisuke; Tagawa, Yoh-Ichi; Nara, Futoshi; Nagayama, Takahiro

    2017-01-15

    It has been elucidated that mineralocorticoid receptor antagonists reduce mortality in patients with congestive heart failure and post-acute myocardial infarction. A direct inhibition of aldosterone synthase (CYP11B2) is also expected to have therapeutic benefits equal in quality to mineralocorticoid receptor antagonists in terms of reducing mineralocorticoid receptor signaling. Therefore, we have screened our chemical libraries and identified a novel and potent aldosterone synthase inhibitor, 2,2,2-trifluoro-1-{4-[(4-fluorophenyl)amino]pyrimidin-5-y}-1-[1-(methylsulfonyl)piperidin-4-yl]ethanol (compound 1), by lead optimization. Pharmacological properties of compound 1 were examined in in vitro cell-based assays and an in vivo mouse model of pressure-overload hypertrophy by transverse aortic constriction (TAC). Compound 1 showed potent CYP11B2 inhibition against human and mouse enzymes (IC50; 0.003μM and 0.096μM, respectively) in a cell-based assay. The oral administration of 0.06% compound 1 in the food mixture of a mouse TAC model significantly reduced the plasma aldosterone level and ameliorated mortality rate. This study is the first to demonstrate that a CYP11B2 inhibitor improved survival rates of heart failure induced by pressure-overload in mice. The treatment of 0.06% compound 1 did not elevate plasma potassium level in this model, although further evaluation of hyperkalemia is needed. These results suggest that compound 1 can be developed as a promising oral CYP11B2 inhibitor for pharmaceutical applications. Compound 1 could also be a useful compound for clarifying the role of aldosterone in cardiac hypertrophy.

  3. Discovery of Potential Inhibitors of Aldosterone Synthase from Chinese Herbs Using Pharmacophore Modeling, Molecular Docking, and Molecular Dynamics Simulation Studies

    PubMed Central

    Lu, Fang; Qiao, Liansheng; Chen, Xi; Li, Gongyu

    2016-01-01

    Aldosterone synthase (CYP11B2) is a key enzyme for the biosynthesis of aldosterone, which plays a significant role for the regulation of blood pressure. Excess aldosterone can cause the dysregulation of the renin-angiotensin-aldosterone system (RAAS) and lead to hypertension. Therefore, research and development of CYP11B2 inhibitor are regarded as a novel approach for the treatment of hypertension. In this study, the pharmacophore models of CYP11B2 inhibitors were generated and the optimal model was used to identify potential CYP11B2 inhibitors from the Traditional Chinese Medicine Database (TCMD, Version 2009). The hits were further refined by molecular docking and the interactions between compounds and CYP11B2 were analyzed. Compounds with high Fitvalue, high docking score, and expected interactions with key residues were selected as potential CYP11B2 inhibitors. Two most promising compounds, ethyl caffeate and labiatenic acid, with high Fitvalue and docking score were reserved for molecular dynamics (MD) study. All of them have stability of ligand binding which suggested that they might perform the inhibitory effect on CYP11B2. This study provided candidates for novel drug-like CYP11B2 inhibitors by molecular simulation methods for the hypertension treatment. PMID:27781210

  4. Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of Aldosterone Synthase (CYP11B2)

    PubMed Central

    Schwab, Dietmar; Delporte, Marie-Laure; Palermo, Giuseppe; Amrein, Kurt; Mohr, Susanne; De Vera Mudry, Maria Cristina; Brown, Morris J.; Ferber, Philippe

    2017-01-01

    Primary hyperaldosteronism is a common cause of resistant hypertension. Aldosterone is produced in the adrenal by aldosterone synthase (AS, encoded by the gene CYP11B2). AS shares 93% homology to 11β-hydroxylase (encoded by the gene CYP11B1), responsible for cortisol production. This homology has hitherto impeded the development of a drug, which selectively suppresses aldosterone but not cortisol production, as a new treatment for primary hyperaldosteronism. We now report the development of RO6836191 as a potent (Ki 13 nmol/L) competitive inhibitor of AS, with in vitro selectivity >100-fold over 11β-hydroxylase. In cynomolgus monkeys challenged with synthetic adrenocorticotropic hormone, single doses of RO6836191 inhibited aldosterone synthesis without affecting the adrenocorticotropic hormone–induced rise in cortisol. In repeat-dose toxicity studies in monkeys, RO6836191 reproduced the adrenal changes of the AS−/− mouse: expansion of the zona glomerulosa; increased expression of AS (or disrupted green fluorescent protein gene in the AS−/− mouse); hypertrophy, proliferation, and apoptosis of zona glomerulosa cells. These changes in the monkey were partially reversible and partially preventable by electrolyte supplementation and treatment with an angiotensin-converting enzyme inhibitor. In healthy subjects, single doses of RO6836191, across a 360-fold dose range, reduced plasma and urine aldosterone levels with maximum suppression at a dose of 10 mg, but unchanged cortisol, on adrenocorticotropic hormone challenge, up to 360 mg, and increase in the precursors 11-deoxycorticosterone and 11-deoxycortisol only at or >90 mg. In conclusion, RO6836191 demonstrates that it is possible to suppress aldosterone production completely in humans without affecting cortisol production. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01995383. PMID:27872236

  5. In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.

    PubMed

    Lucas, Simon; Heim, Ralf; Ries, Christina; Schewe, Katarzyna E; Birk, Barbara; Hartmann, Rolf W

    2008-12-25

    Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats.

  6. Molecular identity and gene expression of aldosterone synthase cytochrome P450

    SciTech Connect

    Okamoto, Mitsuhiro . E-mail: mokamoto@mr-mbio.med.osaka-u.ac.jp; Nonaka, Yasuki; Takemori, Hiroshi; Doi, Junko

    2005-12-09

    11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.

  7. Association of Aldosterone Synthase Polymorphism (CYP11B2 -344T>C) and Genetic Ancestry with Atrial Fibrillation and Serum Aldosterone in African Americans with Heart Failure

    PubMed Central

    Bress, Adam; Han, Jin; Patel, Shitalben R.; Desai, Ankit A.; Mansour, Ibrahim; Groo, Vicki; Progar, Kristin; Shah, Ebony; Stamos, Thomas D.; Wing, Coady; Garcia, Joe G. N.; Kittles, Rick; Cavallari, Larisa H.

    2013-01-01

    The objective of this study was to examine the extent to which aldosterone synthase genotype (CYP11B2) and genetic ancestry correlate with atrial fibrillation (AF) and serum aldosterone in African Americans with heart failure. Clinical data, echocardiographic measurements, and a genetic sample for determination of CYP11B2 -344T>C (rs1799998) genotype and genetic ancestry were collected from 194 self-reported African Americans with chronic, ambulatory heart failure. Genetic ancestry was determined using 105 autosomal ancestry informative markers. In a sub-set of patients (n = 126), serum was also collected for determination of circulating aldosterone. The CYP11B2 −344C allele frequency was 18% among the study population, and 19% of patients had AF. Multiple logistic regression revealed that the CYP11B2 −344CC genotype was a significant independent predictor of AF (OR 12.7, 95% CI 1.60–98.4, p = 0.0150, empirical p = 0.011) while holding multiple clinical factors, left atrial size, and percent European ancestry constant. Serum aldosterone was significantly higher among patients with AF (p = 0.036), whereas increased West African ancestry was inversely correlated with serum aldosterone (r = −0.19, p = 0.037). The CYP11B2 −344CC genotype was also overrepresented among patients with extreme aldosterone elevation (≥90th percentile, p = 0.0145). In this cohort of African Americans with chronic ambulatory heart failure, the CYP11B2 −344T>C genotype was a significant independent predictor of AF while holding clinical, echocardiographic predictors, and genetic ancestry constant. In addition, increased West African ancestry was associated with decreased serum aldosterone levels, potentially providing an explanation for the lower risk for AF observed among African Americans. PMID:23936266

  8. Inhibitors of specific ceramide synthases.

    PubMed

    Schiffmann, Susanne; Hartmann, Daniela; Fuchs, Sina; Birod, Kerstin; Ferreiròs, Nerea; Schreiber, Yannick; Zivkovic, Aleksandra; Geisslinger, Gerd; Grösch, Sabine; Stark, Holger

    2012-02-01

    Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C(14:0)-Cer - C(26:0)-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure-activity relationships and the potential biological impact of these compounds are discussed.

  9. Pharmacological treatment of aldosterone excess.

    PubMed

    Deinum, Jaap; Riksen, Niels P; Lenders, Jacques W M

    2015-10-01

    Primary aldosteronism, caused by autonomous secretion of aldosterone by the adrenals, is estimated to account for at least 5% of hypertension cases. Hypertension explains the considerable cardiovascular morbidity caused by aldosteronism only partly, calling for specific anti-aldosterone drugs. The pharmacology of aldosterone is complex due to high homology with other steroids, the resemblance of steroid receptors, and the common pathways of steroid synthesis. Classically, pharmacological treatment of aldosteronism relied on the mineralocorticoid receptor (MR) antagonist spironolactone, which is highly effective, but causes considerable, mainly sexual side-effects due to limited selectivity for the MR. New agents have been developed or are being developed that aim at higher selectivity for MR antagonists (eplerenone, dihydropyridine-derived calcium channel blockers (CCB)), or inhibition of aldosterone synthesis. Eplerenone is less potent than spironolactone, but causes fewer adverse effects due to its selectivity for the MR. Non-steroidal MR antagonists have been developed from dihydropyridine CCBs, having lost their CCB activity and being highly selective for the MR. The first clinical studies with these drugs are underway. Aldosterone synthase inhibitors are an attractive alternative, but are prone to interference with cortisol synthesis due to the inhibition of 11-β-hydroxylation, an essential step in both cortisol and aldosterone synthesis, and accumulation of mineralocorticoid precursors. In coming years clinical research will provide the answers as to which drugs and strategies to treat high-aldosterone states are the most effective.

  10. Chitin synthase inhibitors as antifungal agents.

    PubMed

    Chaudhary, Preeti M; Tupe, Santosh G; Deshpande, Mukund V

    2013-02-01

    Increased risk of fungal diseases in immunocompromised patients, emerging fungal pathogens, limited repertoire of antifungal drugs and resistance development against the drugs demands for development of new and effective antifungal agents. With greater knowledge of fungal metabolism efforts are being made to inhibit specific enzymes involved in different biochemical pathways for the development of antifungal drugs. Chitin synthase is one such promising target as it is absent in plants and mammals. Nikkomycin Z, a chitin synthase inhibitor is under clinical development. Chitin synthesis in fungi, chitin synthase as a target for antifungal agent development, different chitin synthase inhibitors isolated from natural sources, randomly synthesized and modified from nikkomycin and polyoxin are discussed in this review.

  11. Modulation of cytochromes P450 with xanthone-based molecules: from aromatase to aldosterone synthase and steroid 11β-hydroxylase inhibition.

    PubMed

    Gobbi, Silvia; Hu, Qingzhong; Negri, Matthias; Zimmer, Christina; Belluti, Federica; Rampa, Angela; Hartmann, Rolf W; Bisi, Alessandra

    2013-02-28

    Imidazolylmethylflavones previously reported by us as aromatase inhibitors proved to be able to interact with aldosterone synthase (CYP11B2), a cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual screening of a small compounds library of our earlier synthesized aromatase inhibitors was performed and, according to the results and the corresponding biological data, led to the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl substituent in position 1 and different substituents in position 4. Some very potent inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be directed to different targets when appropriately functionalized.

  12. [Prothrombotic aldosterone action--a new side of the hormone].

    PubMed

    Gromotowicz, Anna; Osmólska, Urszula; Mantur, Maria; Szoka, Piotr; Zakrzeska, Agnieszka; Szemraj, Janusz; Chabielska, Ewa

    2010-10-18

    Recent studies have focused on a new wave of interest in aldosterone due mainly to its growing profile as a local messenger in pathology of the cardiovascular system, rather than its hormonal action. In the last few years strong evidence for a correlation between raised aldosterone level and haemostasis disturbances leading to increased risk of cardiovascular events has been provided. It has been demonstrated that aldosterone contributes to endothelial dysfunction, fibrinolytic disorders and oxidative stress augmentation. It was also shown that chronic aldosterone treatment results in enhanced experimental arterial thrombosis. Our study in a venous model of thrombosis in normotensive rats confirmed that even a short-lasting increase in aldosterone level intensified thrombus formation. One-hour aldosterone infusion shortened bleeding time; increased platelet adhesion to collagen; reduced tissue factor, thrombin activatable fibrinolysis inhibitor, and plasminogen activator inhibitor; and increased plasminogen activator plasma level. A fall in plasma nitric oxide metabolite concentration with a decrease in aortic nitric oxide synthase mRNA level was also observed. Moreover, aldosterone increased hydrogen peroxide and malonyl dialdehyde plasma concentration and augmented NADPH oxidase and superoxide dismutase aortic expression. Therefore, the mechanism of aldosterone prothrombotic action is multiple and involves primary haemostasis activation, procoagulative and antifibrinolytic action, NO bioavailability impairment and oxidative stress augmentation. The effects of aldosterone were not fully abolished by mineralocorticoid receptor blockade, suggesting the involvement of alternative mechanisms in the prothrombotic aldosterone action.

  13. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    PubMed

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism.

  14. Effect of aldosterone breakthrough on albuminuria during treatment with a direct renin inhibitor and combined effect with a mineralocorticoid receptor antagonist.

    PubMed

    Sato, Atsuhisa; Fukuda, Seiichi

    2013-10-01

    We have reported observing aldosterone breakthrough in the course of relatively long-term treatment with renin-angiotensin (RA) system inhibitors, where the plasma aldosterone concentration (PAC) increased following an initial decrease. Aldosterone breakthrough has the potential to eliminate the organ-protective effects of RA system inhibitors. We therefore conducted a study in essential hypertensive patients to determine whether aldosterone breakthrough occurred during treatment with the direct renin inhibitor (DRI) aliskiren and to ascertain its clinical significance. The study included 40 essential hypertensive patients (18 men and 22 women) who had been treated for 12 months with aliskiren. Aliskiren significantly decreased blood pressure and plasma renin activity (PRA). The PAC was also decreased significantly at 3 and 6 months; however, the significant difference disappeared after 12 months. Aldosterone breakthrough was observed in 22 of the subjects (55%). Urinary albumin excretion differed depending on whether breakthrough occurred. For the subjects in whom aldosterone breakthrough was observed, eplerenone was added. A significant decrease in urinary albumin excretion was observed after 1 month, independent of changes in blood pressure. In conclusion, this study demonstrated that aldosterone breakthrough occurs in some patients undergoing DRI therapy. Aldosterone breakthrough affects the drug's ability to improve urinary albumin excretion, and combining a mineralocorticoid receptor antagonist with the DRI may be useful for decreasing urinary albumin excretion. When the objective is organ protection in hypertensive patients, a two-pronged approach using combination therapy to inhibit both the RA system and aldosterone may be highly effective.

  15. Renin-angiotensin-aldosterone system inhibition: overview of the therapeutic use of angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, mineralocorticoid receptor antagonists, and direct renin inhibitors.

    PubMed

    Mercier, Kelly; Smith, Holly; Biederman, Jason

    2014-12-01

    Angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) therapy in hypertensive diabetic patients with macroalbuminuria, microalbuminuria, or normoalbuminuria has been repeatedly shown to improve cardiovascular mortality and reduce the decline in glomerular filtration rate. Renin-angiotensin-aldosterone system (RAAS) blockade in normotensive diabetic patients with normoalbuminuria or microalbuminuria cannot be advocated at present. Dual RAAS inhibition with ACE inhibitors plus ARBs or ACE inhibitors plus direct renin inhibitors has failed to improve cardiovascular or renal outcomes but has predisposed patients to serious adverse events.

  16. Substituted 2-aminopyridines as inhibitors of nitric oxide synthases.

    PubMed

    Hagmann, W K; Caldwell, C G; Chen, P; Durette, P L; Esser, C K; Lanza, T J; Kopka, I E; Guthikonda, R; Shah, S K; MacCoss, M; Chabin, R M; Fletcher, D; Grant, S K; Green, B G; Humes, J L; Kelly, T M; Luell, S; Meurer, R; Moore, V; Pacholok, S G; Pavia, T; Williams, H R; Wong, K K

    2000-09-04

    A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

  17. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    PubMed

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  18. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  19. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  20. Synthesis of antifungal glucan synthase inhibitors from enfumafungin.

    PubMed

    Zhong, Yong-Li; Gauthier, Donald R; Shi, Yao-Jun; McLaughlin, Mark; Chung, John Y L; Dagneau, Philippe; Marcune, Benjamin; Krska, Shane W; Ball, Richard G; Reamer, Robert A; Yasuda, Nobuyoshi

    2012-04-06

    An efficient, new, and scalable semisynthesis of glucan synthase inhibitors 1 and 2 from the fermentation product enfumafungin 3 is described. The highlights of the synthesis include a high-yielding ether bond-forming reaction between a bulky sulfamidate 17 and alcohol 4 and a remarkably chemoselective, improved palladium(II)-mediated Corey-Yu allylic oxidation at the highly congested C-12 position of the enfumafungin core. Multi-hundred gram quantities of the target drug candidates 1 and 2 were prepared, in 12 linear steps with 25% isolated yield and 13 linear steps with 22% isolated yield, respectively.

  1. Structure-based design of bacterial nitric oxide synthase inhibitors.

    PubMed

    Holden, Jeffrey K; Kang, Soosung; Hollingsworth, Scott A; Li, Huiying; Lim, Nathan; Chen, Steven; Huang, He; Xue, Fengtian; Tang, Wei; Silverman, Richard B; Poulos, Thomas L

    2015-01-22

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial ( Holden , , Proc. Natl. Acad. Sci. U.S.A. 2013 , 110 , 18127 ). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.

  2. Structure-Based Design of Bacterial Nitric Oxide Synthase Inhibitors

    PubMed Central

    2015-01-01

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial (Holden, , Proc. Natl. Acad. Sci. U.S.A.2013, 110, 1812724145412). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors. PMID:25522110

  3. Structure-based design of bacterial nitric oxide synthase inhibitors

    SciTech Connect

    Holden, Jeffrey K.; Kang, Soosung; Hollingsworth, Scott A.; Li, Huiying; Lim, Nathan; Chen, Steven; Huang, He; Xue, Fengtian; Tang, Wei; Silverman, Richard B.; Poulos, Thomas L.

    2014-12-18

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial. Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Lastly, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.

  4. Structure-based design of bacterial nitric oxide synthase inhibitors

    DOE PAGES

    Holden, Jeffrey K.; Kang, Soosung; Hollingsworth, Scott A.; ...

    2014-12-18

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial. Here wemore » present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Lastly, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.« less

  5. Structure-Based Discovery of Inhibitors of Thymidylate Synthase

    NASA Astrophysics Data System (ADS)

    Shoichet, Brian K.; Stroud, Robert M.; Santi, Daniel V.; Kuntz, Irwin D.; Perry, Kathy M.

    1993-03-01

    A molecular docking computer program (DOCK) was used to screen the Fine Chemical Directory, a database of commercially available compounds, for molecules that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compounds inhibited Lactobacillus caser TS at submillimolar concentrations. One of these inhibitors, sulisobenzone, crystallized with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with molecules sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK.

  6. Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase

    SciTech Connect

    Hevener, Kirk E.; Yun, Mi-Kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephan W.; Lee, Richard E.

    2010-01-12

    Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.

  7. Primary Aldosteronism

    MedlinePlus

    ... Endocrinology Find an Endocrinologist Value of an Endocrinologist Learn About Clinical Trials Keep Your Body in Balance › Primary Aldosteronism Fact Sheet Primary Aldosteronism March 2012 Download PDFs English Espanol Editors Paul Stewart, MD, FRCP William Young, ...

  8. Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments

    PubMed Central

    King, Margaret K.; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S.; Beurel, Eléonore

    2013-01-01

    Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions. PMID:23916593

  9. [The influence of inhibitors of neuronal and inducible NO-synthases on experimental hemorrhagic stroke].

    PubMed

    Krushinskiĭ, A L; Kuzenkov, V S; D'iakonova, V E; Reutov, V P

    2014-01-01

    Objectives. To study the effect of inhibitors of neuronal and inducible NO-synthase on the development of hemorrhagic stroke in rats Krushinsky-Molodkina (KM) without adaptation to hypoxia and with short-term adaptation to hypobaric hypoxia. Material and methods. Ninety rats were included in the study. Experiments with short-term adaptation to hypobaric hypoxia were performed on 48 rats. The inhibitor of inducible NO-synthase (aminoguanidine, "Sigma") or the inhibitor of neuronal NO-synthase (7-nitroindasol, "Sigma") were injected in dosage 2.5 mg/100g intraperitoneally. Results. Selective inhibitors of neuronal and inducible NO-synthase had a protective effect on stress injuries in KM rats. The inhibitor of neuronal NO-synthase was more effective than the inhibitor of inducible NO-synthase in the experiments without adaptation to hypoxia. Markedly greater protective effect was achieved by the simultaneous introduction of inhibitors of neuronal and inducible NO-synthase. The greatest protective effect in the development of stress damage in rats of KM was observed in short-term adaptation to hypobaric hypoxia with simultaneous introduction of both inhibitors. Conclusions. It can be assumed that an excessive amount of NO produced by neuronal and inducible NO-synthases during the acoustic exposure in KM rats leads to stress damage. Use of selective inhibitors reduce the excess NO synthesis and the development of audiogenic stress damage caused by hemorrhagic stroke.

  10. Combination therapy with renin-angiotensin-aldosterone system inhibitor telmisartan and serine protease inhibitor camostat mesilate provides further renoprotection in a rat chronic kidney disease model.

    PubMed

    Narita, Yuki; Ueda, Miki; Uchimura, Kohei; Kakizoe, Yutaka; Miyasato, Yoshikazu; Mizumoto, Teruhiko; Morinaga, Jun; Hayata, Manabu; Nakagawa, Terumasa; Adachi, Masataka; Miyoshi, Taku; Sakai, Yoshiki; Kadowaki, Daisuke; Hirata, Sumio; Mukoyama, Masashi; Kitamura, Kenichiro

    2016-02-01

    We previously reported that camostat mesilate (CM) had renoprotective and antihypertensive effects in rat CKD models. In this study, we examined if CM has a distinct renoprotective effect from telmisartan (TE), a renin-angiotensin-aldosterone system (RAS) inhibitor, on the progression of CKD. We evaluated the effect of CM (400 mg/kg/day) and/or TE (10 mg/kg/day) on renal function, oxidative stress, renal fibrosis, and RAS components in the adenine-induced rat CKD model following 5-weeks treatment period. The combination therapy with CM and TE significantly decreased the adenine-induced increase in serum creatinine levels compared with each monotherapy, although all treatment groups showed similar reduction in blood pressure. Similarly, adenine-induced elevation in oxidative stress markers and renal fibrosis markers were significantly reduced by the combination therapy relative to each monotherapy. Furthermore, the effect of the combination therapy on plasma renin activity (PRA) and plasma aldosterone concentration (PAC) was similar to that of TE monotherapy, and CM had no effect on both PRA and PAC, suggesting that CM has a distinct pharmacological property from RAS inhibition. Our findings indicate that CM could be a candidate drug for an add-on therapy for CKD patients who had been treated with RAS inhibitors.

  11. Renin-angiotensin-aldosterone system inhibitors improve membrane stability and change gene-expression profiles in dystrophic skeletal muscles.

    PubMed

    Chadwick, Jessica A; Bhattacharya, Sayak; Lowe, Jeovanna; Weisleder, Noah; Rafael-Fortney, Jill A

    2017-02-01

    Angiotensin-converting enzyme inhibitors (ACEi) and mineralocorticoid receptor (MR) antagonists are FDA-approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the ACEi lisinopril and the nonspecific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy (DMD) mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current DMD standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared with untreated mice. Short-term treatments of dystrophic mice with specific and nonspecific MR antagonists combined with lisinopril led to overlapping gene-expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic, and chemokine gene expression that was not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in DMD.

  12. Structure-based inhibitor discovery of Helicobacter pylori dehydroquinate synthase.

    PubMed

    Liu, Jai-Shin; Cheng, Wen-Chi; Wang, Hung-Jung; Chen, Yen-Cheng; Wang, Wen-Ching

    2008-08-15

    Dehydroquinate synthase (DHQS) is a nicotinamide adenine dinucleotide (NAD)-dependent enzyme that converts 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) into 3-dehydroquinate (DHQ). Since it catalyzes the second key step in the shikimate pathway, which is crucial for the aromatic amino acid metabolism in bacteria, fungi, and plants, but not in mammals, DHQS is a potential target for new antimicrobial agents, anti-parasitic agents and herbicides. The crystal structure of Helicobacter pylori DHQS (HpDHQS) complexed with NAD has been determined at 2.4-A resolution and was found to possess an N-terminal Rossmann-fold domain and a C-terminal alpha-helical domain. Structural comparison reveals that the binary complex adopts an open-state conformation and shares conserved residues in the binding pocket. Virtual docking of compounds into the active site of the HpDHQS structure using the GOLD docking program led to the identification of several inhibitors. The most active compound had an IC(50) value of 61 microM, which may serve as a lead for potent inhibitors.

  13. Aminodeoxychorismate synthase inhibitors from one-bead one-compound combinatorial libraries: "staged" inhibitor design.

    PubMed

    Dixon, Seth; Ziebart, Kristin T; He, Ze; Jeddeloh, Melissa; Yoo, Choong Leol; Wang, Xiaobing; Lehman, Alan; Lam, Kit S; Toney, Michael D; Kurth, Mark J

    2006-12-14

    4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in the conversion of chorismate into p-aminobenzoate, which is incorporated into folic acid. We aim to discover compounds that inhibit ADCS and serve as leads for a new class of antimicrobial compounds. This report presents (1) synthesis of a mass-tag encoded library based on a "staged" design, (2) massively parallel fluorescence-based on-bead screening, (3) rapid structural identification of hits, and (4) full kinetic analysis of ADCS. All inhibitors are competitive against chorismate and Mg(2+). The most potent ADCS inhibitor identified has a K(i) of 360 microM. We show that the combinatorial diversity elements add substantial binding affinity by interacting with residues outside of but proximal to the active site. The methods presented here constitute a paradigm for inhibitor discovery through active site targeting, enabled by rapid library synthesis, facile massively parallel screening, and straightforward hit identification.

  14. Studies on 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase using chorismate mutase inhibitors.

    PubMed

    Birck, M R; Husain, A; Sheflyan, G Y; Ganem, B; Woodard, R W

    2001-11-05

    The proposed cyclic mechanism of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and the mechanism of chorismate mutase share certain structural and electronic similarities. In this report, we examine several inhibitors of chorismate mutase for their efficacy against KDO 8-P synthase.

  15. Efficacy and Safety of Complete RAAS Blockade with ALISKIREN in Patients with Refractory Proteinuria Who were already on Combined ACE Inhibitor, ARB, and Aldosterone Antagonist

    PubMed Central

    Sreelatha, M

    2016-01-01

    Introduction Proteinuria is always associated with intrinsic kidney disese and is a strong predictor of later development of End Stage Renal Disease (ESRD). As Renin Angiotensin Aldosterone System (RAAS) has a role in mediating proteinuria, inhibitors of this system are renoprotective and patients with refractory proteinuria are put on a combination of these agents. The routinely employed triple blockade of RAAS with Angiotensin Converting Enzyme (ACE) inhibitor, ARB and Aldosterone antagonist has many limitations. Addition of Aliskiren to this combination suppresses the RAAS at the earliest stage and can offset many of these limitations. Aim This study was conducted to assess the safety and efficacy of complete RAAS blockade by the addition of Aliskiren in those patients with refractory proteinuria who were already on triple blockade with ACE inhibitor, ARB and Aldosterone antagonist. Settings This study was conducted in Nephrology Department, Calicut Medical College. Materials and Methods A total of 36 patients with refractory proteinuria who were already on ACE inhibitor, ARB and Aldosterone antagonist were divided in to two groups A and B. Group A received Aliskiren in addition to the above combination whereas group B continued the same treatment for 12 weeks. Efficacy of the treatment was assessed by recording 24hr urine protein and safety by S.Creatinine, S.Potassium every 2 weeks of the treatment period. Statistical Analysis Statistical analysis of the lab values was done using SPSS software. Unpaired t-test, Paired t-test and Chi-square test were done for data analysis. Results Statistical analysis revealed that addition of Aliskiren to the combination therapy with ACE inhibitor+ ARB+ Aldosterone antagonist offers no advantage. But mean reduction in proteinuria was more with Group A than Group B. There is no statistically significant change in S.Creatinine and S.Potassium at the end of treatment. Conclusion As proteinuria is a strong risk factor for

  16. [Primary aldosteronism].

    PubMed

    Amar, Laurence

    2015-06-01

    Primary aldosteronism affects 6% of hypertensive patients. The diagnosis should be suspected in any patient with severe or resistant hypertension or hypertension associated with hypokalemia. The screening test consists on the assessment of the aldosterone to renin ratio. In case of an elevated ratio, the diagnosis of primary aldosteronism is confirmed by either elevated concentrations of basal plasma and/or urinary aldosterone or absence of suppression of aldosterone during dynamic test (including the saline infusion test). CT aims to ensure the absence of adrenal carcinoma and to study the morphology of the adrenals. The unilateral or bilateral type of aldosterone secretion is based on the realization of an adrenal venous sampling. When the hypersecretion is unilateral, the treatment consists of adrenalectomy leading to cure of hypertension in 42% of cases, improvement in 40% of cases. For patient with bilateral disease or who don't want to undergo surgery, treatment is based on spironolactone usually at doses of 25 or 50 mg in combination with other antihypertensives drugs such as diuretics or calcium channel blockers.

  17. Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure

    PubMed Central

    Cao, Guoqing; Beyer, Thomas P.; Zhang, Youyan; Schmidt, Robert J.; Chen, Yan Q.; Cockerham, Sandra L.; Zimmerman, Karen M.; Karathanasis, Sotirios K.; Cannady, Ellen A.; Fields, Todd; Mantlo, Nathan B.

    2011-01-01

    Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC50) and CETP activity in human plasma (36 nM IC50) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED50 of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development. PMID:21957197

  18. Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria.

    PubMed

    Morii, Hiroyuki; Okauchi, Tatsuo; Nomiya, Hiroki; Ogawa, Midori; Fukuda, Kazumasa; Taniguchi, Hatsumi

    2013-03-01

    We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.

  19. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site.

  20. Nitric oxide synthase inhibitors do not alter functional hyperemia in canine skeletal muscle.

    PubMed

    Barclay, J K; Woodley, N E

    1994-09-01

    To test the hypothesis that endothelium-derived products contribute to functional hyperemia in skeletal muscle, we infused nitric oxide synthase inhibitors, either 200 microM N omega-nitro-L-arginine (NNA) (N = 4) or 1 mM N gamma-monomethyl-L-arginine (NMMA) (N = 4), before and during 6 min of 4 Hz stimulation of canine gastrocnemius in situ. We infused saline (N = 4) as a control. NNA significantly decreased steady-level resting flow by 3.8 +/- 0.4 mL.kg-1.s-1. The increase in flow from rest to 5 min of stimulation was not changed by the nitric oxide synthase inhibitors. We also stimulated muscles for 60 min either with saline infusion (N = 4) or with the infusion of saline during the first 15 min and NNA for the remaining 45 min (n = 4). There was no difference in the flow during contractions. To clarify the effect of these inhibitors on canine vessels, we challenged rings of canine femoral artery with and without endothelium with acetylcholine and bradykinin (both 1 microM) before and after the addition of NNA and NMMA (both 10 microM). The nitric oxide synthase inhibitors decreased the relaxation accompanying acetylcholine. Both inhibitors caused only endothelium-intact rings to contract. Thus, the presence of a nitric oxide synthase inhibitor identified an endothelium-dependent contribution to the regulation of blood flow to skeletal muscle at rest but had no effect on functional hyperemia.

  1. Reviewing Ligand-Based Rational Drug Design: The Search for an ATP Synthase Inhibitor

    PubMed Central

    Lee, Chia-Hsien; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2011-01-01

    Following major advances in the field of medicinal chemistry, novel drugs can now be designed systematically, instead of relying on old trial and error approaches. Current drug design strategies can be classified as being either ligand- or structure-based depending on the design process. In this paper, by describing the search for an ATP synthase inhibitor, we review two frequently used approaches in ligand-based drug design: The pharmacophore model and the quantitative structure-activity relationship (QSAR) method. Moreover, since ATP synthase ligands are potentially useful drugs in cancer therapy, pharmacophore models were constructed to pave the way for novel inhibitor designs. PMID:21954360

  2. Role of angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and aldosterone antagonists in the prevention of atrial and ventricular arrhythmias.

    PubMed

    Makkar, Kathy M; Sanoski, Cynthia A; Spinler, Sarah A

    2009-01-01

    Atrial arrhythmias, ventricular arrhythmias, and sudden cardiac death (SCD) are significant health problems and an economic burden to society. The renin-angiotensin-aldosterone system (RAAS) may play a key role in the occurrence of structural and electrical remodeling, potentially explaining the development of atrial and ventricular arrhythmias. Angiotensin II has been shown to regulate cardiac cell proliferation and to modulate cardiac myocyte ion channels. Results of post hoc analyses from prospective clinical trials appear to show that angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) are most effective in the prevention of new-onset atrial fibrillation in patients with heart failure. It is difficult to determine if these agents are useful in the prevention of new-onset atrial fibrillation after myocardial infarction, and available evidence suggests that the benefit of ACE inhibitors and ARBs for prevention of new-onset atrial fibrillation in patients with hypertension appears limited to those with left ventricular hypertrophy. Patients with structural changes in cardiac muscle, such as those with heart failure and left ventricular hypertrophy, appear to benefit the most from RAAS blockade, possibly due to the theory of reversal of cardiac remodeling. There is no evidence, to our knowledge, that either ACE inhibitors or ARBs facilitate direct electrical current cardioversion in patients with atrial fibrillation; however, it appears that RAAS blockade may be useful in the prevention of recurrent atrial fibrillation after direct electrical current cardioversion. Whether ACE inhibitors may prevent life-threatening ventricular arrhythmias or SCD is unclear. Aldosterone antagonists appear to be useful for the prevention of SCD in patients with left ventricular systolic dysfunction. Results from ongoing clinical trials are anticipated to provide further insight on the potential roles of RAAS inhibitors for the prevention of

  3. Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening.

    PubMed

    Magellan, Hervé; Boccara, Martine; Drujon, Thierry; Soulié, Marie-Christine; Guillou, Catherine; Dubois, Joëlle; Becker, Hubert F

    2013-09-01

    Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC50 values of 1.8 and 10μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties.

  4. The leaf extract of Siberian Crabapple (Malus baccata (Linn.) Borkh) contains potential fatty acid synthase inhibitors.

    PubMed

    Wei, Xiang; Zhao, Ran; Sun, Ying-Hui; Cong, Jian-Ping; Meng, Fan-Guo; Zhou, Hai-Meng

    2009-02-01

    The present work focused on the kinetics of the inhibitory effects of the leaf extract of Siberian Crabapple, named Shan jingzi in China, on chicken liver fatty acid synthase. The results showed that this extract had much stronger inhibitory ability on fatty acid synthase than that from green teas described in many previous reports. The inhibitory ability of this extract is closely related to the extracting solvent, and the time of extraction was also an important influencing factor. The inhibitory types of this extract on diffeerent substrates of chicken liver fatty acid synthase, acetyl-CoA, malonyl-CoA and NADPH, were found to be noncompetitive, uncompetitive and mixed, respectively. The studies here shed a new light on the exploration for inhibitors of fatty acid synthase.

  5. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase.

    PubMed

    Farha, Maya A; Czarny, Tomasz L; Myers, Cullen L; Worrall, Liam J; French, Shawn; Conrady, Deborah G; Wang, Yang; Oldfield, Eric; Strynadka, Natalie C J; Brown, Eric D

    2015-09-01

    Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.

  6. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

    PubMed Central

    Farha, Maya A.; Czarny, Tomasz L.; Myers, Cullen L.; Worrall, Liam J.; French, Shawn; Conrady, Deborah G.; Wang, Yang; Oldfield, Eric; Strynadka, Natalie C. J.; Brown, Eric D.

    2015-01-01

    Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery. PMID:26283394

  7. Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

    PubMed

    Concha, Nestor; Huang, Jianzhong; Bai, Xiaopeng; Benowitz, Andrew; Brady, Pat; Grady, LaShadric C; Kryn, Luz Helena; Holmes, David; Ingraham, Karen; Jin, Qi; Pothier Kaushansky, Laura; McCloskey, Lynn; Messer, Jeffrey A; O'Keefe, Heather; Patel, Amish; Satz, Alexander L; Sinnamon, Robert H; Schneck, Jessica; Skinner, Steve R; Summerfield, Jennifer; Taylor, Amy; Taylor, J David; Evindar, Ghotas; Stavenger, Robert A

    2016-08-11

    Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

  8. Synthesis of 5-ethynyl-2'-deoxyuridine-5'-boranomono phosphate as a potential thymidylate synthase inhibitor.

    PubMed

    Khan, Shoeb I; Dobrikov, Mikhail I; Shaw, Barbara Ramsay

    2005-01-01

    The 5-ethynyl-2'-deoxyuridine nucleoside and the 5'-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2'-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.

  9. Structural and biological studies on bacterial nitric oxide synthase inhibitors

    PubMed Central

    Holden, Jeffrey K.; Li, Huiying; Jing, Qing; Kang, Soosung; Richo, Jerry; Silverman, Richard B.; Poulos, Thomas L.

    2013-01-01

    Nitric oxide (NO) produced by bacterial NOS functions as a cytoprotective agent against oxidative stress in Staphylococcus aureus, Bacillus anthracis, and Bacillus subtilis. The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds impede the growth of B. subtilis under oxidative stress, and crystal structures show that each compound exhibits a unique binding mode. Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria. PMID:24145412

  10. Aldosterone blood test

    MedlinePlus

    ... hypotension) Aldosterone is a hormone released by the adrenal glands . It helps the body regulate blood pressure. Aldosterone ... may be due to Bartter syndrome (extremely rare) Adrenal glands release too much aldosterone hormone ( primary hyperaldosteronism - usually ...

  11. Synthesis of Bi-substrate State Mimics of Dihydropteroate Synthase as Potential Inhibitors and Molecular Probes

    PubMed Central

    Qi, Jianjun; Virga, Kristopher G.; Das, Sourav; Zhao, Ying; Yun, Mi-Kyung; White, Stephen W.; Lee, Richard E.

    2010-01-01

    The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics. PMID:21216602

  12. Natural fatty acid synthase inhibitors as potent therapeutic agents for cancers: A review.

    PubMed

    Zhang, Jia-Sui; Lei, Jie-Ping; Wei, Guo-Qing; Chen, Hui; Ma, Chao-Ying; Jiang, He-Zhong

    2016-09-01

    Context Fatty acid synthase (FAS) is the only mammalian enzyme to catalyse the synthesis of fatty acid. The expression level of FAS is related to cancer progression, aggressiveness and metastasis. In recent years, research on natural FAS inhibitors with significant bioactivities and low side effects has increasingly become a new trend. Herein, we present recent research progress on natural fatty acid synthase inhibitors as potent therapeutic agents. Objective This paper is a mini overview of the typical natural FAS inhibitors and their possible mechanism of action in the past 10 years (2004-2014). Method The information was collected and compiled through major databases including Web of Science, PubMed, and CNKI. Results Many natural products induce cancer cells apoptosis by inhibiting FAS expression, with fewer side effects than synthetic inhibitors. Conclusion Natural FAS inhibitors are widely distributed in plants (especially in herbs and foods). Some natural products (mainly phenolics) possessing potent biological activities and stable structures are available as lead compounds to synthesise promising FAS inhibitors.

  13. Differential in radiosensitizing potency of enantiomers of the fatty acid synthase inhibitor C75.

    PubMed

    Rae, Colin; Babich, John W; Mairs, Robert J

    2017-01-01

    The elevated activity of fatty acid synthase has been reported in a number of cancer types. Inhibition of this enzyme has been demonstrated to induce cancer cell death and reduce tumor growth. In addition, the fatty acid synthase inhibitor drug C75 has been reported to synergistically enhance the cancer-killing ability of ionizing radiation. However, clinical use of C75 has been limited due to its producing weight loss, believed to be caused by alterations in the activity of carnitine palmitoyltransferase-1. C75 is administered in the form of a racemic mixture of (-) and (+) enantiomers that may differ in their regulation of fatty acid synthase and carnitine palmitoyltransferase-1. Therefore, we assessed the relative cancer-killing potency of different enantiomeric forms of C75 in prostate cancer cells. These results suggest that (-)-C75 is the more cytotoxic enantiomer and has greater radiosensitizing capacity than (+)-C75. These observations will stimulate the development of fatty acid synthase inhibitors that are selective for cancer cells and enhance the tumor-killing activity of ionizing radiation, while minimizing weight loss in cancer patients.

  14. Discovery of Novel Antifungal (1,3)-β-d-Glucan Synthase Inhibitors

    PubMed Central

    Onishi, J.; Meinz, M.; Thompson, J.; Curotto, J.; Dreikorn, S.; Rosenbach, M.; Douglas, C.; Abruzzo, G.; Flattery, A.; Kong, L.; Cabello, A.; Vicente, F.; Pelaez, F.; Diez, M. T.; Martin, I.; Bills, G.; Giacobbe, R.; Dombrowski, A.; Schwartz, R.; Morris, S.; Harris, G.; Tsipouras, A.; Wilson, K.; Kurtz, M. B.

    2000-01-01

    The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-β-d-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin Bo, is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-β-d-glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-β-d-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1, the gene which encodes the large subunit of glucan synthase. PMID:10639364

  15. Differential in radiosensitizing potency of enantiomers of the fatty acid synthase inhibitor C75

    PubMed Central

    Babich, John W.; Mairs, Robert J.

    2016-01-01

    Abstract The elevated activity of fatty acid synthase has been reported in a number of cancer types. Inhibition of this enzyme has been demonstrated to induce cancer cell death and reduce tumor growth. In addition, the fatty acid synthase inhibitor drug C75 has been reported to synergistically enhance the cancer‐killing ability of ionizing radiation. However, clinical use of C75 has been limited due to its producing weight loss, believed to be caused by alterations in the activity of carnitine palmitoyltransferase‐1. C75 is administered in the form of a racemic mixture of (−) and (+) enantiomers that may differ in their regulation of fatty acid synthase and carnitine palmitoyltransferase‐1. Therefore, we assessed the relative cancer‐killing potency of different enantiomeric forms of C75 in prostate cancer cells. These results suggest that (−)‐C75 is the more cytotoxic enantiomer and has greater radiosensitizing capacity than (+)‐C75. These observations will stimulate the development of fatty acid synthase inhibitors that are selective for cancer cells and enhance the tumor‐killing activity of ionizing radiation, while minimizing weight loss in cancer patients. PMID:27901292

  16. Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.

    PubMed

    Richie, Daryl L; Thompson, Katherine V; Studer, Christian; Prindle, Vivian C; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N Rao; Tallarico, John A; Ryder, Neil S; Hoepfner, Dominic

    2013-05-01

    High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.

  17. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    SciTech Connect

    Morgunova, Ekaterina; Illarionov, Boris; Saller, Sabine; Popov, Aleksander; Sambaiah, Thota; Bacher, Adelbert; Cushman, Mark; Fischer, Markus; Ladenstein, Rudolf

    2010-09-01

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

  18. In search of potent and selective inhibitors of neuronal nitric oxide synthase with more simple structures

    PubMed Central

    Jing, Qing; Li, Huiying; Fang, Jianguo; Roman, Linda J.; Martásek, Pavel; Poulos, Thomas L.; Silverman, Richard B.

    2013-01-01

    In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper functioning of the cardiovascular system. While we have been successful in developing potent and isoform-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhibitor complexes that integrate the advantages of easy synthesis and good biological properties compared to the lead compounds. These results provide the basis for additional structure–activity relationship (SAR) studies to guide further improvement of isozyme selective inhibitors. PMID:23867386

  19. Therapeutic perspectives in hypertension: novel means for renin-angiotensin-aldosterone system modulation and emerging device-based approaches.

    PubMed

    Unger, Thomas; Paulis, Ludovit; Sica, Domenic A

    2011-11-01

    The conventional antihypertensive therapies including renin-angiotensin-aldosterone system antagonists (converting enzyme inhibitors, receptor blockers, renin inhibitors, and mineralocorticoid receptor blockers), diuretics, β-blockers, and calcium channel blockers are variably successful in achieving the challenging target blood pressure values in hypertensive patients. Difficult to treat hypertension is still a commonly observed problem world-wide. A number of drugs are considered to be used as novel therapies for hypertension. Renalase supplementation, vasopeptidase inhibitors, endothelin antagonists, and especially aldosterone antagonists (aldosterone synthase inhibitors and novel selective mineralocorticoid receptor blockers) are considered an option in resistant hypertension. In addition, the aldosterone antagonists as well as (pro)renin receptor blockers or AT(2) receptor agonists might attenuate end-organ damage. This array of medications has now been complemented by a number of new approaches of non-pharmacological strategies including vaccination, genomic interference, controlled breathing, baroreflex activation, and probably most successfully renal denervation techniques. However, the progress on innovative therapies seems to be slow and the problem of resistant hypertension and proper blood pressure control appears to be still persisting. Therefore the regimens of currently available drugs are being fine-tuned, resulting in the establishment of several novel fixed-dose combinations including triple combinations with the aim to facilitate proper blood pressure control. It remains an exciting question which approach will confer the best blood pressure control and risk reduction in this tricky disease.

  20. Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs

    SciTech Connect

    McDaniel, M.D.; Huntsman, W.T.; Miett, T.O.; Cronenwett, J.L.

    1987-08-01

    This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study.

  1. Arginine-Based Inhibitors of Nitric Oxide Synthase: Therapeutic Potential and Challenges

    PubMed Central

    Víteček, Jan; Lojek, Antonín; Valacchi, Giuseppe; Kubala, Lukáš

    2012-01-01

    In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted. PMID:22988346

  2. Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia

    PubMed Central

    Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

    2016-01-01

    The challenge for Glycogen Synthase Kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML) may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far, but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy. PMID:26496242

  3. Geranyl and Neryl Triazole Bisphosphonates as Inhibitors of Geranylgeranyl Diphosphate Synthase

    PubMed Central

    Zhou, Xiang; Ferree, Sarah D.; Wills, Veronica S.; Born, Ella J.; Tong, Huaxiang; Holstein, Sarah A.

    2014-01-01

    When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase. PMID:24726306

  4. Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

    PubMed

    Wang, Yang; Desai, Janish; Zhang, Yonghui; Malwal, Satish R; Shin, Christopher J; Feng, Xinxin; Sun, Hong; Liu, Guizhi; Guo, Rey-Ting; Oldfield, Eric

    2016-10-19

    We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED50 ∼0.15 μg mL(-1) ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition.

  5. Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.

    PubMed

    Skovpen, Yulia V; Conly, Cuylar J T; Sanders, David A R; Palmer, David R J

    2016-02-17

    Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development.

  6. ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

    PubMed Central

    Hong, Sangjin; Pedersen, Peter L.

    2008-01-01

    Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

  7. Structure-based inhibitors exhibit differential activities against Helicobacter pylori and Escherichia coli undecaprenyl pyrophosphate synthases.

    PubMed

    Kuo, Chih-Jung; Guo, Rey-Ting; Lu, I-Lin; Liu, Hun-Ge; Wu, Su-Ying; Ko, Tzu-Ping; Wang, Andrew H-J; Liang, Po-Huang

    2008-01-01

    Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.

  8. Potent, Highly Selective, and Orally Bioavailable Gem-Difluorinated Monocationic Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    Xue, Fengtian; Li, Huiying; Delker, Silvia L.; Fang, Jianguo; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    In our efforts to discover neuronal isoform selective nitric oxide synthase (NOS) inhibitors we have developed a series of compounds containing a pyrrolidine ring with two stereogenic centers. The enantiomerically pure compounds, (S,S) vs. (R,R), exhibited two different binding orientations, with (R,R) inhibitors showing much better potency and selectivity. To improve the bioavailability of these inhibitors we have introduced a CF2 moiety geminal to an amino group in the long tail of one of these inhibitors, which reduced its basicity, resulting in compounds with monocationic character under physiological pH conditions. Biological evaluations have led to a nNOS inhibitor with a Ki of 36 nM and high selectivity for nNOS over eNOS (3800-fold) and iNOS (1400-fold). MM-PBSA calculations indicated that the low pKa NH is, at least, partially protonated when bound to the active site. A comparison of rat oral bioavailability of the difluorinated compound to the parent molecule shows 22% for the difluorinated compound versus essentially no oral bioavailability for the parent compound. This indicates that the goal of this research to make compounds with only one protonated nitrogen atom at physiological pH to allow for membrane permeability, but which can become protonated when bound to NOS, has been accomplished. PMID:20843082

  9. Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis

    PubMed Central

    Begley, Darren W.; Edwards, Thomas E.; Raymond, Amy C.; Smith, Eric R.; Hartley, Robert C.; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D.; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J.

    2011-01-01

    Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research. PMID:21904052

  10. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  11. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    PubMed Central

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  12. [Hematopoietic prostaglandin D synthase inhibitors for the treatment of duchenne muscular dystrophy].

    PubMed

    Kamauchi, Shinya; Urade, Yoshihiro

    2011-11-01

    Duchenne muscular dystrophy (DMD) is a severe X-linked muscle disease, characterized by progressive skeletal muscle atrophy and weakness. DMD is caused by mutations in the dystrophin gene, which encodes for the cytoskeletal protein dystrophin. DMD is one of the most common types of muscular dystrophies, affecting approximately 1 in 3,500 boys. There is no complete cure for this disease. Clinical trials for gene transfer therapy as a treatment for DMD have been performed but mainly in animal models. Hematopoietic prostaglandin (PG) D synthase (H-PGDS) was found to be induced in grouped necrotic muscle fibers of DMD patients and animal models, mdx mice, and DMD dogs. We found an orally active H-PGDS inhibitor (HQL-79) and determined the 3D structure of the inhibitor-human H-PGDS complex by X-ray crystallography. Oral administration of HQL-79 markedly suppressed prostaglandin D2 (PGD2) production, reduced necrotic muscle volume, and improved muscle strength in mdx dystrophic mice. Based on the high-resolution 3D structures of the inhibitor-H-PGDS complex, we designed alternative H-PGDS inhibitors, which were 100- to 3000-times more potent than HQL-79, as assessed by in vitro and in vivo analyses. We used these novel inhibitors for the treatment of DMD dogs and confirmed that oral administration of these inhibitors prevented skeletal muscle atrophy and weakness by decreasing PGD2 production. These results indicate that PGD2, synthesized by H-PGDS, is involved in the expansion of muscle necrosis in DMD. Thus, inhibition of H-PGDS by using inhibitors is a novel therapy for DMD.

  13. Identification and Characterization of Novel Microsomal Prostaglandin E Synthase-1 Inhibitors for Analgesia.

    PubMed

    Chandrasekhar, Srinivasan; Harvey, Anita K; Yu, Xiao-Peng; Chambers, Mark G; Oskins, Jennifer L; Lin, Chaohua; Seng, Thomas W; Thibodeaux, Stefan J; Norman, Bryan H; Hughes, Norman E; Schiffler, Matthew A; Fisher, Matthew J

    2016-03-01

    Prostaglandin (PG) E2 plays a critical role in eliciting inflammation. Nonsteroidal anti-inflammatory drugs and selective inhibitors of cyclooxygenase, which block PGE2 production, have been used as key agents in treating inflammation and pain associated with arthritis and other conditions. However, these agents have significant side effects such as gastrointestinal bleeding and myocardial infarction, since they also block the production of prostanoids that are critical for other normal physiologic functions. Microsomal prostaglandin E2 synthase-1 is a membrane-bound terminal enzyme in the prostanoid pathway, which acts downstream of cyclooxygenase 2 and is responsible for PGE2 production during inflammation. Thus, inhibition of this enzyme would be expected to block PGE2 production without inhibiting other prostanoids and would provide analgesic efficacy without the side effects. In this report, we describe novel microsomal prostaglandin E2 synthase-1 inhibitors that are potent in blocking PGE2 production and are efficacious in a guinea pig monoiodoacetate model of arthralgia. These molecules may be useful in treating the signs and symptoms associated with arthritis.

  14. Monte Carlo method based QSAR modeling of maleimide derivatives as glycogen synthase kinase-3β inhibitors.

    PubMed

    Živković, Jelena V; Trutić, Nataša V; Veselinović, Jovana B; Nikolić, Goran M; Veselinović, Aleksandar M

    2015-09-01

    The Monte Carlo method was used for QSAR modeling of maleimide derivatives as glycogen synthase kinase-3β inhibitors. The first QSAR model was developed for a series of 74 3-anilino-4-arylmaleimide derivatives. The second QSAR model was developed for a series of 177 maleimide derivatives. QSAR models were calculated with the representation of the molecular structure by the simplified molecular input-line entry system. Two splits have been examined: one split into the training and test set for the first QSAR model, and one split into the training, test and validation set for the second. The statistical quality of the developed model is very good. The calculated model for 3-anilino-4-arylmaleimide derivatives had following statistical parameters: r(2)=0.8617 for the training set; r(2)=0.8659, and r(m)(2)=0.7361 for the test set. The calculated model for maleimide derivatives had following statistical parameters: r(2)=0.9435, for the training, r(2)=0.9262 and r(m)(2)=0.8199 for the test and r(2)=0.8418, r(av)(m)(2)=0.7469 and ∆r(m)(2)=0.1476 for the validation set. Structural indicators considered as molecular fragments responsible for the increase and decrease in the inhibition activity have been defined. The computer-aided design of new potential glycogen synthase kinase-3β inhibitors has been presented by using defined structural alerts.

  15. A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases.

    PubMed

    Christensen, Quin H; Grove, Tyler L; Booker, Squire J; Greenberg, E Peter

    2013-08-20

    Many Proteobacteria use N-acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S-adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a Ki of 0.7 µM and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, anti-quorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach.

  16. Conformationally-Restricted Dipeptide Amides as Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors

    PubMed Central

    Ji, Haitao; Gómez-Vidal, José A.; Martásek, Pavel; Roman, Linda J.; Silverman, Richard B.

    2008-01-01

    Four new conformationally-restricted analogues of the potent and selective neuronal nitric oxide synthase inhibitor, L-nitroargininyl-L-2,4-diaminobutyramide (1), have been synthesized. Nα-Methyl and Nα-benzyl derivatives (3 and 4, respectively) of 4N-(L-ArgNO2)-trans-4-amino-L-prolineamide (2) are also selective inhibitors, but the potency and selectivity of 3 are weak. Analogue 4 has only one-third the potency and one-half to one-third the selectivity of 2 against iNOS and eNOS, respectively. 3-N-(L-ArgNO2)-trans-3-amino-L-prolineamide (6) is as potent an inhibitor of nNOS as is 2; selectivity for nNOS over iNOS is half of that for 2 but the selectivity for nNOS over eNOS is almost double that for 2. The corresponding cis-isomer (5) is a weak inhibitor of nNOS. These results are supported by computer modeling. PMID:17034131

  17. A Small-Molecule Screening Platform for the Discovery of Inhibitors of Undecaprenyl Diphosphate Synthase.

    PubMed

    Czarny, Tomasz L; Brown, Eric D

    2016-07-08

    The bacterial cell wall has long been a celebrated target for antibacterial drug discovery due to its critical nature in bacteria and absence in mammalian systems. At the heart of the cell wall biosynthetic pathway lies undecaprenyl phosphate (Und-P), the lipid-linked carrier upon which the bacterial cell wall is built. This study exploits recent insights into the link between late-stage wall teichoic acid inhibition and Und-P production, in Gram-positive organisms, to develop a cell-based small-molecule screening platform that enriches for inhibitors of undecaprenyl diphosphate synthase (UppS). Screening a chemical collection of 142,000 small molecules resulted in the identification of 6 new inhibitors of UppS. To date, inhibitors of UppS have generally shown off-target effects on membrane potential due to their physical-chemical characteristics. We demonstrate that MAC-0547630, one of the six inhibitors identified, exhibits selective, nanomolar inhibition against UppS without off-target effects on membrane potential. Such characteristics make it a unique chemical probe for exploring the inhibition of UppS in bacterial cell systems.

  18. Selective Monocationic Inhibitors of Neuronal Nitric Oxide Synthase. Binding Mode Insights from Molecular Dynamics Simulations

    PubMed Central

    Huang, He; Ji, Haitao; Li, Huiying; Jing, Qing; Labby, Kristin Jansen; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2012-01-01

    The reduction of pathophysiologic levels of nitric oxide through inhibition of neuronal nitric oxide synthase (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. We have developed a series of pyrrolidine-based nNOS inhibitors that exhibit excellent potencies and isoform selectivities (J. Am. Chem. Soc. 2010, 132, 5437). However, there are still important challenges, such as how to decrease the multiple positive charges derived from basic amino groups, which contribute to poor bioavailability, without losing potency and/or selectivity. Here we present an interdisciplinary study combining molecular docking, crystallography, molecular dynamics simulations, synthesis, and enzymology to explore potential pharmacophoric features of nNOS inhibitors and to design potent and selective monocationic nNOS inhibitors. The simulation results indicate that different hydrogen bond patterns, electrostatic interactions, hydrophobic interactions, and a water molecule bridge are key factors for stabilizing ligands and controlling ligand orientation. We find that a heteroatom in the aromatic head or linker chain of the ligand provides additional stability and blocks the substrate binding pocket. Finally, the computational insights are experimentally validated with double-headed pyridine analogs. The compounds reported here are among the most potent and selective monocationic pyrrolidine-based nNOS inhibitors reported to date, and 10 shows improved membrane permeability. PMID:22731813

  19. Recent Advances in the Development of Undecaprenyl Pyrophosphate Synthase Inhibitors as Potential Antibacterials.

    PubMed

    Jukic, Marko; Rozman, Kaja; Gobec, Stanislav

    2016-01-01

    Expanding antibiotic use in clinical practice and emergence of bacterial resistance are fueling research efforts for the development of novel antibacterials. Underexploited or completely novel mechanistic approaches and biological targets are of especial interest. Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in the biosynthesis of the bacterial cell wall. Although UppS is a validated target, no selective inhibitors occur in materia medica. Nevertheless, several native substrate analogues have been reported and used in enzyme kinetics studies or as pharmacological probes. The majority of small-molecule UppS inhibitors belong to the well-known class of bisphosphonates that are primarily used for treatment of bone resorption disorders. The most potent compound of this class has an IC50 of 0.59 µM. Inherently, the selectivity and suitability of such compounds for antimicrobial drug design can be questioned. Therefore, highthroughput and virtual screenings for non-bisphosphonate inhibitors were performed, and nanomolar inhibitors of UppS were identified, some with antimicrobial activities towards clinically relevant strains. The reported scaffolds belong to tetramic and tetronic acids with IC50 in the 100-nM range, and to dihydropyridines with IC50 down to 40 nM, all with antibacterial activity. Aryl-diketo acids are also potent inhibitors with MRSA antimicrobial activity, with the allosteric inhibitor methylisoxazole-4-carboxamide (IC50, 50 nM) active on several pathogenic Streptococcus pneumoniae strains. Clomiphene is a well-known oestrogen receptor modulator, and it has been reported to inhibit UppS. Although conclusions on the structure activity relationships cannot be drawn from all these data, these compound series represent an important contribution to the field of antibiotics.

  20. Binding and Inhibition of Spermidine Synthase from Plasmodium falciparum and Implications for In Vitro Inhibitor Testing

    PubMed Central

    Sprenger, Janina; Carey, Jannette; Svensson, Bo; Wengel, Verena

    2016-01-01

    The aminopropyltransferase spermidine synthase (SpdS) is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS) transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet) site must be occupied before the aminopropyl acceptor (putrescine) site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD−IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism. PMID:27661085

  1. Inhibition of cholesterol synthesis by squalene synthase inhibitors does not induce myotoxicity in vitro.

    PubMed

    Flint, O P; Masters, B A; Gregg, R E; Durham, S K

    1997-07-01

    The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previous in vitro study, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-induced in vitro myotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis ([3H]leucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis ([14C]acetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-induced in vitro myotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that

  2. Role of ACTH and Other Hormones in the Regulation of Aldosterone Production in Primary Aldosteronism

    PubMed Central

    El Ghorayeb, Nada; Bourdeau, Isabelle; Lacroix, André

    2016-01-01

    aldosteronism, a chimeric CYP11B2 becomes regulated by ACTH activating its chimeric CYP11B1 promoter of aldosterone synthase in bilateral adrenal fasciculate-like hyperplasia. This review will focus on the role of ACTH on excess aldosterone secretion in PA with particular focus on the aberrant expression of MC2R in comparison with other aberrant ligands and their GPCRs in this frequent pathology. PMID:27445975

  3. Quinazolinones, Quinazolinthiones, and Quinazolinimines as Nitric Oxide Synthase Inhibitors: Synthetic Study and Biological Evaluation.

    PubMed

    Camacho, M Encarnación; Chayah, Mariem; García, M Esther; Fernández-Sáez, Nerea; Arias, Fabio; Gallo, Miguel A; Carrión, M Dora

    2016-08-01

    The synthesis of different compounds with a quinazolinone, quinazolinthione, or quinazolinimine skeleton and their in vitro biological evaluation as inhibitors of inducible and neuronal nitric oxide synthase (iNOS and nNOS) isoforms are described. These derivatives were obtained from substituted 2-aminobenzylamines, using diverse cyclization procedures. Furthermore, the diamines were synthesized by two routes: A conventional pathway and an efficient one-pot synthesis in a continuous-flow hydrogenator. The structures of these heterocycles were confirmed by (1) H and (13) C nuclear magnetic resonance and high-resolution mass spectroscopy data. The structure-activity relationships of the target molecules are discussed in terms of the effects of both the R radical and the X heteroatom in the 2-position. In general, the assayed compounds behave as better iNOS than nNOS inhibitors, with the quinazolinone 11e being the most active inhibitor of all tested compounds and the most iNOS/nNOS selective one.

  4. Immediate Force Loss after Eccentric Contractions is Increased with L-NAME Administration, a Nitric Oxide Synthase Inhibitor

    DTIC Science & Technology

    2013-02-01

    IMMEDIATE FORCE LOSS AFTER ECCENTRIC CONTRACTIONS IS INCREASED WITH L-NAME ADMINISTRATION, A NITRIC OXIDE SYNTHASE INHIBITOR BENJAMIN T. CORONA, PhD...performance of eccentric contractions. Methods—Wild-type mouse extensor digitorum longus (EDL) muscles underwent in vitro functional testing in the...presence or absence of a non-specific NOS inhibitor (L-NAME, 10 mM) before and after performance of 10 eccentric contractions. Results—After eccentric

  5. The effect of triton concentration on the activity of undecaprenyl pyrophosphate synthase inhibitors.

    PubMed

    Li, Hu; Huang, Jianzhong; Jiang, Xinhe; Seefeld, Mark; McQueney, Michael; Macarron, Ricardo

    2003-12-01

    Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of 8 molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C55-undecaprenyl pyrophosphate, which is required for bacterial cell wall synthesis. UPPS is found in both gram-positive and gram-negative bacteria, and based on the differences between bacterial variants of UPPS and their human counterpart, dolicopyrophosphate synthase, it was identified as an attractive antibacterial target. An assay, which monitors the release of Pi by coupling the UPPS catalyzed reaction with inorganic pyrophosphatase, was employed to conduct an HTS campaign using an inhouse collection of compounds. A direct assay measuring the incorporation of 14C-IPP (isopentenyl pyrophosphate) was used as a secondary assay to evaluate the high-throughput screening (HTS) hits. From the HTS campaign, a few classes of UPPS inhibitors were identified. During the process of hit evaluation by the direct assay, the authors observed that Triton, an essential factor for the enzyme activity and accurate formation of the natural product, dramatically altered the inhibitory activity of a particular class of compounds. Above its critical micellar concentration (CMC), Triton abolished the inhibitory activity of these compounds. Further research will be required to establish the biophysical phenomenon that causes this effect. Meanwhile, it can be speculated that Triton (and other detergents) above CMC may hinder the identification in screening compounds of certain classes of hits.

  6. Presence of fatty acid synthase inhibitors in the rhizome of Alpinia officinarum hance.

    PubMed

    Li, Bing-Hui; Tian, Wei-Xi

    2003-08-01

    The galangal (the rhizome of Alpinia officinarum, Hance) is popular in Asia as a traditional herbal medicine. The present study reports that the galangal extract (GE) can potently inhibit fatty-acid synthase (FAS, E.C.2.3.1.85). The inhibition consists of both reversible inhibition with an IC50 value of 1.73 microg dried GE/ml, and biphasic slow-binding inactivation. Subsequently the reversible inhibition and slow-binding inactivation to FAS were further studied. The inhibition of FAS by galangin, quercetin and kaempferol, which are the main flavonoids existing in the galangal, showed that quercetin and kaempferol had potent reversible inhibitory activity, but all three flavonoids had no obvious slow-binding inactivation. Analysis of the kinetic results led to the conclusion that the inhibitory mechanism of GE is totally different from that of some other previously reported inhibitors of FAS, such as cerulenin, EGCG (epigallocatechin gallate) and C75.

  7. Imidazopyridine-Based Fatty Acid Synthase Inhibitors That Show Anti-HCV Activity and in Vivo Target Modulation

    PubMed Central

    2012-01-01

    Potent imidazopyridine-based inhibitors of fatty acid synthase (FASN) are described. The compounds are shown to have antiviral (HCV replicon) activities that track with their biochemical activities. The most potent analogue (compound 19) also inhibits rat FASN and inhibits de novo palmitate synthesis in vitro (cell-based) as well as in vivo. PMID:24900571

  8. Synthesis of 2-arylindole derivatives and evaluation as nitric oxide synthase and NFκB inhibitors.

    PubMed

    Yu, Xufen; Park, Eun-Jung; Kondratyuk, Tamara P; Pezzuto, John M; Sun, Dianqing

    2012-11-28

    Development of small molecule drug-like inhibitors blocking both nitric oxide synthase and NFκB could offer a synergistic therapeutic approach in the prevention and treatment of inflammation and cancer. During the course of evaluating the biological potential of a commercial compound library, 2-phenylindole (1) displayed inhibitory activity against nitrite production and NFκB with IC(50) values of 38.1 ± 1.8 and 25.4 ± 2.1 μM, respectively. Based on this lead, synthesis and systematic optimization have been undertaken in an effort to find novel and more potent nitric oxide synthase and NFκB inhibitors with antiinflammatory and cancer preventive potential. First, chemical derivatizations of 1 and 2-phenylindole-3-carboxaldehyde (4) were performed to generate a panel of N-alkylated indoles and 3-oxime derivatives 2–7. Second, a series of diversified 2-arylindole derivatives (10) were synthesized from an array of substituted 2-iodoanilines (8) and terminal alkynes (9) by applying a one-pot palladium catalyzed Sonogashira-type alkynylation and base-assisted cycloaddition. Subsequent biological evaluations revealed 3-carboxaldehyde oxime and cyano substituted 2-phenylindoles 5 and 7 exhibited the strongest nitrite inhibitory activities (IC(50) = 4.4 ± 0.5 and 4.8 ± 0.4 μM, respectively); as well as NFκB inhibition (IC(50) = 6.9 ± 0.8 and 8.5 ± 2.0 μM, respectively). In addition, the 6′-MeO-naphthalen-2′-yl indole derivative 10at displayed excellent inhibitory activity against NFκB with an IC(50) value of 0.6 ± 0.2 μM.

  9. Aldosterone and the conquest of land.

    PubMed

    Colombo, L; Dalla Valle, L; Fiore, C; Armanini, D; Belvedere, P

    2006-04-01

    The sequence of the phylogenetic events that preceded the appearance of aldosterone in vertebrates is described, starting from the ancestral conversion of cytochrome P450s from oxygen detoxification to xenobiotic detoxification and synthesis of oxygenated endobiotics with useful functions in intercellular signalling, such as steroid hormones. At the end of the Silurian period [438-408 million yr ago, (Mya)], a complete set of cytochrome P450s for corticoid synthesis was presumably already available, except for mitochondrial cytochrome P450c18 or aldosterone synthase encoded by CYP11B2. This gene arose by duplication of the CYP11B gene in the sarcopterygian or lobe-finned fish/tetrapod line after its divergence from the actinopterygian or ray-finned fish line 420 Mya, but before the beginning of the colonization of land by tetrapods in the late Devonian period, around 370 Mya. The fact that aldosterone is already present in Dipnoi, which occupy an evolutionary transition between water- and air-breathing but are fully aquatic, suggests that the role of this steroid was to potentiate the corticoid response to hypoxia, rather than to prevent dehydration out of the water. In terrestrial amphibians, there is no differentiation between the secretion rates and gluco- and mineralocorticoid effects of aldosterone and corticosterone. In sauropsids, plasma aldosterone concentrations are much lower than in amphibians, but regulation of salt/water balance is dependent upon both aldosterone and corticosterone, though sometimes with opposed actions. In terrestrial mammals, aldosterone acquires a specific mineralocorticoid function, because its interaction with the mineralocorticoid receptor is protected by the coexpression of the enzyme 11beta-hydroxysteroid dehydrogenase type 2, which inactivates both cortisol and corticosterone. There is evidence that aldosterone can be also synthesized extra-adrenally in brain neurons and cardiac myocytes, which lack this protection and where

  10. Correction of Cystathionine β-synthase Deficiency in Mice by Treatment with Proteasome Inhibitors

    PubMed Central

    Gupta, Sapna; Wang, Liqun; Anderl, Janet; Slifker, Michael J.; Kirk, Christopher; Kruger, Warren D.

    2013-01-01

    Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism characterized by extremely elevated levels of plasma total homocysteine. The vast majority of CBS-deficient patients have missense mutations located in the CBS gene that result in the production of either misfolded or unstable protein. Here, we examine the effect of proteasome inhibitors on mutant CBS function using two different mouse models of CBS deficiency. These mice lack mouse CBS and express a missense mutant human CBS enzyme (either p.I278T or p.S466L) that has less than 5% of normal liver CBS activity, resulting in a 10–30 fold elevation in plasma homocysteine levels. We show that treatment of these mice with two different proteasome inhibitors can induce liver Hsp70, Hsp40, and Hsp27, increase levels of active CBS, and lower plasma homocysteine levels to within the normal range. However, response rates varied, with 100% (8/8) of the p.S466L animals showing correction, but only 38% (10/26) of the p.I278T animals. In total, our data shows that treatment with proteostasis modulators can restore significant enzymatic activity to mutant misfolded CBS enzymes and suggests that they may be useful in treating certain types of genetic diseases caused by missense mutations. PMID:23592311

  11. Discovery of Highly Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase by Fragment Hopping

    PubMed Central

    Ji, Haitao; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2009-01-01

    Selective inhibition of neuronal nitric oxide synthase (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. This study shows that not only greater inhibitory potency and isozyme selectivity, but more drug-like properties can be achieved by fragment hopping. Based on the structure of lead molecule 6, fragment hopping effectively extracted the minimal pharmacophoric elements in the active site of nNOS for ligand hydrophobic and steric interactions and generated appropriate lipophilic fragments for lead optimization. More potent and selective inhibitors with better drug-like properties were obtained within the design of 20 derivatives (compounds 7-26). Our structure-based inhibitor design for nNOS and SAR analysis reveal the robustness and efficiency of fragment hopping in lead discovery and structural optimization, which implicates a broad application of this approach to many other therapeutic targets for which known drug-like small-molecule modulators are still limited. PMID:19125620

  12. Effects of the inducible nitric oxide synthase inhibitor aminoguanidine in two different rat models of schizophrenia.

    PubMed

    Lafioniatis, Anastasios; Orfanidou, Martha A; Papadopoulou, Evangelia S; Pitsikas, Nikolaos

    2016-08-01

    Several lines evidence indicate that the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist ketamine and the mixed dopamine (DA) D1/D2 receptor agonist apomorphine induce schizophrenia-like symptoms in rodents, including memory impairments and social withdrawal. Nitric oxide (NO) has been proposed to act as an intracellular messenger in the brain and its overproduction is associated with schizophrenia. The current study was designed to investigate the ability of the inducible NO synthase (iNOS) inhibitor aminoguanidine (AG) to counteract schizophrenia-like behavioural deficits produced by ketamine and apomorphine in rats. The efficacy of AG to antagonize extinction of recognition memory, ketamine and apomorphine-induced recognition memory impairments was tested utilizing the novel object recognition task (NORT). Further, the efficacy of AG to attenuate ketamine-induced social withdrawal was examined in the social interaction test. AG (25 and 50mg/kg) antagonized extinction of recognition memory and reversed ketamine (3mg/kg) and apomorphine (1mg/kg)-induced recognition memory deficits. In contrast, AG (50 and 100mg/kg) did not counteract the ketamine (8mg/kg)-induced social isolation. The present data show that the iNOS inhibitor AG counteracted extinction of recognition memory and reversed recognition memory deficits produced by dysfunction of the glutamatergic and the dopaminergic (DAergic) system in rats. Therefore, AG may be efficacious in attenuating memory impairments often observed in schizophrenia patients.

  13. New drug therapies interfering with the renin-angiotensin-aldosterone system for resistant hypertension.

    PubMed

    Monge, Matthieu; Lorthioir, Aurélien; Bobrie, Guillaume; Azizi, Michel

    2013-12-01

    There is a persistent need for the development of new antihypertensive drugs, because the control of blood pressure is still not achievable in a significant proportion of hypertensive patients. Since the approval in 2007 of aliskiren, no other new antihypertensive based on new mechanism(s) of action have been approved. In fact, the development of promising novel drugs has been stopped for safety, efficacy or marketing reasons. Despite these difficulties, the pipeline is not dry and different new antihypertensive strategies targeting the renin-angiotensin-aldosterone pathway, are in clinical development stage. The dual angiotensin II receptor-neprilysin inhibitor LCZ696, a single molecule synthetized by cocrystallisation of valsartan and the neprilysin inhibitor prodrug AHU377 is in development for resistant hypertension and for heart failure. Daglutril is a dual neprylisin-endothelin converting enzyme inhibitor which was shown to decrease BP in patients with type 2 diabetic nephropathy. Aldosterone synthase inhibitors and the third and fourth generation non-steroidal dihydropyridine based mineralocorticoid receptors blockers are new ways to target the multiple noxious effects of aldosterone in the kidney, vessels and heart. Centrally acting aminopeptidase A inhibitors block brain angiotensin III formation, one of the main effector peptides of the brain renin angiotensin system. However, a long time will be still necessary to evaluate extensively the efficacy and safety of these new approaches. In the mean time, using appropriate and personalized daily doses of available drugs, decreasing physician inertia, improving treatment adherence, improving access to healthcare and reducing treatment costs remain major objectives to reduce the incidence of resistant hypertension.

  14. Molecular Docking Analysis of Selected Clinacanthus nutans Constituents as Xanthine Oxidase, Nitric Oxide Synthase, Human Neutrophil Elastase, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9 and Squalene Synthase Inhibitors

    PubMed Central

    Narayanaswamy, Radhakrishnan; Isha, Azizul; Wai, Lam Kok; Ismail, Intan Safinar

    2016-01-01

    Background: Clinacanthus nutans (Burm. f.) Lindau has gained popularity among Malaysians as a traditional plant for anti-inflammatory activity. Objective: This prompted us to carry out the present study on a selected 11 constituents of C. nutans which are clinacoside A–C, cycloclinacoside A1, shaftoside, vitexin, orientin, isovitexin, isoorientin, lupeol and β-sitosterol. Materials and Methods: Selected 11 constituents of C. nutans were evaluated on the docking behavior of xanthine oxidase (XO), nitric oxide synthase (NOS), human neutrophil elastase (HNE), matrix metalloproteinase (MMP 2 and 9), and squalene synthase (SQS) using Discovery Studio Version 3.1. Also, molecular physicochemical, bioactivity, absorption, distribution, metabolism, excretion, and toxicity (ADMET), and toxicity prediction by computer assisted technology analyzes were also carried out. Results: The molecular physicochemical analysis revealed that four ligands, namely clinacoside A–C and cycloclinacoside A1 showed nil violations and complied with Lipinski's rule of five. As for the analysis of bioactivity, all the 11 selected constituents of C. nutans exhibited active score (>0) toward enzyme inhibitors descriptor. ADMET analysis showed that the ligands except orientin and isoorientin were predicted to have Cytochrome P4502D6 inhibition effect. Docking studies and binding free energy calculations revealed that clinacoside B exhibited the least binding energy for the target enzymes except for XO and SQS. Isovitexin and isoorientin showed the potentials in the docking and binding with all of the six targeted enzymes, whereas vitexin and orientin docked and bound with only NOS and HNE. Conclusion: This present study has paved a new insight in understanding these 11 C. nutans ligands as potential inhibitors against XO, NOS, HNE, MMP 2, MMP 9, and SQS. SUMMARY Isovitexin and isoorientin (Clinacanthus nutans constituent) showed potentials in the docking and binding with all of the six targeted

  15. Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors.

    PubMed

    Grant, S K; Green, B G; Stiffey-Wilusz, J; Durette, P L; Shah, S K; Kozarich, J W

    1998-03-24

    Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NADPH-dependent oxidation of one of the free guanidino nitrogens of L-Arg to form nitric oxide and L-citrulline. Analogues of L-Arg and the inhibitor, L-N6-(1-iminoethyl)lysine, were used to define structural elements required for the binding and catalysis of compounds. L-Arg analogues with sequentially shorter methylene spacing between the guanidino group and the amino acid portion of the molecule were not iNOS substrates but were reversible inhibitors. L-Arg analogues such as agmatine with a hydroxyl substitution at the 2-amino position were substrates. Desaminoarginine was not a substrate but a reversible inhibitor. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give type I difference spectra similar to that of L-Arg. The amidino compounds L-N6-(1-iminoethyl)lysine, L-N5-(1-iminoethyl)ornithine, and N5-(1-iminoethyl)cadaverdine, but not N6-(1-iminoethyl)-6-aminocaproic acid, were NADPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N6-(1-iminoethyl)lysine analogues, the 2-amino group appeared to play an important role in catalytic events leading to either substrate turnover or mechanism-based inactivation. Inactivation of iNOS by L-N6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with DL--4, 5-3H]-N6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.

  16. Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis

    PubMed Central

    Iyer, Malliga R.; Liu, Ziyi; Cao, Zongxian; Jourdan, Tony; Erdelyi, Katalin; Godlewski, Grzegorz; Szanda, Gergő; Liu, Jie; Park, Joshua K.; Mukhopadhyay, Bani; Rosenberg, Avi Z.; Liow, Jeih-San; Lorenz, Robin G.; Pacher, Pal; Innis, Robert B.

    2016-01-01

    Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1–/– but not in nos2–/– mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis. PMID:27525312

  17. Primary aldosteronism and pregnancy.

    PubMed

    Morton, Adam

    2015-10-01

    Primary aldosteronism is the most common cause of secondary hypertension. Less than 50 cases of pregnancy in women with primary aldosteronism have been reported, suggesting the disorder is significantly underdiagnosed in confinement. Accurate diagnosis is complicated by physiological changes in the renin-angiotensin-aldosterone axis in pregnancy, leading to a risk of false negative results on screening tests. The course of primary aldosteronism during pregnancy is highly variable, although overall it is associated with a very high risk of fetal and maternal morbidity and mortality. The optimal management of primary aldosteronism during pregnancy is unclear, with uncertainty regarding the safety of mineralocorticoid antagonists and amiloride, their relative efficacy compared with the antihypertensive medications commonly used during pregnancy, and as to whether prognosis is improved by laparoscopic adrenalectomy where an adrenal adenoma can be demonstrated.

  18. Nitric Oxide Synthase Inhibitor Improves De Novo and Long-Term l-DOPA-Induced Dyskinesia in Hemiparkinsonian Rats

    PubMed Central

    Padovan-Neto, Fernando Eduardo; Echeverry, Marcela Bermúdez; Chiavegatto, Silvana; Del-Bel, Elaine

    2011-01-01

    Inhibitors of neuronal and endothelial nitric oxide synthase decrease l-3,4-dihidroxifenilalanine (l-DOPA)-induced dyskinesias in rodents. The mechanism of nitric oxide inhibitor action is unknown. The aims of the present study were to investigate the decrease of l-DOPA-induced abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA)-lesioned rats by nitric oxide inhibitors following either acute or chronic treatment. The primary findings of this study were that NG-nitro-l-Arginine, an inhibitor of endothelial and neuronal nitric oxide synthase, attenuated AIMs induced by chronic and acute l-DOPA. In contrast, rotational behavior was attenuated only after chronic l-DOPA. The 6-OHDA lesion and the l-DOPA treatment induced a bilateral increase (1.5 times) in the neuronal nitric oxide synthase (nNOS) protein and nNOS mRNA in the striatum and in the frontal cortex. There was a parallel increase, bilaterally, of the FosB/ΔFosB, primarily in the ipsilateral striatum. The exception was in the contralateral striatum and the ipsilateral frontal cortex, where chronic l-DOPA treatment induced an increase of approximately 10 times the nNOS mRNA. Our results provided further evidence of an anti-dyskinetic effect of NOS inhibitor. The effect appeared under l-DOPA acute and chronic treatment. The l-DOPA treatment also revealed an over-expression of the neuronal NOS in the frontal cortex and striatum. Our results corroborated findings that l-DOPA-induced rotation differs between acute and chronic treatment. The effect of the NOS inhibitor conceivably relied on the l-DOPA structural modifications in the Parkinsonian brain. Taken together, these data provided a rationale for further evaluation of NOS inhibitors in the treatment of l-DOPA-induced dyskinesia. PMID:21713068

  19. Circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, feeding and activity in rats.

    PubMed

    Kamerman, Peter; Mitchell, Duncan; Laburn, Helen

    2002-02-01

    We have investigated whether there is circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, physical activity and feeding. We used nocturnally active Sprague-Dawley rats, housed at approximately 24 degrees C with a 12:12 h light:dark cycle (lights on 07:00 hours) and provided with food and water ad libitum. Nitric oxide synthesis was inhibited by intraperitoneal injection of the unspecific nitric oxide synthase inhibitor N-nitro- L-arginine methyl ester ( L-NAME, 100, 50, 25, 10 mg/kg), or the relatively selective inducible nitric oxide synthase inhibitor aminoguanidine (100, 50 mg/kg), during the day ( approximately 09:00 hours) or night ( approximately 21:00 hours). Body temperature and physical activity were measured using radiotelemetry, while food intake was calculated by weighing each animal's food before as well as 12 and 24 h after each injection. We found that daytime injection of L-NAME and aminoguanidine had no effect on daytime body temperature. However, daytime injection of both drugs did decrease nocturnal food intake ( P<0.05) and activity ( P<0.05). When injected at night, L-NAME reduced night-time body temperature ( P<0.01), activity ( P<0.05) and food intake ( P<0.05) in a dose-dependent manner, but night-time injection of aminoguanidine inhibited only night-time activity ( P<0.05). The effects of nitric oxide synthase inhibition on body temperature, feeding and activity therefore are primarily a consequence of inhibiting constitutively expressed nitric oxide synthase, and are subject to circadian variation.

  20. Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

    PubMed Central

    Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

    2014-01-01

    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

  1. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    SciTech Connect

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  2. Identification of a glycogen synthase kinase-3β inhibitor that attenuates hyperactivity in CLOCK mutant mice.

    PubMed

    Kozikowski, Alan P; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N; Walter, Richard L; Ketcherside, Ariel; McClung, Colleen A; Mesecar, Andrew D; Caldarone, Barbara

    2011-09-05

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called "mood-stabilizing drugs", such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3β (GSK-3β) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3β. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC₅₀ values in the range of 4 to 680 nM against human GSK-3β. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg⁻¹ resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg⁻¹) and the antipsychotic haloperidol (1 mg kg⁻¹). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3β in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3β as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  3. Identification of a Glycogen Synthase Kinase-3β Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    PubMed Central

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara

    2012-01-01

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called “mood-stabilizing drugs”, such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3β (GSK-3β) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3β. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC50 values in the range of 4 to 680 nm against human GSK-3β. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mgkg−1 resulted in the attenuation of hyperactivity in amphetamine/ chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mgkg−1) and the antipsychotic haloperidol (1 mgkg−1). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3β in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3β as a relevant therapeutic target in the identification of new therapies for bipolar patients. PMID:21732538

  4. Cell-permeable inhibitors of neuronal nitric oxide synthase open new prospects for the treatment of neurological disorders.

    PubMed

    Annedi, Subhash C

    2015-02-12

    The disubstituted pyrimidines based SAR study by Silverman et al. ( J. Med. Chem. 2014 , DOI: 10.1021/jm501719e ) was focused on improving bioavailability and physicochemical properties of the designed inhibitors while retaining the potency for neural nitric oxide synthase (nNOS) and selectivity over the other two nitric oxide synthase (NOS) isoforms (endothelial NOS and inducible NOS). One of the new promising lead compounds, compound 7, displayed nanomolar potency for nNOS (Ki = 19 nM), good selectivity over endothelial (260-fold), and inducible (41-fold) NOS isoforms and also showed potential for oral bioavailability (good cell permeability with efflux ratio of 1.8) and broad safety profile with minimal off-target activities at 50 CNS based receptors. This remarkable achievement not only serves as a template for next generation selective NOS inhibitor design but also opens new prospects for the treatment of neurological disorders.

  5. CNS-accessible Inhibitor of Glucosylceramide Synthase for Substrate Reduction Therapy of Neuronopathic Gaucher Disease

    PubMed Central

    Marshall, John; Sun, Ying; Bangari, Dinesh S; Budman, Eva; Park, Hyejung; Nietupski, Jennifer B; Allaire, Amy; Cromwell, Mary A; Wang, Bing; Grabowski, Gregory A; Leonard, John P; Cheng, Seng H

    2016-01-01

    Gaucher disease (GD) is caused by a deficiency of glucocerebrosidase and the consequent lysosomal accumulation of unmetabolized glycolipid substrates. Enzyme-replacement therapy adequately manages the visceral manifestations of nonneuronopathic type-1 Gaucher patients, but not the brain disease in neuronopathic types 2 and 3 GD. Substrate reduction therapy through inhibition of glucosylceramide synthase (GCS) has also been shown to effectively treat the visceral disease. Here, we evaluated the efficacy of a novel small molecule inhibitor of GCS with central nervous system (CNS) access (Genz-682452) to treat the brain disease. Treatment of the conduritol β epoxide-induced mouse model of neuronopathic GD with Genz-682452 reduced the accumulation of liver and brain glycolipids (>70% and >20% respectively), extent of gliosis, and severity of ataxia. In the genetic 4L;C* mouse model, Genz-682452 reduced the levels of substrate in the brain by >40%, the extent of gliosis, and paresis. Importantly, Genz-682452-treated 4L;C* mice also exhibited an ~30% increase in lifespan. Together, these data indicate that an orally available antagonist of GCS that has CNS access is effective at attenuating several of the neuropathologic and behavioral manifestations associated with mouse models of neuronopathic GD. Therefore, Genz-682452 holds promise as a potential therapeutic approach for patients with type-3 GD. PMID:26948439

  6. Selective Neuronal Nitric Oxide Synthase Inhibitors and the Prevention of Cerebral Palsy

    PubMed Central

    Ji, Haitao; Tan, Sidhartha; Igarashi, Jotaro; Li, Huiying; Derrick, Matthew; Martásek, Pavel; Roman, Linda J.; Vásquez-Vivar, Jeannette; Poulos, Thomas L.; Silverman, Richard B.

    2008-01-01

    Objective To design a new class of selective neuronal nitric oxide synthase (nNOS) inhibitors and demonstrate that administration in a rabbit model for cerebral palsy (CP) prevents hypoxia-ischemia induced deaths and reduces the number of newborn kits exhibiting signs of CP. Methods We used a novel computer-based drug design method called fragment hopping to identify new chemical entities, synthesized them, carried out in vitro enzyme inhibition studies with the three isozymes of NOS and in vivo experiments to monitor cardiovascular effects on pregnant rabbit dams, NOS activity and NOx concentration in fetal brain, and assess neurobehavioral effects on kits born to saline- and compound treated dams. Results The computer-based design led to the development of powerful and highly selective compounds for inhibition of nNOS over the other isozymes. Following maternal administration in a rabbit model of CP, these compounds were found to distribute to fetal brain, to be non-toxic, without cardiovascular effects, inhibit fetal brain NOS activity in vivo, reduce NO concentration in fetal brain, and dramatically ameliorate deaths and number of newborn kits exhibiting signs of CP. Interpretation This approach may lead to new preventive strategies for cerebral palsy. PMID:19235180

  7. Discovery and Characterization of 2-Acylaminoimidazole Microsomal Prostaglandin E Synthase-1 Inhibitors.

    PubMed

    Schiffler, Matthew A; Antonysamy, Stephen; Bhattachar, Shobha N; Campanale, Kristina M; Chandrasekhar, Srinivasan; Condon, Bradley; Desai, Prashant V; Fisher, Matthew J; Groshong, Christopher; Harvey, Anita; Hickey, Michael J; Hughes, Norman E; Jones, Scott A; Kim, Euibong J; Kuklish, Steven L; Luz, John G; Norman, Bryan H; Rathmell, Richard E; Rizzo, John R; Seng, Thomas W; Thibodeaux, Stefan J; Woods, Timothy A; York, Jeremy S; Yu, Xiao-Peng

    2016-01-14

    As part of a program aimed at the discovery of antinociceptive therapy for inflammatory conditions, a screening hit was found to inhibit microsomal prostaglandin E synthase-1 (mPGES-1) with an IC50 of 17.4 μM. Structural information was used to improve enzyme potency by over 1000-fold. Addition of an appropriate substituent alleviated time-dependent cytochrome P450 3A4 (CYP3A4) inhibition. Further structure-activity relationship (SAR) studies led to 8, which had desirable potency (IC50 = 12 nM in an ex vivo human whole blood (HWB) assay) and absorption, distribution, metabolism, and excretion (ADME) properties. Studies on the formulation of 8 identified 8·H3PO4 as suitable for clinical development. Omission of a lipophilic portion of the compound led to 26, a readily orally bioavailable inhibitor with potency in HWB comparable to celecoxib. Furthermore, 26 was selective for mPGES-1 inhibition versus other mechanisms in the prostanoid pathway. These factors led to the selection of 26 as a second clinical candidate.

  8. Aggravation of experimental allergic encephalomyelitis (EAE) by administration of nitric oxide (NO) synthase inhibitors

    PubMed Central

    RUULS, S. R.; VAN DER LINDEN, S.; SONTROP, K.; HUITINGA, I.; DIJKSTRA, C. D.

    1996-01-01

    Macrophages constitute a large proportion of the inflammatory cells that infiltrate the central nervous system (CNS) of animals with EAE. Through the production of inflammatory mediators these infiltrating macrophages can contribute to the regulation of the immune reaction within the CNS, that eventually results in neurological deficits associated with EAE. NO, a free radical produced by macrophages and other cell types, has been put forward as such an immune mediator. In the present study we show that macrophages isolated from the CNS of Lewis rats with clinical signs of EAE produce elevated amounts of NO. We treated rats, in which EAE was induced, with Nw-nitro-L-arginine-methylester or NG-monomethyl-L-arginine, inhibitors of NO synthase, either systemically via intraperitoneal injection, or intracerebrally via a cannula placed in the lateral ventricle. Both treatments resulted in a marked aggravation of clinical signs of EAE. These data point to an important role of NO, produced by infiltrating macrophages, as an immune-suppressor in the disease process during EAE. PMID:8608648

  9. Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase.

    PubMed

    Lee, Yu-Ting; Cui, Chang-Jun; Chow, Eve W L; Pue, Nason; Lonhienne, Thierry; Wang, Jian-Guo; Fraser, James A; Guddat, Luke W

    2013-01-10

    The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell.

  10. Temperature-dependent spin crossover in neuronal nitric oxide synthase bound with the heme-coordinating thioether inhibitors.

    PubMed

    Doukov, Tzanko; Li, Huiying; Sharma, Ajay; Martell, Jeffrey D; Soltis, S Michael; Silverman, Richard B; Poulos, Thomas L

    2011-06-01

    A series of L-arginine analogue nitric oxide synthase inhibitors with a thioether tail have been shown to form an Fe-S thioether interaction as evidenced by continuous electron density between the Fe and S atoms. Even so, the Fe-S thioether interaction was found to be far less important for inhibitor binding than the hydrophobic interactions between the alkyl group in the thioether tail and surrounding protein (Martell et al. J. Am. Chem. Soc. 2010 , 132 , 798). However, among the few thioether inhibitors that showed Fe-S thioether interaction in crystal structures, variations in spin state (high-spin or low-spin) were observed dependent upon the heme iron oxidation state and temperature. Since modern synchrotron X-ray data collection is typically carried out at cryogenic temperatures, we reasoned that some of the discrepancies between cryo-crystal structures and room-temperature UV-visible spectroscopy could be the result of temperature-dependent spin-state changes. We, therefore, have characterized some of these neuronal nitric oxide synthase (nNOS)-thioether inhibitor complexes in both crystal and solution using EPR and UV-visible absorption spectrometry as a function of temperature and the heme iron redox state. We found that some thioether inhibitors switch from high to low spin at lower temperatures similar to the "spin crossover" phenomenon observed in many transition metal complexes.

  11. Identification of lead small molecule inhibitors of glycogen synthase kinase-3 beta using a fragment-linking strategy.

    PubMed

    Kim, Jinhee; Moon, Yonghoon; Hong, Sungwoo

    2016-12-01

    Glycogen synthase kinase-3 beta (GSK3β) kinase serves as a promising therapeutic target for the treatment of various human diseases, such as diabetes, obesity, and Alzheimer's disease. In this study, we report lead GSK3β inhibitors identified using a fragment-linking strategy. Through the systematic exploration, a six-atom chain unit bearing the rigid double bond was found to be a suitable linker connecting two fragments, which enables favorable contacts with backbone groups of residues in the pockets. As a consequence, potent GSK3β inhibitor 9i was found with IC50 values of 19nM. The binding mode analysis indicates that the activities of the inhibitors appear to be achieved by the establishment of multiple hydrogen bonds and hydrophobic interactions in the ATP-binding site of GSK3β. The good biochemical potencies and structural uniqueness of the inhibitors support consideration in the further study to optimize the biological activity.

  12. Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis.

    PubMed

    Liu, Zheng; Liu, Feng; Aldrich, Courtney C

    2015-07-02

    Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis, and biochemical evaluation of an inhibitor based on the putative transition state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge buildup at C-4 of chorismate in the TS as well as C-O bond formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps.

  13. An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.

    PubMed

    Marzabadi, M R; Gruys, K J; Pansegrau, P D; Walker, M C; Yuen, H K; Sikorski, J A

    1996-04-02

    A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described

  14. Aldosterone and Renin Test

    MedlinePlus

    ... the kidney (renal vein renin levels). If the value is significantly higher in one side, this indicates where the narrowing of the artery is present. Low aldosterone (hypoaldosteronism) usually occurs as part of ...

  15. Molecular Heterogeneity in Aldosterone-Producing Adenomas

    PubMed Central

    Nanba, Kazutaka; Chen, Andrew X.; Omata, Kei; Vinco, Michelle; Giordano, Thomas J.; Else, Tobias; Hammer, Gary D.

    2016-01-01

    Context: The use of next-generation sequencing has resulted in the identification of recurrent somatic mutations underlying primary aldosteronism (PA). However, significant gaps remain in our understanding of the relationship between tumor aldosterone synthase (CYP11B2) expression and somatic mutation status. Objective: The objective of the study was to investigate tumor CYP11B2 expression and somatic aldosterone-driver gene mutation heterogeneity. Methods: Fifty-one adrenals from 51 PA patients were studied. Immunohistochemistry for CYP11B2 was performed. Aldosterone-producing adenomas with intratumor CYP11B2 heterogeneity were analyzed for mutation status using targeted next-generation sequencing. DNA was isolated from CYP11B2-positive, CYP11B2-negative, and adjacent normal areas from formalin-fixed, paraffin-embedded sections. Results: Of 51 adrenals, seven (14 %) showed distinct heterogeneity in CYP11B2 by immunohistochemistry, including six adenomas with intratumor heterogeneity and one multinodular hyperplastic adrenal with both CYP11B2-positive and -negative nodules. Of the six adrenocortical adenomas with CYP11B2 heterogeneity, three had aldosterone-regulating mutations (CACNA1D p.F747C, KCNJ5 p.L168R, ATP1A1 p.L104R) only in CYP11B2-positive regions, and one had two different mutations localized to two histologically distinct CYP11B2-positive regions (ATP2B3 p.L424_V425del, KCNJ5 p.G151R). Lastly, one adrenal with multiple CYP11B2-expressing nodules showed different mutations in each (CACNA1D p.F747V and ATP1A1 p.L104R), and no mutations were identified in CYP11B2-negative nodule or adjacent normal adrenal. Conclusions: Adrenal tumors in patients with PA can demonstrate clear heterogeneity in CYP11B2 expression and somatic mutations in driver genes for aldosterone production. These findings suggest that aldosterone-producing adenoma tumorigenesis can occur within preexisting nodules through the acquisition of somatic mutations that drive aldosterone

  16. Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.

    PubMed

    Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

    2008-10-01

    Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.

  17. Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro.

    PubMed Central

    Papapetropoulos, A.; Desai, K. M.; Rudic, R. D.; Mayer, B.; Zhang, R.; Ruiz-Torres, M. P.; García-Cardeña, G.; Madri, J. A.; Sessa, W. C.

    1997-01-01

    Angiogenesis is a complex process involving endothelial cell (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-beta 1. Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-beta 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs. Images Figure 2 Figure 4 Figure 5 PMID:9137106

  18. Attenuation of malonate-induced degeneration of the nigrostriatal pathway by inhibitors of nitric oxide synthase.

    PubMed

    Connop, B P; Boegman, R J; Beninger, R J; Jhamandas, K

    1996-04-01

    Focal infusions of the succinate dehydrogenase inhibitor, malonate, into the substantia nigra pars compacta (SNc) of adult Sprague-Dawley rats resulted in a substantial depletion of ipsilateral striatal tyrosine hydroxylase (TH) activity. The percentage decrease in striatal TH activity following intranigral malonate (0.5 mumol/0.5 microliter) infusion was similar at 4 (58%) and 7 days (62%) post-infusion. To assess the role of N-methyl-D-aspartate (NMDA) receptor activation in malonate neurotoxicity, animals were pretreated with the NMDA receptor antagonist MK-801 (2 x 5 mg/kg, i.p.). Four days post-infusion of malonate (0.5 mumol/0.5 microliter) into the SNc, striatal TH activity was depleted by 58% in vehicle pretreated animals and 14% in the presence of MK-801 indicating a significant neuroprotective effect of MK-801 on malonate action. To determine the role of nitric oxide (NO) in malonate-induced nigral toxicity, the actions of malonate were evaluated in the presence of the nitric oxide synthase (NOS) inhibitors, 7-nitro indazole (7-NI) and N omega-nitro-L-arginine methyl ester (L- NAME). Systemic injections of 7-NI (20, 30, 40, 50 and 75 mg/kg, i.p.) produced a dose-related inhibition of nigral NOS activity which was maximal at a dose of 40 mg/kg. Intranigral infusion of malonate with 20 and 50 mg/kg 7-NI pretreatment produced a 46 and 31% decrease in striatal TH activity, respectively. Thus, a significant protective effect at the higher but not lower dose of 7-NI was observed. Pretreatment with a L- NAME regimen (2 x 250 mg/kg; i.p.), previously shown to inhibit brain NOS activity by greater than 86%, also produced a significant neuroprotective effect against malonate-induced neurotoxicity (30% decrease). The results of this study suggest that malonate-induced toxicity to the dopaminergic neurons of the nigrostriatal pathway is mediated, at least in part, by NMDA receptor activation and the formation of NO.

  19. Identification of natural inhibitors of Entamoeba histolytica cysteine synthase from microbial secondary metabolites

    PubMed Central

    Mori, Mihoko; Jeelani, Ghulam; Masuda, Yui; Sakai, Kazunari; Tsukui, Kumiko; Waluyo, Danang; Tarwadi; Watanabe, Yoshio; Nonaka, Kenichi; Matsumoto, Atsuko; Ōmura, Satoshi; Nozaki, Tomoyoshi; Shiomi, Kazuro

    2015-01-01

    Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC50 values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC50 values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC50 values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to

  20. Structure-guided Discovery of Phenyl diketo-acids as Potent Inhibitors of M. tuberculosis Malate Synthase

    PubMed Central

    Krieger, Inna V.; Freundlich, Joel S.; Gawandi, Vijay B.; Roberts, Justin P.; Gawandi, Vidyadhar B.; Sun, Qingan; Owen, Joshua L.; Fraile, Maria T.; Huss, Sofia I.; Lavandera, Jose-Luis; Ioerger, Thomas R.; Sacchettini, James C.

    2012-01-01

    Summary The glyoxylate shunt plays an important role in fatty-acid metabolism, and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of novel phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and over-expression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors were used to guide optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics. PMID:23261599

  1. Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI) inhibitors designed to probe plasticity in the active site.

    PubMed

    Manos-Turvey, Alexandra; Cergol, Katie M; Salam, Noeris K; Bulloch, Esther M M; Chi, Gamma; Pang, Angel; Britton, Warwick J; West, Nicholas P; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2012-12-14

    Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro.

  2. Marine Fungi as Producers of Benzocoumarins, a New Class of Inhibitors of Glycogen-Synthase-Kinase 3β

    PubMed Central

    Wiese, Jutta; Imhoff, Johannes F.; Gulder, Tobias A. M.; Labes, Antje; Schmaljohann, Rolf

    2016-01-01

    The glycogen-synthase-kinase 3 (GSK-3) is an important target in drug discovery. This enzyme is involved in the signaling pathways of type 2 diabetes, neurological disorders, cancer, and other diseases. Therefore, inhibitors of GSK-3 are promising drug candidates for the treatment of a broad range of diseases. Here we report pannorin (1), alternariol (2), and alternariol-9-methylether (3) to be promising inhibitors of the isoform GSK-3β showing sub-μM IC50 values. The in vitro inhibition is in the range of the known highly active GSK-3β inhibitor TDZD-8. Compounds 1–3 have a highly oxygenated benzocoumarin core structure in common, which suggests that this may be a new structural feature for efficient GSK-3β inhibition. PMID:27801816

  3. Intracellular mediators of potassium-induced aldosterone secretion

    SciTech Connect

    Ganguly, A.; Chiou, S.; Davis, J.S. )

    1990-01-01

    We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) in {sup 3}H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium.

  4. Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis.

    PubMed

    Chi, Gamma; Manos-Turvey, Alexandra; O'Connor, Patrick D; Johnston, Jodie M; Evans, Genevieve L; Baker, Edward N; Payne, Richard J; Lott, J Shaun; Bulloch, Esther M M

    2012-06-19

    MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.

  5. Mechanistic analysis of a synthetic inhibitor of the Pseudomonas aeruginosa LasI quorum-sensing signal synthase

    PubMed Central

    Lidor, O.; Al-Quntar, A.; Pesci, E. C.; Steinberg, D.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for many human infections. LasI is an acyl-homoserine lactone synthase that produces a quorum-sensing (QS) signal that positively regulates numerous P. aeruginosa virulence determinants. The inhibition of the LasI protein is therefore an attractive drug target. In this study, a novel in silico to in vitro complementation was applied to screen thiazolidinedione-type compounds for their ability to inhibit biofilm formation at concentrations not affecting bacterial growth. The compound (z)-5-octylidenethiazolidine-2, 4-dione (TZD-C8) was a strong inhibitor of biofilm formation and chosen for further study. Structural exploration of in silico docking predicted that the compound had high affinity for the LasI activity pocket. The TZD-C8 compound was also predicted to create hydrogen bonds with residues Arg30 and Ile107. Site-directed mutagenesis (SDM) of these two sites demonstrated that TZD-C8 inhibition was abolished in the lasI double mutant PAO-R30D, I107S. In addition, in vitro swarming motility and quorum sensing signal production were affected by TZD-C 8, confirming this compound alters the cell to cell signalling circuitry. Overall, this novel inhibitor of P. aeruginosa quorum sensing shows great promise and validates our mechanistic approach to discovering inhibitors of LuxI-type acyl-homoserine lactone synthases. PMID:26593271

  6. Fatty Acid Synthase Inhibitors Engage the Cell Death Program Through the Endoplasmic Reticulum

    DTIC Science & Technology

    2007-12-01

    any capacity. The goal of this proposal was two- fold. One was to determine the mechanism by which the ER might initiate death following FAS inhibition...acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 2000;16:202–8. 8. Kuhajda FP, Jenner K, Wood FD, et al. Fatty...flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase . Biochemistry 44, 4100–4107

  7. Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2014-06-01

    Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families.

  8. New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

    PubMed

    Sebastián, Víctor; Manoli, Maria-Tsampika; Pérez, Daniel I; Gil, Carmen; Mellado, Emilia; Martínez, Ana; Espeso, Eduardo A; Campillo, Nuria E

    2016-06-30

    Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3β inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3β inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA.

  9. Aldosterone: effects on the kidney and cardiovascular system.

    PubMed

    Briet, Marie; Schiffrin, Ernesto L

    2010-05-01

    Aldosterone, a steroid hormone with mineralocorticoid activity, is mainly recognized for its action on sodium reabsorption in the distal nephron of the kidney, which is mediated by the epithelial sodium channel (ENaC). Beyond this well-known action, however, aldosterone exerts other effects on the kidney, blood vessels and the heart, which can have pathophysiological consequences, particularly in the presence of a high salt intake. Aldosterone is implicated in renal inflammatory and fibrotic processes, as well as in podocyte injury and mesangial cell proliferation. In the cardiovascular system, aldosterone has specific hypertrophic and fibrotic effects and can alter endothelial function. Several lines of evidence support the existence of crosstalk between aldosterone and angiotensin II in vascular smooth muscle cells. The deleterious effects of aldosterone on the cardiovascular system require concomitant pathophysiological conditions such as a high salt diet, increased oxidative stress, or inflammation. Large interventional trials have confirmed the benefits of adding mineralocorticoid-receptor antagonists to standard therapy, in particular to angiotensin-converting-enzyme inhibitor and angiotensin II receptor blocker therapy, in patients with heart failure. Small interventional studies in patients with chronic kidney disease have shown promising results, with a significant reduction of proteinuria associated with aldosterone antagonism, but large interventional trials that test the efficacy and safety of mineralocorticoid-receptor antagonists in chronic kidney disease are needed.

  10. Reevaluating glyphosate as a transition-state inhibitor of EPSP synthase: identification of an EPSP synthase.EPSP.glyphosate ternary complex.

    PubMed

    Sammons, R D; Gruys, K J; Anderson, K S; Johnson, K A; Sikorski, J A

    1995-05-16

    Numerous studies have confirmed that glyphosate forms a tight ternary complex with EPSP synthase and shikimate 3-phosphate. It has been proposed [Anton, D., Hedstrom, L., Fish, S., & Abeles, R. (1983) Biochemistry 22, 5903-5908; Steinrücken, H. C., & Amrhein, N. (1984) Eur. J. Biochem. 143, 351-357] that in this complex glyphosate functions as a transition-state analog of the putative phosphoenolpyruvoyl oxonium ion. For this to be true, glyphosate must occupy the space in the enzyme active site that is normally associated with PEP and, through turnover, the carboxyvinyl group of the product EPSP. According to this model, one would predict that, in the reverse EPSP synthase reaction with EPSP and phosphate as substrates, there should be little if any interaction of glyphosate with enzyme or enzyme.substrate complexes. In contrast to this expectation, rapid gel filtration experiments provided direct evidence that glyphosate could be trapped on the enzyme in the presence of EPSP to form a ternary complex of EPSPS.EPSP.glyphosate. The experimentally determined stoichiometry for this complex, 0.62 equiv of glyphosate/mole of EPSPS, is similar to that found for the EPSPS.S3P.glyphosate ternary complex (0.66). This direct binding result was corroborated and quantitated by fluorescence titration experiments which demonstrated that glyphosate forms a reasonably tight (Kd = 56 +/- 1 microM) ternary complex with enzyme and EPSP. This finding was further verified, and its impact on substrate turnover analyzed, by steady-state kinetics. Glyphosate was found to be an uncompetitive inhibitor versus EPSP with Kii(app) = 54 +/- 2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Use of structure-based drug design approaches to obtain novel anthranilic acid acyl carrier protein synthase inhibitors.

    PubMed

    Joseph-McCarthy, Diane; Parris, Kevin; Huang, Adrian; Failli, Amedeo; Quagliato, Dominick; Dushin, Elizabeth Glasfeld; Novikova, Elena; Severina, Elena; Tuckman, Margareta; Petersen, Peter J; Dean, Charles; Fritz, Christian C; Meshulam, Tova; DeCenzo, Maureen; Dick, Larry; McFadyen, Iain J; Somers, William S; Lovering, Frank; Gilbert, Adam M

    2005-12-15

    Acyl carrier protein synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheinyl group from the coenzyme A to a serine residue in acyl carrier protein (ACP), thereby activating ACP, an important step in cell wall biosynthesis. The structure-based design of novel anthranilic acid inhibitors of AcpS, a potential antibacterial target, is presented. An initial high-throughput screening lead and numerous analogues were modeled into the available AcpS X-ray structure, opportunities for synthetic modification were identified, and an iterative process of synthetic modification, X-ray complex structure determination with AcpS, biological testing, and further modeling ultimately led to potent inhibitors of the enzyme. Four X-ray complex structures of representative anthranilic acid ligands bound to AcpS are described in detail.

  12. Use of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to evaluate the role of nitric oxide in periapical inflammation.

    PubMed

    Farhad, Ali R; Razavi, Seyedmohammad; Jahadi, Sanaz; Saatchi, Masoud

    2011-06-01

    The purpose of this study was to evaluate the effects of aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS) on the degree of inflammatory response in periapical lesions in the canine teeth of cats. Root canals from 52 cat canine teeth were exposed to the oral cavity and sealed after 7 days. One day before pulp exposure, cats were administered either AG (experimental group) or normal saline (control group), which was continued on a daily basis until the day of sacrifice. Animals were sacrificed at 28 days after pulp exposure. Inflammatory response in the periapical zones was analyzed histologically. The degree of periapical inflammation in the AG group was significantly lower than that in the control group (P < 0.05). Selective iNOS inhibitors such as AG thus reduce the intensity of inflammatory responses in periapical lesions.

  13. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    SciTech Connect

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  14. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    SciTech Connect

    Sharma, Bhupesh Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in

  15. L-NAME, a nitric oxide synthase inhibitor, as a potential countermeasure to post-suspension hypotension in rats

    NASA Technical Reports Server (NTRS)

    Bayorh, M. A.; Socci, R. R.; Watts, S.; Wang, M.; Eatman, D.; Emmett, N.; Thierry-Palmer, M.

    2001-01-01

    A large number of astronauts returning from spaceflight experience orthostatic hypotension. This hypotension may be due to overproduction of vasodilatory mediators, such as nitric oxide (NO) and prostaglandins. To evaluate the role of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) as a countermeasure against the post-suspension reduction in mean arterial pressure (MAP), we assessed the cardiovascular responses and vascular reactivity to 7-day 30 degrees tail-suspension and a subsequent 6 hr post-suspension period in conscious rats. After a pre-suspension reading, direct MAP and heart rate (HR) were measured daily and every 2 hrs post-suspension. The NO synthase inhibitor L-NAME (20 mg/kg, i.v.), or saline, were administered after the 7th day reading prior to release from suspension and at 2 and 4 hrs post-suspension. At 6 hrs post-suspension, vascular reactivity was assessed. While MAP did not change during the suspension period, it was reduced post-suspension. Heart rate was not significantly altered. L-NAME administration reversed the post-suspension reduction in MAP. In addition, the baroreflex sensitivity for heart rate was modified by L-NAME. Thus, the post-suspension reduction in MAP may be due to overproduction of NO and altered baroreflex activity.

  16. Protective effects of a squalene synthase inhibitor, lapaquistat acetate (TAK-475), on statin-induced myotoxicity in guinea pigs

    SciTech Connect

    Nishimoto, Tomoyuki; Ishikawa, Eiichiro; Anayama, Hisashi; Hamajyo, Hitomi; Nagai, Hirofumi; Hirakata, Masao; Tozawa, Ryuichi

    2007-08-15

    High-dose statin treatment has been recommended as a primary strategy for aggressive reduction of LDL cholesterol levels and protection against coronary artery disease. The effectiveness of high-dose statins may be limited by their potential for myotoxic side effects. There is currently little known about the molecular mechanisms of statin-induced myotoxicity. Previously we showed that T-91485, an active metabolite of the squalene synthase inhibitor lapaquistat acetate (lapaquistat: a previous name is TAK-475), attenuated statin-induced cytotoxicity in human skeletal muscle cells [Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y., Sugiyama, Y., 2003a. Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A. Biochem. Pharmacol. 66, 2133-2139]. In the current study, we investigated the effects of lapaquistat administration on statin-induced myotoxicity in vivo. Guinea pigs were treated with either high-dose cerivastatin (1 mg/kg) or cerivastatin together with lapaquistat (30 mg/kg) for 14 days. Treatment with cerivastatin alone decreased plasma cholesterol levels by 45% and increased creatine kinase (CK) levels by more than 10-fold (a marker of myotoxicity). The plasma CK levels positively correlated with the severity of skeletal muscle lesions as assessed by histopathology. Co-administration of lapaquistat almost completely prevented the cerivastatin-induced myotoxicity. Administration of mevalonolactone (100 mg/kg b.i.d.) prevented the cerivastatin-induced myotoxicity, confirming that this effect is directly related to HMG-CoA reductase inhibition. These results strongly suggest that cerivastatin-induced myotoxicity is due to depletion of mevalonate derived isoprenoids. In addition, squalene synthase inhibition could potentially be used clinically to prevent statin-induced myopathy.

  17. Aldosterone Activates NF-κB in the Collecting Duct

    PubMed Central

    Leroy, Valérie; De Seigneux, Sophie; Agassiz, Victor; Hasler, Udo; Rafestin-Oblin, Marie-Edith; Vinciguerra, Manlio; Martin, Pierre-Yves; Féraille, Eric

    2009-01-01

    Besides its classical effects on salt homeostasis in renal epithelial cells, aldosterone promotes inflammation and fibrosis and modulates cell proliferation. The proinflammatory transcription factor NF-κB has been implicated in cell proliferation, apoptosis, and regulation of transepithelial sodium transport. The effect of aldosterone on the NF-κB pathway in principal cells of the cortical collecting duct, a major physiologic target of aldosterone, is unknown. Here, in both cultured cells and freshly isolated rat cortical collecting duct, aldosterone activated the canonical NF-κB signaling pathway, leading to increased expression of several NF-κB–targeted genes (IκBα, plasminogen activator inhibitor 1, monocyte chemoattractant protein 1, IL-1β, and IL-6). Small interfering RNA–mediated knockdown of the serum and glucocorticoid-inducible kinase SGK1, a gene induced early in the response to aldosterone, but not pharmacologic inhibition of extracellular signal–regulated kinase and p38 kinase, attenuated aldosterone-induced NF-κB activation. Pharmacologic antagonism or knockdown of the mineralocorticoid receptor prevented aldosterone-induced NF-κB activity. In addition, activation of the glucocorticoid receptor inhibited the transactivation of NF-κB by aldosterone. In agreement with these in vitro findings, spironolactone prevented NF-κB–induced transcriptional activation observed in cortical collecting ducts of salt-restricted rats. In summary, aldosterone activates the canonical NF-κB pathway in principal cells of the cortical collecting duct by activating the mineralocorticoid receptor and by inducing SGK1. PMID:18987305

  18. The current state of resistance to acetohydroxyacid/acetolactate synthase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The acetohydroxyacid/acetolactate synthase (ALS) inhibiting herbicides are used for weed management in multiple crop and non-crop situations. Herbicides with this mechanism of action were introduced in the early 1980s and quickly came to dominate many cropping situations due to their broad spectrum...

  19. Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis

    PubMed Central

    Liu, Zheng; Liu, Feng; Aldrich, Courtney C.

    2015-01-01

    Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis and biochemical evaluation of an inhibitor based on the putative transition-state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge build-up at C-4 of chorismate in the TS as well as C-O bond-formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side-chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps. PMID:26035083

  20. Novel 2,4-Disubstituted Pyrimidines as Potent, Selective, and Cell-Permeable Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    2014-01-01

    Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure–activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes. PMID:25489882

  1. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    SciTech Connect

    Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

    2015-03-01

    In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.

  2. Transcriptome Pathway Analysis of Pathological and Physiological Aldosterone-Producing Human Tissues

    PubMed Central

    Zhou, Junhua; Lam, Brian; Neogi, Sudeshna G.; Yeo, Giles S.H.; Brown, Morris J.

    2016-01-01

    Primary aldosteronism is present in ≈10% of hypertensives. We previously performed a microarray assay on aldosterone-producing adenomas and their paired zona glomerulosa and fasciculata. Confirmation of top genes validated the study design and functional experiments of zona glomerulosa selective genes established the role of the encoded proteins in aldosterone regulation. In this study, we further analyzed our microarray data using AmiGO 2 for gene ontology enrichment and Ingenuity Pathway Analysis to identify potential biological processes and canonical pathways involved in pathological and physiological aldosterone regulation. Genes differentially regulated in aldosterone-producing adenoma and zona glomerulosa were associated with steroid metabolic processes gene ontology terms. Terms related to the Wnt signaling pathway were enriched in zona glomerulosa only. Ingenuity Pathway Analysis showed "NRF2-mediated oxidative stress response pathway" and "LPS (lipopolysaccharide)/IL-1 (interleukin-1)–mediated inhibition of RXR (retinoid X receptor) function" were affected in both aldosterone-producing adenoma and zona glomerulosa with associated genes having up to 21- and 8-fold differences, respectively. Comparing KCNJ5-mutant aldosterone-producing adenoma, zona glomerulosa, and zona fasciculata samples with wild-type samples, 138, 56, and 59 genes were differentially expressed, respectively (fold-change >2; P<0.05). ACSS3, encoding the enzyme that synthesizes acetyl-CoA, was the top gene upregulated in KCNJ5-mutant aldosterone-producing adenoma compared with wild-type. NEFM, a gene highly upregulated in zona glomerulosa, was upregulated in KCNJ5 wild-type aldosterone-producing adenomas. NR4A2, the transcription factor for aldosterone synthase, was highly expressed in zona fasciculata adjacent to a KCNJ5-mutant aldosterone-producing adenoma. Further interrogation of these genes and pathways could potentially provide further insights into the pathology of primary

  3. Factors affecting the aldosterone/renin ratio.

    PubMed

    Stowasser, M; Ahmed, A H; Pimenta, E; Taylor, P J; Gordon, R D

    2012-03-01

    Although the aldosterone/renin ratio (ARR) is the most reliable screening test for primary aldo-steronism, false positives and negatives occur. Dietary salt restriction, concomitant malignant or renovascular hypertension, pregnancy and treatment with diuretics (including spironolactone), dihydropyridine calcium blockers, angiotensin converting enzyme inhibitors, and angiotensin receptor antagonists can produce false negatives by stimulating renin. We recently reported selective serotonin reuptake inhibitors lower the ratio. Because potassium regulates aldosterone, uncorrected hypokalemia can lead to false negatives. Beta-blockers, alpha-methyldopa, clonidine, and nonsteroidal anti-inflammatory drugs suppress renin, raising the ARR with potential for false positives. False positives may occur in patients with renal dysfunction or advancing age. We recently showed that (1) females have higher ratios than males, and (2) false positive ratios can occur during the luteal menstrual phase and while taking an oral ethynylestradiol/drospirenone (but not implanted subdermal etonogestrel) contraceptive, but only if calculated using direct renin concentration and not plasma renin activity. Where feasible, diuretics should be ceased at least 6 weeks and other interfering medications at least 2 before ARR measurement, substituting noninterfering agents (e. g., verapamil slow-release±hydralazine and prazosin or doxazosin) were required. Hypokalemia should be corrected and a liberal salt diet encouraged. Collecting blood midmorning from seated patients following 2-4 h upright posture improves sensitivity. The ARR is a screening test only and should be repeated once or more before deciding whether to proceed to confirmatory suppression testing. Liquid chromatography-tandem mass spectrometry aldosterone assays represent a major advance towards addressing inaccuracies inherent in other available methods.

  4. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    PubMed Central

    Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

    2015-01-01

    The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthio­adenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethyl­aniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS. PMID:25760598

  5. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    PubMed

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, José A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

  6. Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I.

    PubMed

    Sayahi, Halimah; Zimhony, Oren; Jacobs, William R; Shekhtman, Alexander; Welch, John T

    2011-08-15

    Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.

  7. Characterization of 3,3-dimethyl substituted N-aryl piperidines as potent microsomal prostaglandin E synthase-1 inhibitors.

    PubMed

    Kuklish, Steven L; Antonysamy, Stephen; Bhattachar, Shobha N; Chandrasekhar, Srinivasan; Fisher, Matthew J; Fretland, Adrian J; Gooding, Karen; Harvey, Anita; Hughes, Norman E; Luz, John G; Manninen, Peter R; McGee, James E; Navarro, Antonio; Norman, Bryan H; Partridge, Katherine M; Quimby, Steven J; Schiffler, Matthew A; Sloan, Ashley V; Warshawsky, Alan M; York, Jeremy S; Yu, Xiao-Peng

    2016-10-01

    Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine inhibitors of microsomal prostaglandin E synthases-1(mPGES-1). Example 14 potently inhibited PGE2 synthesis in an ex vivo human whole blood (HWB) assay with an IC50 of 7nM. In addition, 14 had no activity in human COX-1 or COX-2 assays at 30μM, and failed to inhibit human mPGES-2 at 62.5μM in a microsomal prep assay. These data are consistent with selective mPGES-1-mediated reduction of PGE2. In dog, 14 had oral bioavailability (74%), clearance (3.62mL/(min*kg)) and volume of distribution (Vd,ss=1.6L/kg) values within our target ranges. For these reasons, 14 was selected for further study.

  8. Inhibitors of the Salicylate Synthase (MbtI) from Mycobacterium tuberculosis Discovered by High-Throughput Screening

    PubMed Central

    Vasan, Mahalakshmi; Neres, João; Williams, Jessica; Wilson, Daniel J.; Teitelbaum, Aaron M.; Remmel, Rory P.; Aldrich, Courtney C.

    2010-01-01

    A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure–activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition. PMID:21053346

  9. Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening.

    PubMed

    Vasan, Mahalakshmi; Neres, João; Williams, Jessica; Wilson, Daniel J; Teitelbaum, Aaron M; Remmel, Rory P; Aldrich, Courtney C

    2010-12-03

    A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure-activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition.

  10. Design and structure-activity relationships of potent and selective inhibitors of undecaprenyl pyrophosphate synthase (UPPS): tetramic, tetronic acids and dihydropyridin-2-ones.

    PubMed

    Peukert, Stefan; Sun, Yingchuan; Zhang, Rui; Hurley, Brian; Sabio, Mike; Shen, Xiaoyu; Gray, Christen; Dzink-Fox, JoAnn; Tao, Jianshi; Cebula, Regina; Wattanasin, Sompong

    2008-03-15

    Based on a pharmacophore hypothesis substituted tetramic and tetronic acid 3-carboxamides as well as dihydropyridin-2-one-3-carboxamides were investigated as inhibitors of undecaprenyl pyrophosphate synthase (UPPS) for use as novel antimicrobial agents. Synthesis and structure-activity relationship patterns for this class of compounds are discussed. Selectivity data and antibacterial activities for selected compounds are provided.

  11. Effect of inhibitors of inducible and neuronal NO synthases on the development of audiogenic stress-induced damage in Krushinskii-Molodkina rats.

    PubMed

    Krushinskii, A L; Kuzenkov, V S; D'yakonova, V E; Reutov, V P

    2010-12-01

    Experiments on the models of epileptiform seizure and hemorrhagic stroke (Krushinskii-Molodkina rats) showed that selective inhibitors of inducible and neuronal NO synthases (aminoguanidine and 7-nitroindazole) significantly decrease the mortality rate, reduce the severity of motor disorders, and prevent the development of intracranial hemorrhages under conditions of audiogenic stress.

  12. Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.

    PubMed

    Rossato, Franco A; Zecchin, Karina G; La Guardia, Paolo G; Ortega, Rose M; Alberici, Luciane C; Costa, Rute A P; Catharino, Rodrigo R; Graner, Edgard; Castilho, Roger F; Vercesi, Aníbal E

    2014-01-01

    The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce

  13. Fatty Acid Synthase Inhibitors Induce Apoptosis in Non-Tumorigenic Melan-A Cells Associated with Inhibition of Mitochondrial Respiration

    PubMed Central

    Rossato, Franco A.; Zecchin, Karina G.; La Guardia, Paolo G.; Ortega, Rose M.; Alberici, Luciane C.; Costa, Rute A. P.; Catharino, Rodrigo R.; Graner, Edgard; Castilho, Roger F.; Vercesi, Aníbal E.

    2014-01-01

    The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat

  14. Pharmacological characterization of KLYP961, a dual inhibitor of inducible and neuronal nitric-oxide synthases.

    PubMed

    Symons, Kent T; Nguyen, Phan M; Massari, Mark E; Anzola, John V; Staszewski, Lena M; Wang, Li; Yazdani, Nahid; Dorow, Steven; Muhammad, Jerry; Sablad, Marciano; Rozenkrants, Natasha; Bonefous, Celine; Payne, Joseph E; Rix, Peter J; Shiau, Andrew K; Noble, Stewart A; Smith, Nicholas D; Hassig, Christian A; Zhang, Yan; Rao, Tadimeti S

    2011-02-01

    Nitric oxide (NO) derived from neuronal nitric-oxide synthase (nNOS) and inducible nitric-oxide synthase (iNOS) plays a key role in various pain and inflammatory states. KLYP961 (4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one) inhibits the dimerization, and hence the enzymatic activity of human, primate, and murine iNOS and nNOS (IC(50) values 50-400 nM), with marked selectivity against endothelial nitric-oxide synthase (IC(50) >15,000 nM). It has ideal drug like-properties, including excellent rodent and primate pharmacokinetics coupled with a minimal off-target activity profile. In mice, KLYP961 attenuated endotoxin-evoked increases in plasma nitrates, a surrogate marker of iNOS activity in vivo, in a sustained manner (ED(50) 1 mg/kg p.o.). KLYP961 attenuated pain behaviors in a mouse formalin model (ED(50) 13 mg/kg p.o.), cold allodynia in the chronic constriction injury model (ED(50) 25 mg/kg p.o.), or tactile allodynia in the spinal nerve ligation model (ED(50) 30 mg/kg p.o.) with similar efficacy, but superior potency relative to gabapentin, pregabalin, or duloxetine. Unlike morphine, the antiallodynic activity of KLYP961 did not diminish upon repeated dosing. KLYP961 also attenuated carrageenin-induced edema and inflammatory hyperalgesia and writhing response elicited by phenylbenzoquinone with efficacy and potency similar to those of celecoxib. In contrast to gabapentin, KLYP961 did not impair motor coordination at doses as high as 1000 mg/kg p.o. KLYP961 also attenuated capsaicin-induced thermal allodynia in rhesus primates in a dose-related manner with a minimal effective dose (≤ 10 mg/kg p.o.) and a greater potency than gabapentin. In summary, KLYP961 represents an ideal tool with which to probe the physiological role of NO derived from iNOS and nNOS in human pain and inflammatory states.

  15. The Design and Synthesis of Potent and Selective Inhibitors of Trypanosoma brucei Glycogen Synthase Kinase 3 for the Treatment of Human African Trypanosomiasis

    PubMed Central

    2014-01-01

    Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3β enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture. PMID:25198388

  16. A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.

    PubMed

    Burkholder, Kristin M; Perry, Jeffrey W; Wobus, Christiane E; Donato, Nicholas J; Showalter, Hollis D; Kapuria, Vaibhav; O'Riordan, Mary X D

    2011-12-01

    Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.

  17. Pharmacophore Modeling and Virtual Screening for Novel Acidic Inhibitors of Microsomal Prostaglandin E2 Synthase-1 (mPGES-1)

    PubMed Central

    2011-01-01

    Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes prostaglandin E2 formation and is considered as a potential anti-inflammatory pharmacological target. To identify novel chemical scaffolds active on this enzyme, two pharmacophore models for acidic mPGES-1 inhibitors were developed and theoretically validated using information on mPGES-1 inhibitors from literature. The models were used to screen chemical databases supplied from the National Cancer Institute (NCI) and the Specs. Out of 29 compounds selected for biological evaluation, nine chemically diverse compounds caused concentration-dependent inhibition of mPGES-1 activity in a cell-free assay with IC50 values between 0.4 and 7.9 μM, respectively. Further pharmacological characterization revealed that also 5-lipoxygenase (5-LO) was inhibited by most of these active compounds in cell-free and cell-based assays with IC50 values in the low micromolar range. Together, nine novel chemical scaffolds inhibiting mPGES-1 are presented that may possess anti-inflammatory properties based on the interference with eicosanoid biosynthesis. PMID:21466167

  18. Catechol-based substrates of chalcone synthase as a scaffold for novel inhibitors of PqsD.

    PubMed

    Allegretta, Giuseppe; Weidel, Elisabeth; Empting, Martin; Hartmann, Rolf W

    2015-01-27

    A new strategy for treating Pseudomonas aeruginosa infections could be disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to impair communication among the cells and, hence, reduce the expression of virulence factors and the formation of biofilms. PqsD is an essential enzyme for the synthesis of PQS and shares some features with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa. Both proteins are quite similar concerning the size of the active site, the catalytic residues and the electrostatic surface potential at the entrance of the substrate tunnel. Hence, we evaluated selected substrates of the vegetable enzyme as potential inhibitors of the bacterial protein. This similarity-guided approach led to the identification of a new class of PqsD inhibitors having a catechol structure as an essential feature for activity, a saturated linker with two or more carbons and an ester moiety bearing bulky substituents. The developed compounds showed PqsD inhibition with IC50 values in the single-digit micromolar range. The binding mode of these compounds was investigated by Surface Plasmon Resonance (SPR) experiments revealing that their interaction with the protein is not influenced by the presence of the anthranilic acid bound to active site cysteine. Importantly, some compounds reduced the signal molecule production in cellulo.

  19. Phenyl Ether- and Aniline-Containing 2-Aminoquinolines as Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase.

    PubMed

    Cinelli, Maris A; Li, Huiying; Pensa, Anthony V; Kang, Soosung; Roman, Linda J; Martásek, Pavel; Poulos, Thomas L; Silverman, Richard B

    2015-11-12

    Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.

  20. Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology

    PubMed Central

    Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pelà, Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D’Arca, Domenico

    2016-01-01

    Demonstrating a candidate drug’s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding. PMID:27250901

  1. Biophysical investigation of the mode of inhibition of tetramic acids, the allosteric inhibitors of undecaprenyl pyrophosphate synthase.

    PubMed

    Lee, Lac V; Granda, Brian; Dean, Karl; Tao, Jianshi; Liu, Eugene; Zhang, Rui; Peukert, Stefan; Wattanasin, Sompong; Xie, Xiaoling; Ryder, Neil S; Tommasi, Ruben; Deng, Gejing

    2010-06-29

    Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C(55) undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS.TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS.TA complex, but the quaternary complex, UPPS.TA.FPP.IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.

  2. The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys

    PubMed Central

    Nawrocki, Andrea R.; Zhou, Dan; Wu, Margaret; Previs, Stephen; Miller, Corey; Liu, Haiying; Hines, Catherine D. G.; Madeira, Maria; Cao, Jin; Herath, Kithsiri; Wang, Liangsu; Kelley, David E.; Li, Cai

    2016-01-01

    Objectives Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. Methods We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. Results The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. Conclusions These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic

  3. Discovery and characterization of [(cyclopentyl)ethyl]benzoic acid inhibitors of microsomal prostaglandin E synthase-1.

    PubMed

    Partridge, Katherine M; Antonysamy, Stephen; Bhattachar, Shobha N; Chandrasekhar, Srinivasan; Fisher, Matthew J; Fretland, Adrian; Gooding, Karen; Harvey, Anita; Hughes, Norman E; Kuklish, Steven L; Luz, John G; Manninen, Peter R; McGee, James E; Mudra, Daniel R; Navarro, Antonio; Norman, Bryan H; Quimby, Steven J; Schiffler, Matthew A; Sloan, Ashley V; Warshawsky, Alan M; Weller, Jennifer M; York, Jeremy S; Yu, Xiao-Peng

    2017-03-15

    We describe a novel class of acidic mPGES-1 inhibitors with nanomolar enzymatic and human whole blood (HWB) potency. Rational design in conjunction with structure-based design led initially to the identification of anthranilic acid 5, an mPGES-1 inhibitor with micromolar HWB potency. Structural modifications of 5 improved HWB potency by over 1000×, reduced CYP2C9 single point inhibition, and improved rat clearance, which led to the selection of [(cyclopentyl)ethyl]benzoic acid compound 16 for clinical studies. Compound 16 showed an IC80 of 24nM for inhibition of PGE2 formation in vitro in LPS-stimulated HWB. A single oral dose resulted in plasma concentrations of 16 that exceeded its HWB IC80 in both rat (5mg/kg) and dog (3mg/kg) for over twelve hours.

  4. Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

    PubMed Central

    Wallace, Joselynn; Bowlin, Nicholas O.; Mills, Debra M.; Saenkham, Panatda; Kwasny, Steven M.; Opperman, Timothy J.; Williams, John D.; Rock, Charles O.; Bowlin, Terry L.

    2015-01-01

    Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. PMID:26169404

  5. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer

    PubMed Central

    Sadowski, Martin C.; Pouwer, Rebecca H.; Gunter, Jennifer H.; Lubik, Amy A.; Quinn, Ronald J.; Nelson, Colleen C.

    2014-01-01

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer. PMID:25313139

  6. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer.

    PubMed

    Sadowski, Martin C; Pouwer, Rebecca H; Gunter, Jennifer H; Lubik, Amy A; Quinn, Ronald J; Nelson, Colleen C

    2014-10-15

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

  7. Calpain-10 Activity Underlies Angiotensin II-Induced Aldosterone Production in an Adrenal Glomerulosa Cell Model

    PubMed Central

    Seremwe, Mutsa; Schnellmann, Rick G.

    2015-01-01

    Aldosterone is a steroid hormone important in the regulation of blood pressure. Aberrant production of aldosterone results in the development and progression of diseases including hypertension and congestive heart failure; therefore, a complete understanding of aldosterone production is important for developing more effective treatments. Angiotensin II (AngII) regulates steroidogenesis, in part through its ability to increase intracellular calcium levels. Calcium can activate calpains, proteases classified as typical or atypical based on the presence or absence of penta-EF-hands, which are involved in various cellular responses. We hypothesized that calpain, in particular calpain-10, is activated by AngII in adrenal glomerulosa cells and underlies aldosterone production. Our studies showed that pan-calpain inhibitors reduced AngII-induced aldosterone production in 2 adrenal glomerulosa cell models, primary bovine zona glomerulosa and human adrenocortical carcinoma (HAC15) cells, as well as CYP11B2 expression in the HAC15 cells. Although AngII induced calpain activation in these cells, typical calpain inhibitors had no effect on AngII-elicited aldosterone production, suggesting a lack of involvement of classical calpains in this process. However, an inhibitor of the atypical calpain, calpain-10, decreased AngII-induced aldosterone production. Consistent with this result, small interfering RNA (siRNA)-mediated knockdown of calpain-10 inhibited aldosterone production and CYP11B2 expression, whereas adenovirus-mediated overexpression of calpain-10 resulted in increased AngII-induced aldosterone production. Our results indicate that AngII-induced activation of calpain-10 in glomerulosa cells underlies aldosterone production and identify calpain-10 or its downstream pathways as potential targets for the development of drug therapies for the treatment of hypertension. PMID:25836666

  8. Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica.

    PubMed

    An, Hyo-Jin; Nugroho, Agung; Song, Byong-Min; Park, Hee-Juhn

    2015-12-01

    Phytochemical studies on the constituents of the rhizomes of Imperata cylindrica (Gramineae) were performed using high-performance liquid chromatography (HPLC). We also aimed to search for any biologically active substance capable of inhibiting nitric oxide (NO) formation in lipopolysaccharide (LPS)-activated macrophage 264.7 cells, by testing four compounds isolated from this plant. Four compounds, including a new chromone, isoeugenin, along with ferulic acid, p-coumaric acid, and caffeic acid were isolated and identified by NMR spectroscopy. The structure of isoeugenin was determined as 7-hydroxy-5-methoxy-2-methylchromone by the 2D-NMR technique. Among the four compounds, isoeugenin has the lowest IC50 value on the inhibition of NO production in LPS-activated macrophage RAW264.7 cells (IC50, 9.33 μg/mL). In addition, isoeugenin significantly suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines mRNA levels. Taken together, these results suggest that the anti-inflammatory activity of isoeugenin is associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines in RAW264.7 cells. Accordingly, our results suggest that the new chromone isoegenin should be considered a potential treatment for inflammatory disease.

  9. Bcl2L13 is a ceramide synthase inhibitor in glioblastoma

    PubMed Central

    Jensen, Samuel A.; Calvert, Andrea E.; Volpert, Giora; Kouri, Fotini M.; Hurley, Lisa A.; Luciano, Janina P.; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H.; Stegh, Alexander H.

    2014-01-01

    Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13–CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use. PMID:24706805

  10. Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.

    PubMed

    Jensen, Samuel A; Calvert, Andrea E; Volpert, Giora; Kouri, Fotini M; Hurley, Lisa A; Luciano, Janina P; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H; Stegh, Alexander H

    2014-04-15

    Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.

  11. (-)-UB006: A new fatty acid synthase inhibitor and cytotoxic agent without anorexic side effects.

    PubMed

    Makowski, Kamil; Mir, Joan Francesc; Mera, Paula; Ariza, Xavier; Asins, Guillermina; Hegardt, Fausto G; Herrero, Laura; García, Jordi; Serra, Dolors

    2017-05-05

    C75 is a synthetic anticancer drug that inhibits fatty acid synthase (FAS) and shows a potent anorexigenic side effect. In order to find new cytotoxic compounds that do not impact food intake, we synthesized a new family of C75 derivatives. The most promising anticancer compound among them was UB006 ((4SR,5SR)-4-(hydroxymethyl)-3-methylene-5-octyldihydrofuran-2(3H)-one). The effects of this compound on cytotoxicity, food intake and body weight were studied in UB006 racemic mixture and in both its enantiomers separately. The results showed that both enantiomers inhibit FAS activity and have potent cytotoxic effects in several tumour cell lines, such as the ovarian cell cancer line OVCAR-3. The (-)-UB006 enantiomer's cytotoxic effect on OVCAR-3 was 40-fold higher than that of racemic C75, and 2- and 38-fold higher than that of the racemic mixture and its opposite enantiomer, respectively. This cytotoxic effect on the OVCAR-3 cell line involves mechanisms that reduce mitochondrial respiratory capacity and ATP production, DDIT4/REDD1 upregulation, mTOR activity inhibition, and caspase-3 activation, resulting in apoptosis. In addition, central and peripheral administration of (+)-UB006 or (-)-UB006 into rats and mice did not affect food intake or body weight. Altogether, our data support the discovery of a new potential anticancer compound (-)-UB006 that has no anorexigenic side effects.

  12. Aldosterone breakthrough during aliskiren, valsartan, and combination (aliskiren + valsartan) therapy.

    PubMed

    Bomback, Andrew S; Rekhtman, Yelena; Klemmer, Philip J; Canetta, Pietro A; Radhakrishnan, Jai; Appel, Gerald B

    2012-01-01

    Aldosterone levels increase in 30%-40% of patients on angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers over the long term. This "aldosterone breakthrough" may carry important clinical consequences given aldosterone's nonepithelial, pro-fibrotic actions. The renin inhibitor, aliskiren, by suppressing the renin-angiotensin-aldosterone system (RAAS) proximally, may limit breakthrough compared to conventional RAAS blockade. This open-label study (NCT01129557) randomized subjects to aliskiren 300 mg daily (A), valsartan 320 mg daily (V), or aliskiren 150 mg + valsartan 160 mg daily (A+V) for 9 months. Eligible subjects had proteinuria >300 mg/day, estimated glomerular filtration rate (eGFR) >45 mL/min/1.73 m(2), and systolic blood pressure (BP) >130 or diastolic BP >80 mm Hg. Serum and 24-hour urine aldosterone (indexed to 24-hour urine Na) were checked before initiation of therapy and at 3, 6, and 9 months. Aldosterone breakthrough was defined as a sustained increase from baseline aldosterone by study end. The study was intended to enroll 120 subjects but was terminated early by the sponsor. We present here the results of 33 subjects who completed the protocol, of which 12 were randomized to A, 11 were randomized to V, and 10 were randomized to A+V. Mean baseline eGFR was 75.5 (±23.3) mL/min/1.73 m(2); baseline proteinuria was 3104 (±2943) mg/day; and baseline BP was 134.7 (±10.5)/84.8 (±8.4) mm Hg. Three (27%) subjects on V, three (25%) subjects on A, and three (30%) subjects on A+V had aldosterone breakthrough. Mean proteinuria reduction was 31% from baseline in all subjects: 30% in subjects with breakthrough vs. 32% in subjects without breakthrough. Mean BP reduction was 11.0/8.8 mm Hg in all subjects: 8.4/6.1 mm Hg in subjects with breakthrough vs. 12.0/9.8 mm Hg in subjects without breakthrough. Aliskiren, alone or in combination with valsartan, did not reduce the incidence of aldosterone breakthrough in subjects with hypertension

  13. Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands.

    PubMed

    Nishimoto, Koshiro; Tomlins, Scott A; Kuick, Rork; Cani, Andi K; Giordano, Thomas J; Hovelson, Daniel H; Liu, Chia-Jen; Sanjanwala, Aalok R; Edwards, Michael A; Gomez-Sanchez, Celso E; Nanba, Kazutaka; Rainey, William E

    2015-08-18

    Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

  14. Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages.

    PubMed

    Zhang, Yumei; Pan, Yu; Bian, Zhixiang; Chen, Peihua; Zhu, Shijian; Gu, Huiyi; Guo, Liping; Hu, Chun

    2016-01-01

    Here, we studied the underlying mechanism of aldosterone (Aldo)-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L) inhibited human umbilical vein endothelial cells (HUVEC) survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18) production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P), an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS) inhibitor PDMP or the ceramide (C6) potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR) antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1) is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.

  15. Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages

    PubMed Central

    Zhang, Yumei; Pan, Yu; Bian, Zhixiang; Chen, Peihua; Zhu, Shijian; Gu, Huiyi; Guo, Liping; Hu, Chun

    2016-01-01

    Here, we studied the underlying mechanism of aldosterone (Aldo)-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L) inhibited human umbilical vein endothelial cells (HUVEC) survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18) production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P), an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS) inhibitor PDMP or the ceramide (C6) potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR) antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1) is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo. PMID:26788916

  16. Characterization of Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitors as Stimulators of Steroidogenesis

    PubMed Central

    Gunosewoyo, Hendra; Midzak, Andrew; Gaisina, Irina N.; Sabath, Emily V.; Fedolak, Allison; Hanania, Taleen; Brunner, Dani; Papadopoulos, Vassilios; Kozikowski, Alan P.

    2013-01-01

    Inhibition of GSK-3β has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3β resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3β inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these GSK-3β inhibitors, in particular analogs 1 and 9, were able to stimulate progesterone production in the MA-10 mouse tumor Leydig cell model of steroidogenesis without any significant toxicity. These two compounds were tested in the SmartCube® behavioral assay and showed anxiolytic-like signatures following daily dose administration (50 mg/kg, i.p.) for 13 days. Taken together, these results support the hypothesis that GSK-3β inhibition could influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo. PMID:23725591

  17. Characterization of maleimide-based glycogen synthase kinase-3 (GSK-3) inhibitors as stimulators of steroidogenesis.

    PubMed

    Gunosewoyo, Hendra; Midzak, Andrew; Gaisina, Irina N; Sabath, Emily V; Fedolak, Allison; Hanania, Taleen; Brunner, Dani; Papadopoulos, Vassilios; Kozikowski, Alan P

    2013-06-27

    Inhibition of GSK-3β has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3β resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3β inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these GSK-3β inhibitors, in particular analogues 1 and 9, were able to stimulate progesterone production in the MA-10 mouse tumor Leydig cell model of steroidogenesis without any significant toxicity. These two compounds were tested in the SmartCube behavioral assay and showed anxiolytic-like signatures following daily dose administration (50 mg/kg, ip) for 13 days. Taken together, these results support the hypothesis that GSK-3β inhibition could influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo.

  18. Fluorinated indazoles as novel selective inhibitors of nitric oxide synthase (NOS): synthesis and biological evaluation.

    PubMed

    Claramunt, Rosa M; López, Concepción; Pérez-Medina, Carlos; Pérez-Torralba, Marta; Elguero, José; Escames, Germaine; Acuña-Castroviejo, Darío

    2009-09-01

    In order to find new compounds with neuroprotective activity and NOS-I/NOS-II selectivity, we have designed, synthesized, and characterized 14 new NOS inhibitors with an indazole structure. The first group corresponds to 4,5,6,7-tetrahydroindazoles (4-8), the second to the N-methyl derivatives (9-12) of 7-nitro-1H-indazole (1) and 3-bromo-7-nitro-1H-indazole (2), and the latter to 4,5,6,7-tetrafluoroindazoles (13-17). Compound 13 (4,5,6,7-tetrafluoro-3-methyl-1H-indazole) inhibited NOS-I by 63% and NOS-II by 83%. Interestingly, compound 16 (4,5,6,7-tetrafluoro-3-perfluorophenyl-1H-indazole) inhibited NOS-II activity by 80%, but it did not affect to NOS-I activity. Structural comparison between these new indazoles further supports the importance of the aromatic indazole skeleton for NOS inhibition and indicate that bulky groups or N-methylation of 1 and 2 diminish their effect on NOS activity. The fluorination of the aromatic ring increased the inhibitory potency and NOS-II selectivity, suggesting that this is a promising strategy for NOS selective inhibitors.

  19. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    PubMed

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  20. Aldosterone-to-Renin Ratio Is Associated With Reduced 24-Hour Heart Rate Variability and QTc Prolongation in Hypertensive Patients.

    PubMed

    Grübler, Martin R; Kienreich, Katharina; Gaksch, Martin; Verheyen, Nicolas; Hartaigh, Bríain Ó; Fahrleitner-Pammer, Astrid; März, Winfried; Schmid, Johannes; Oberreither, Eva-Maria; Wetzel, Julia; Catena, Cristiana; Sechi, Leonardo A; Pieske, Burkert; Tomaschitz, Andreas; Pilz, Stefan

    2016-02-01

    Aldosterone is considered to exert direct effects on the myocardium and the sympathetic nervous system. Both QT time and heart rate (HR) variability (HRV) are considered to be markers of arrhythmic risk and autonomous dysregulation. In this study, we investigated the associations between aldosterone, QT time, and HRV in patients with arterial hypertension.We recruited 477 hypertensive patients (age: 60.2 ± 10.2 years; 52.3% females) with a mean systolic/diastolic 24-hour ambulatory blood pressure monitoring (ABPM) value of 128 ± 12.8/77.1 ± 9.2 mmHg and with a median of 2 (IQR: 1-3) antihypertensive agents. Patients were recruited from the outpatient clinic at the Department of Internal Medicine of the Medical University of Graz, Austria. Blood samples, 24-hour HRV derived from 24-hour blood pressure monitoring (ABPM) and ECG's were obtained. Plasma aldosterone and plasma renin concentrations were measured by means of a radioimmunoassay. Twenty-four-hour urine specimens were collected in parallel with ABPM.Mean QTc was 423.3 ± 42.0 milliseconds for males and 434.7 ± 38.3 milliseconds for females. Mean 24H-HR and 24H-HRV was 71.9 ± 9.8 and 10.0 ± 3.6 bpm, respectively. In linear regression analyses adjusted for age, sex, body mass index, ABPM, and current medication, aldosterone to active renin ratio (AARR) was significantly associated with the QTc interval, a marker for cardiac repolarization abnormalities (mean = 426 ± 42.4 milliseconds; β-coefficient = 0.121; P = 0.03) as well as with the 24-hour heart rate variability a surrogate for autonomic dysfunction (median = 9.67 [IQR = 7.38-12.22 bpm]; β-coefficient = -0.133; P = 0.01).In hypertensive patients, AARR is significantly related to QTc prolongation as well as HRV. Further studies investigating the effects of mineralocorticoid receptor blocker and aldosterone synthase inhibitors on QTc and HRV are warranted.

  1. Selective Nitric Oxide Synthase Inhibitor 7-Nitroindazole Protects against Cocaine-Induced Oxidative Stress in Rat Brain

    PubMed Central

    Vitcheva, Vessela; Simeonova, Rumyana; Kondeva-Burdina, Magdalena; Mitcheva, Mitka

    2015-01-01

    One of the mechanisms involved in the development of addiction, as well as in brain toxicity, is the oxidative stress. The aim of the current study was to investigate the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (nNOS), on cocaine withdrawal and neurotoxicity in male Wistar rats. The animals were divided into four groups: control; group treated with cocaine (15 mg/kg−1, i.p., 7 days); group treated with 7-NI (25 mg/kg−1, i.p., 7 days); and a combination group (7-NI + cocaine). Cocaine repeated treatment resulted in development of physical dependence, judged by withdrawal symptoms (decreased locomotion, increased salivation and breathing rate), accompanied by an increased nNOS activity and oxidative stress. The latter was discerned by an increased formation of malondialdehyde (MDA), depletion of reduced glutathione (GSH) levels, and impairment of the enzymatic antioxidant defense system measured in whole brain. In synaptosomes, isolated from cocaine-treated rats, mitochondrial activity and GSH levels were also decreased. 7-NI administered along with cocaine not only attenuated the withdrawal, due to its nNOS inhibition, but also reversed both the GSH levels and antioxidant enzyme activities near control levels. PMID:26576217

  2. Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS).

    PubMed

    Edfeldt, Fredrik; Evenäs, Johan; Lepistö, Matti; Ward, Alison; Petersen, Jens; Wissler, Lisa; Rohman, Mattias; Sivars, Ulf; Svensson, Karin; Perry, Matthew; Feierberg, Isabella; Zhou, Xiao-Hong; Hansson, Thomas; Narjes, Frank

    2015-06-15

    Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket.

  3. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics.

  4. Lack of tolerance for the anti-dyskinetic effects of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, in rats.

    PubMed

    Novaretti, N; Padovan-Neto, F E; Tumas, V; da-Silva, C A; Del Bel, E A

    2010-11-01

    7-Nitroindazole (7-NI) inhibits neuronal nitric oxide synthase in vivo and reduces l-DOPA-induced dyskinesias in a rat model of parkinsonism. The aim of the present study was to determine if the anti-dyskinetic effect of 7-NI was subject to tolerance after repeated treatment and if this drug could interfere with the priming effect of l-DOPA. Adult male Wistar rats (200-250 g) with unilateral depletion of dopamine in the substantia nigra compacta were treated with l-DOPA (30 mg/kg) for 34 days. On the 1st day, 6 rats received ip saline and 6 received ip 7-NI (30 mg/kg) before l-DOPA. From the 2nd to the 26th day, all rats received l-DOPA daily and, from the 27th to the 34th day, they also received 7-NI before l-DOPA. Animals were evaluated before the drug and 1 h after l-DOPA using an abnormal involuntary movement scale and a stepping test. All rats had a similar initial motor deficit. 7-NI decreased abnormal involuntary movement induced by l-DOPA and the effect was maintained during the experiment before 7-NI, median (interquartile interval), day 26: 16.75 (15.88-17.00); day 28: 0.00 (0.00-9.63); day 29: 13.75 (2.25-15.50); day 30: 0.5 (0.00-6.25); day 31: 4.00 (0.00-7.13), and day 34: 0.5 (0.00-14.63), Friedman followed by Wilcoxon test,vs day 26, P < 0.05;. The response to l-DOPA alone was not modified by the use of 7-NI before the first administration of the drug (l-DOPA vs time interaction, F1,10 = 1.5, NS). The data suggest that tolerance to the anti-dyskinetic effects of a neuronal nitric oxide synthase inhibitor does not develop over a short-term period of repeated administration. These observations open a possible new therapeutic approach to motor complications of chronic l-DOPA therapy in patients with Parkinson's disease.

  5. Molecular Mechanism of Silver Nanoparticles-Induced Human Osteoblast Cell Death: Protective Effect of Inducible Nitric Oxide Synthase Inhibitor

    PubMed Central

    Zielinska, Ewelina; Tukaj, Cecylia; Radomski, Marek Witold; Inkielewicz-Stepniak, Iwona

    2016-01-01

    Background Silver nanoparticles (AgNPs) show strong antibacterial properties, making them excellent candidates to be used in orthopaedic repair and regeneration. However, there are concerns regarding the cytotoxicity of AgNPs and molecular mechanisms underlying AgNPs-induced bone cells toxicity have not been elucidated. Therefore, the aim of our study was to explore mechanisms of AgNPs-induced osteoblast cell death with particular emphasis on the role of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS). Methods and Result Silver nanoparticles used in this study were 18.3±2.6 nm in size, uncoated, spherical, regular shape and their zeta potential was -29.1±2.4 mV as measured by transmission electron microscopy (TEM) and zetasizer. The release of silver (Ag) from AgNPs was measured in cell culture medium by atomic absorption spectroscopy (AAS). The exposure of human osteoblast cells (hFOB 1.19) to AgNPs at concentration of 30 or 60 μg/mL for 24 or 48 hours, respectively resulted in cellular uptake of AgNPs and changes in cell ultrastructure. These changes were associated with apoptosis and necrosis as shown by flow cytometry and lactate dehydrogenase (LDH) assay as well as increased levels of pro-apoptotic Bax and decreased levels of anti-apoptotic Bcl-2 mRNA and protein. Importantly, we have found that AgNPs elevated the levels of nitric oxide (NO) with concomitant upregulation of inducible nitric oxide synthase (iNOS) mRNA and protein. A significant positive correlation was observed between the concentration of AgNPs and iNOS at protein and mRNA level (r = 0.837, r = 0.721, respectively; p<0.001). Finally, preincubation of osteoblast cells with N-iminoethyl-l-lysine (L-NIL), a selective iNOS inhibitor, as well as treating cells with iNOS small interfering RNAs (siRNA) significantly attenuated AgNPs-induced apoptosis and necrosis. Moreover, we have found that AgNPs-induced cells death is not related to Ag dissolution is cell culture medium

  6. Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic.

    PubMed

    Balachandran, Naresh; To, Frederick; Berti, Paul J

    2017-01-31

    3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with Ki = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KM,MnKM,PEPKM,E4P/kcat) versus log(Ki), revealed a slope, m, of 0.34, with r(2) = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.

  7. Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

    PubMed

    Araujo, Carolina Morais; Hermidorff, Milla Marques; Amancio, Gabriela de Cassia Sousa; Lemos, Denise da Silveira; Silva, Marcelo Estáquio; de Assis, Leonardo Vinícius Monteiro; Isoldi, Mauro César

    2016-10-01

    Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.

  8. Secretin is involved in sodium conservation through the renin-angiotensin-aldosterone system.

    PubMed

    Bai, Juan; Chow, Billy K C

    2017-04-01

    Secretin (SCT) and its receptor (SCTR) are important in fluid regulation at multiple levels via the modulation of expression and translocation of renal aquaporin 2 and functions of central angiotensin II (ANGII). The functional interaction of SCT with peripheral ANGII, however, remains unknown. As the ANGII-aldosterone axis dominates the regulation of renal epithelial sodium channel (ENaC) function, we therefore tested whether SCT/SCTR can regulate sodium homeostasis via the renin-angiotensin-aldosterone system. SCTR-knockout (SCTR(-/-)) mice showed impaired aldosterone synthase (CYP11B2) expression and, consequently, aldosterone release upon intraperitoneal injection of ANGII. Endogenous ANGII production induced by dietary sodium restriction was higher in SCTR(-/-) than in C57BL/6N [wild-type (WT)] mice, but CYP11B2 and aldosterone synthesis were not elevated. Reduced accumulation of cholesteryl ester-the precursor of aldosterone-was observed in adrenal glands of SCTR(-/-) mice that were fed a low-sodium diet. Absence of SCTR resulted in elevated basal transcript levels of adrenal CYP11B2 and renal ENaCs. Although transcript and protein levels of ENaCs were similar in WT and SCTR(-/-) mice under sodium restriction, ENaCs in SCTR(-/-) mice were less sensitive to amiloride hydrochloride. In summary, the SCT/SCTR axis is involved in aldosterone precursor uptake, and the knockout of SCTR results in defective aldosterone biosynthesis/release and altered sensitivity of ENaCs to amiloride.-Bai, J., Chow, B. K. C. Secretin is involved in sodium conservation through the renin-angiotensin-aldosterone system.

  9. Primary aldosteronism and pregnancy.

    PubMed

    Landau, Ester; Amar, Laurence

    2016-06-01

    Hypertension (HT) is a complication of 8% of all pregnancies and 10% of HT cases are due to primary aldosteronism (PA). There is very little data on PA and pregnancy. Given the changes in the renin angiotensin system during pregnancy, the diagnosis of PA is difficult to establish during gestation. It may be suspected in hypertensive patients with hypokalemia. A comprehensive literature review identified reports covering 40 pregnancies in patients suffering from PA. Analysis of these cases shows them to be high-risk pregnancies leading to maternal and fetal complications. Pregnancy must be programmed, and if the patient has a unilateral form of PA, adrenalectomy should be performed prior to conception. It is customary to stop spironolactone prior to conception and introduce antihypertensive drugs that present no risk of teratogenicity. When conventional antihypertensive drugs used during pregnancy fail to control high blood pressure, diuretics, including potassium-sparing diuretics may be prescribed. Adrenalectomy can be considered during the second trimester of pregnancy exclusively in cases of refractory hypertension. A European retrospective study is currently underway to collect a larger number of cases.

  10. Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells

    PubMed Central

    Konopinski, Ryszard; Krishnan, Manickam; Roman, Linda; Bera, Alakesh; Hongying, Zheng; Habib, Samy L.; Mohan, Sumathy

    2015-01-01

    Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-κB kinase-β (IKKβ)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKKβ on Hsp90. Interestingly, IKKβ binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKKβ to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKKβ. The pathophysiological relevance of the IKKβ-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2Akita in vivo model. Our study further defines the preferential involvement of α- vs. β-isoforms of Hsp90 in the IKKβ-eNOS-Hsp90 interaction, even though both Hsp90α and Hsp90β stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKKβ within the cell system that regulates NO production, but they also confirm that the IKKβ-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes. PMID:25652452

  11. Aldosterone and the vascular system.

    PubMed

    Cachofeiro, Victoria; Miana, Maria; de Las Heras, Natalia; Martín-Fernández, Beatriz; Ballesteros, Sandra; Fernández-Tresguerres, Jesús; Lahera, Vicente

    2008-04-01

    Aldosterone can act in different tissues exerting physiological and pathological effects. At the vascular level, aldosterone affects endothelial function since administration of aldosterone impaired endothelium-dependent relaxations. In addition, the administration of mineralocorticoid receptor antagonists ameliorate relaxation to acetylcholine in models of both hypertension and atherosclerosis and in patients with heart failure. A reduction in nitric oxide levels seems to be the main mechanism underlying this effect due to a reduction in its production as well as an increase in its degradation by reactive oxygen species. Aldosterone is a pro-inflammatory factor that can participate in the vascular inflammatory process associated with different pathologies including hypertension through activation of the NFkappaB system, which mediates the vascular production of different cytokines. This mineralocorticoid also participates in the vascular remodeling observed in hypertensive rats since the administration of eplerenone improved the media-to-lumen ratio in these animals. This effect seems to be due to an increase in extracellular matrix. In summary, aldosterone through mineralocorticoid receptors can participate in the vascular damage associated with different pathologies including hypertension through its prooxidant, pro-inflammatory and profibrotic effects that triggered endothelial dysfunction, an inflammatory process and vascular remodeling.

  12. CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

    PubMed Central

    Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

    2015-01-01

    Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

  13. The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats.

    PubMed

    Gocmez, Semil Selcen; Yazir, Yusufhan; Sahin, Deniz; Karadenizli, Sabriye; Utkan, Tijen

    2015-04-01

    Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats.

  14. Use of bacterial surrogates as a tool to explore antimalarial drug interaction: Synergism between inhibitors of malarial dihydrofolate reductase and dihydropteroate synthase.

    PubMed

    Talawanich, Yuwadee; Kamchonwongpaisan, Sumalee; Sirawaraporn, Worachart; Yuthavong, Yongyuth

    2015-09-01

    Interaction between antimalarial drugs is important in determining the outcome of chemotherapy using drug combinations. Inhibitors of dihydrofolate reductase (DHFR) such as pyrimethamine and of dihydropteroate synthase (DHPS) such as sulfa drugs are known to have synergistic interactions. However, studies of the synergism are complicated by the fact that the malaria parasite can also salvage exogenous folates, and the salvage may also be affected by the drugs. It is desirable to have a convenient system to study interaction of DHFR and DHPS inhibitors without such complications. Here, we describe the use of Escherichia coli transformed with malarial DHFR and DHPS, while its own corresponding genes have been inactivated by optimal concentration of trimethoprim and genetic knockout, respectively, to study the interaction of the inhibitors. Marked synergistic effects are observed for all combinations of pyrimethamine and sulfa inhibitors in the presence of trimethoprim. At 0.05μM trimethoprim, sum of fractional inhibitory concentrations, ΣFIC of pyrimethamine with sulfadoxine, pyrimethamine with sulfathiazole, pyrimethamine with sulfamethoxazole, and pyrimethamine with dapsone are in the range of 0.24-0.41. These results show synergism between inhibitors of the two enzymes even in the absence of folate transport and uptake. This bacterial surrogate system should be useful as a tool for assessing the interactions of drug combinations between the DHFR and DHPS inhibitors.

  15. Efficacy of caspofungin, a 1,3-β-D-glucan synthase inhibitor, on Pneumocystis carinii pneumonia in rats.

    PubMed

    Sun, Peipei; Tong, Zhaohui

    2014-11-01

    Pneumocystis carinii pneumonia (PcP) is a common and potentially fatal opportunistic infection in immunosuppressed patients, and the standard trimethoprim-sulfamethoxazole (TMP-SMZ) treatment has serious side effects. The cell wall of the causative fungal pathogen is enriched in 1-3-β-D-glucan, providing an alternative therapeutic target. We directly compared the efficacy of the 1,3-β-D-glucan synthase inhibitor caspofungin to TMP-SMZ for promoting survival and reducing lung cyst number during the early phase of treatment in a rat model of PcP. Rats were immunosuppressed using dexamethasone for 8 weeks and PcP infection confirmed in test animals by lung print smear. The remaining rats were randomly divided into three control groups, a baseline group and two observed for 7 or 14 days, two caspofungin groups treated intravenously for 7 or 14 days (1 mg/kg/d), and 2 TMP-SMZ positive control groups treated by oral gavage for 7 or 14 days (300 mg/kg/d). Mortality was markedly reduced by both caspofungin and TMP-SMZ after 14 days (caspofungin: 20.0%, TMP-SMZ: 13.3%, Control: 40.0%). Body weight gain in caspofungin-treated rats after 7 (3.04 ± 3.54%) and 14 (4.27 ± 2.79%) days was similar to that in TMP-SMZ-treated rats (3.35 ± 1.88% and 5.85 ± 2.78%, respectively), whereas untreated controls showed weight loss. Lung weight to body weight ratio, and mean cyst number per 50 microscopic fields were significantly lower (all P < 0.05) in caspofungin-treated rats than untreated controls at both 7 and 14 days, and similar to those in the TMP-SMZ-treated rats (all P > 0.05 vs. caspofungin). Caspofungin exhibited similar efficacy to TMP-SMZ for enhancing survival and reducing lung edema and cyst load in a rat model of PcP, suggesting potential clinical utility against PcP.

  16. Aldosterone-induced brain MAPK signaling and sympathetic excitation are angiotensin II type-1 receptor dependent.

    PubMed

    Zhang, Zhi-Hua; Yu, Yang; Wei, Shun-Guang; Felder, Robert B

    2012-02-01

    Angiotensin II (ANG II)-induced mitogen-activated protein kinase (MAPK) signaling upregulates angiotensin II type-1 receptors (AT(1)R) in hypothalamic paraventricular nucleus (PVN) and contributes to AT(1)R-mediated sympathetic excitation in heart failure. Aldosterone has similar effects to increase AT(1)R expression in the PVN and sympathetic drive. The present study was undertaken to determine whether aldosterone also activates the sympathetic nervous system via MAPK signaling and, if so, whether its effect is independent of ANG II and AT(1)R. In anesthetized rats, a 4-h intravenous infusion of aldosterone induced increases (P < 0.05) in phosphorylated (p-) p44/42 MAPK in PVN, PVN neuronal excitation, renal sympathetic nerve activity (RSNA), mean blood pressure (MBP), and heart rate (HR). Intracerebroventricular or bilateral PVN microinjection of the p44/42 MAPK inhibitor PD-98059 reduced the aldosterone-induced RSNA, HR, and MBP responses. Intracerebroventricular pretreatment (5 days earlier) with pooled small interfering RNAs targeting p44/42 MAPK reduced total and p-p44/42 MAPK, aldosterone-induced c-Fos expression in the PVN, and the aldosterone-induced increases in RSNA, HR, and MBP. Intracerebroventricular infusion of either the mineralocorticoid receptor antagonist RU-28318 or the AT(1)R antagonist losartan blocked aldosterone-induced phosphorylation of p44/42 MAPK and prevented the increases in RSNA, HR, and MBP. These data suggest that aldosterone-induced sympathetic excitation depends upon that AT(1)R-induced MAPK signaling in the brain. The short time course of this interaction suggests a nongenomic mechanism, perhaps via an aldosterone-induced transactivation of the AT(1)R as described in peripheral tissues.

  17. HUMAN INTERVENTIONS TO CHARACTERIZE NOVEL RELATIONSHIPS BETWEEN THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM AND PARATHYROID HORMONE

    PubMed Central

    Brown, Jenifer M.; Williams, Jonathan S.; Luther, James M.; Garg, Rajesh; Garza, Amanda E.; Pojoga, Luminita H.; Ruan, Daniel T.; Williams, Gordon H.; Adler, Gail K.; Vaidya, Anand

    2014-01-01

    Observational studies in primary hyperaldosteronism (PA) suggest a positive relationship between aldosterone and parathyroid hormone (PTH); however, interventions to better characterize the physiologic relationship between the renin-angiotensin-aldosterone system (RAAS) and PTH are needed. We evaluated the effect of individual RAAS components on PTH using 4 interventions in humans without PA. PTH was measured before and after: Study 1) low-dose angiotensin II [AngII] infusion (1 ng/kg/min) and captopril administration (25 mg × 1); Study 2) high-dose AngII infusion (3 ng/kg/min); Study 3) blinded crossover randomization to aldosterone infusion (0.7 µg/kg/hr) and vehicle; and Study 4) blinded randomization to spironolactone (50mg/daily) or placebo for 6 weeks. Infusion of AngII at 1 ng/kg/min acutely increased aldosterone (+148%) and PTH (+10.3%), while AngII at 3 ng/kg/min induced larger incremental changes in aldosterone (+241%) and PTH (+36%) (P<0.01). Captopril acutely decreased aldosterone (−12%) and PTH (−9.7%) (P<0.01). In contrast, aldosterone infusion robustly raised serum aldosterone (+892%) without modifying PTH. However, spironolactone therapy over 6 weeks modestly lowered PTH when compared to placebo (P<0.05). In vitro studies revealed the presence of AngII type I and mineralocorticoid receptor mRNA and protein expression in normal and adenomatous human parathyroid tissues. We observed novel pleiotropic relationships between RAAS components and the regulation of PTH in individuals without PA: the acute modulation of PTH by the RAAS appears to be mediated by AngII, whereas the long-term influence of the RAAS on PTH may involve aldosterone. Future studies to evaluate the impact of RAAS inhibitors in treating PTH-mediated disorders are warranted. PMID:24191286

  18. Fatty acids are potential endogenous regulators of aldosterone secretion.

    PubMed

    Goodfriend, T L; Ball, D L; Elliott, M E; Morrison, A R; Evenson, M A

    1991-05-01

    Adrenal glomerulosa cells washed with delipidated albumin produced increased amounts of aldosterone in response to angiotensin-II (AII) or (Bu)2cAMP. Albumin treatment also increased binding of 125I-labeled AII to high affinity binding sites on adrenal cells. Lipid extracts of albumin solutions that were used to wash cells inhibited AII binding and aldosterone responses by washed glomerulosa cells. Chromatographic fractionation and mass spectroscopic analysis indicated that the inhibitors removed from cells by albumin were long chain fatty acids. Exogenous fatty acids not only inhibited AII binding, but they inhibited basal aldosterone production and increments in aldosterone caused by AII or dbcAMP, suggesting an effect on postreceptor steps in aldosteronogenesis. The most potent and most abundant fatty acids removed from adrenal cells were oleic, linoleic, and arachidonic. These fatty acids inhibited at micromolar concentrations in the absence of albumin and at somewhat higher concentrations in its presence. Cells that had been washed, then inhibited by exogenous oleic acid in vitro, were restored to their enhanced responsiveness by a second albumin wash, making it unlikely that cell damage is the mechanism of inhibition by fatty acids. Responses of fasciculata cells were not potentiated by albumin washes, and cortisol production was less sensitive than aldosterone production to exogenous fatty acids. Binding of ANP to glomerulosa cells was not affected by albumin or fatty acids. These results combined with clinical correlations make it plausible that unesterified fatty acids are naturally occurring regulators of the adrenal glomerulosa. Insulin's ability to lower plasma levels of fatty acids may be one way that it causes sodium retention.

  19. Sensitivity of Aspergillus nidulans to the cellulose synthase inhibitor dichlobenil: insights from wall-related genes' expression and ultrastructural hyphal morphologies.

    PubMed

    Guerriero, Gea; Silvestrini, Lucia; Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

    2013-01-01

    The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes' expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.

  20. Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

    PubMed Central

    Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

    2013-01-01

    The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis. PMID:24312197

  1. Molecular docking and molecular dynamics simulation study of inositol phosphorylceramide synthaseinhibitor complex in leishmaniasis: Insight into the structure based drug design

    PubMed Central

    Mandlik, Vineetha; Singh, Shailza

    2016-01-01

    Inositol phosphorylceramide synthase (IPCS) has emerged as an important, interesting and attractive target in the sphingolipid metabolism of Leishmania. IPCS catalyzes the conversion of ceramide to IPC which forms the most predominant sphingolipid in Leishmania. IPCS has no mammalian equivalent and also plays an important role in maintaining the infectivity and viability of the parasite. The present study explores the possibility of targeting IPCS; development of suitable inhibitors for the same would serve as a treatment strategy for the infectious disease leishmaniasis. Five coumarin derivatives were developed as inhibitors of IPCS protein. Molecular dynamics simulations of the complexes of IPCS with these inhibitors were performed which provided insights into the binding modes of the inhibitors. In vitro screening of the top three compounds has resulted in the identification of one of the compounds (compound 3) which shows little cytotoxic effects. This compound therefore represents a good starting point for further in vivo experimentation and could possibly serve as an important drug candidate for the treatment of leishmaniasis. PMID:27853511

  2. Microalbuminuria and hypertension in pregnancy: role of aldosterone and inflammation.

    PubMed

    Armanini, Decio; Ambrosini, Guido; Sabbadin, Chiara; Donà, Gabriella; Clari, Giulio; Bordin, Luciana

    2013-09-01

    Women with a history of hypertension in pregnancy are at increased risk of microalbuminuria later in life. Microalbuminuria is a marker of kidney dysfunction frequently related to an inflammatory event. Pregnancy is a dynamic process characterized by immune tolerance, angiogenesis, and hormonal regulation. Menstruation and pregnancy are associated with a physiological inflammation, which is altered in preeclampsia and probably in other hypertensive situations of pregnancy. An imbalance between pro-oxidant factors and the ability to scavenge these factors produces oxidative stress, which has been evaluated in many cells, but leukocytes are the main source of inflammatory cytokines and experimental and clinical evidence support a possible role of aldosterone as a mediator of placental and renal damage mediated by growth factors, reactive oxygen species, and cytokines. Angiotensin-converting enzyme inhibitors and aldosterone receptor blockers are frequently effective in reducing the risk of progression of cardiovascular and renal disease.

  3. An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.

    PubMed

    Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang ping; Lee, Keun Woo

    2013-01-01

    Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS) and human dihydrofolate reductase (hDHFR). These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs.

  4. An Innovative Strategy for Dual Inhibitor Design and Its Application in Dual Inhibition of Human Thymidylate Synthase and Dihydrofolate Reductase Enzymes

    PubMed Central

    Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang ping; Lee, Keun Woo

    2013-01-01

    Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS) and human dihydrofolate reductase (hDHFR). These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs. PMID:23577115

  5. Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson's disease.

    PubMed

    Bortolanza, Mariza; Cavalcanti-Kiwiatkoski, Roberta; Padovan-Neto, Fernando E; da-Silva, Célia Aparecida; Mitkovski, Miso; Raisman-Vozari, Rita; Del-Bel, Elaine

    2015-01-01

    l-3, 4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of L-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with L-DOPA (30 mg/kg plus benserazide 7.5 mg/kg, for 21 days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30 mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with L-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. L-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of L-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinson's disease.

  6. Aldosterone decreases glucose-stimulated insulin secretion in vivo in mice and in murine islets

    PubMed Central

    Luo, P.; Kreger, M. T.; Brissova, M.; Dai, C.; Whitfield, T. T.; Kim, H. S.; Wasserman, D. H.; Powers, A. C.; Brown, N. J.

    2011-01-01

    Aims/hypothesis Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro. Methods We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2]−/−)and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As−/− mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line. Results Fasting glucose concentrations were reduced in As−/−mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As−/− than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As−/− mice even during high sodium intake. There was no difference in insulin sensitivity between As−/− and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol. Conclusions/interpretation We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess. PMID:21519965

  7. Role of calcium in effects of atrial natriuretic peptide on aldosterone production in adrenal glomerulosa cells

    SciTech Connect

    Chartier, L.; Schiffrin, E.L.

    1987-04-01

    Atrial natriuretic peptide (ANP) inhibits the stimulation of aldosterone secretion by isolated adrenal glomerulosa cells produced by angiotensin II (ANG II), ACTH, and potassium. The effect of ANP on the dose-response curve of aldosterone stimulated by ANG II, ACTH, and potassium on isolated rat adrenal glomerulosa cells was studied. In the presence of ANP the maximal response of aldosterone output stimulated by ANG II or potassium decreased and the half-maximum (EC/sub 50/) of the response to ACTH was displaced to the right. Because these effects resemble those of calcium-channel blockers, the authors investigated the effect of different concentrations of nifedipine, a dihydropyridine calcium-channel blocker, on the dose-response curve of aldosterone stimulated by ANG II, ACTH, and potassium. Nifedipine produced effects similar to ANP. The maximal response of aldosterone stimulated by ANG II and potassium was decreased and the dose-response curve to ACTH was displaced to the right. ANP decreased the maximal response of aldosterone to the dihydropyridine derivative BAY K8644, a calcium-channel activator, without change in its EC/sub 50/. In contrast, nifedipine displaced the dose-response curve to BAY K8644 to the right as expected of a competitive inhibitor. The effect of ANP and nifedipine on basal and stimulated /sup 45/Ca influx into isolated rat adrenal glomerulosa cells was studied. ANP may act on the rat adrenal glomerulosa cells at least in part by interference with calcium entry.

  8. Investigating the efficacy of pamidronate, a chemical inhibitor of farnesyl pyrophosphate synthase, in the inhibition of influenza virus infection in vitro and in vivo.

    PubMed

    Tan, Kai Sen; Ng, Wai Chii; Seet, Ju Ee; Olfat, Farzad; Engelward, Bevin P; Chow, Vincent T K

    2014-01-01

    Influenza A virus has caused significant pandemics in the past decades, including the H1N1‑2009 pandemic. Viperin is an interferon‑inducible protein that acts as a broad‑spectrum antiviral protein via the inhibition of farnesyl pyrophosphate synthase (FPPS). To mimic this activity of viperin, the present study investigated the effectiveness of a commercially available FPPS inhibitor (pamidronate) as an inhibitor of influenza virus infection in vitro and in vivo. HeLaM cells were treated with pamidronate to determine its effect on the replication of influenza virus A/H1N1/WSN/1933. C57BL/6 mice were also subjected to intratracheal pamidronate treatment regimes prior to and following lethal influenza challenge. Treatment with the FPPS inhibitor in vitro resulted in a considerable reduction in the viral titer of ~1 log and diminished lipid raft formation without cellular toxicity, thus mimicking the antiviral effect of viperin. However, pamidronate lacked efficacy in vivo and was associated with increased pulmonary damage, most likely due to the complexity of drug‑host interactions in the infected mice. Further studies are warranted on pamidronate treatment in other infectious diseases that are more susceptible to FPPS inhibition.

  9. Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.

    PubMed

    Li, Yunfang; Xu, Jia; Chen, Yu; Mei, Zhinan; Xiao, Yuxiu

    2015-12-18

    Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.

  10. Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization

    PubMed Central

    Boppana, Nithin B.; Kodiha, Mohamed; Stochaj, Ursula; Lin, Ho-sheng; Haimovitz-Friedman, Adrianna; Bielawska, Alicja; Bielawski, Jacek; Divine, George W.; Boyd, John A.; Korbelik, Mladen; Separovic, Duska

    2014-01-01

    The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent. PMID:25266739

  11. Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization.

    PubMed

    Boppana, Nithin B; Kodiha, Mohamed; Stochaj, Ursula; Lin, Ho-sheng; Haimovitz-Friedman, Adriana; Bielawska, Alicja; Bielawski, Jacek; Divine, George W; Boyd, John A; Korbelik, Mladen; Separovic, Duska

    2014-11-01

    The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.

  12. In-silico docking based design and synthesis of [1H,3H] imidazo[4,5-b] pyridines as lumazine synthase inhibitors for their effective antimicrobial activity

    PubMed Central

    Harer, Sunil L.; Bhatia, Manish S.

    2014-01-01

    Purpose: The imidazopyridine moiety is important pharmacophore that has proven to be useful for a number of biologically relevant targets, also reported to display antibacterial, antifungal, antiviral properties. Riboflavin biosynthesis involving catalytic step of Lumazine synthase is absent in animals and human, but present in microorganism, one of marked advantage of this study. Still, this path is not exploited as antiinfective target. Here, we proposed different interactions between [1H,3H] imidazo[4,5-b] pyridine test ligands and target protein Lumazine synthase (protein Data Bank 2C92), one-step synthesis of title compounds and further evaluation of them for in vitro antimicrobial activity. Materials and Methods: Active pocket of the target protein involved in the interaction with the test ligands molecules was found using Biopredicta tools in VLifeMDS 4.3 Suite. In-silico docking suggests H-bonding, hydrophobic interaction, charge interaction, aromatic interaction, and Vanderwaal forces responsible for stabilizing enzyme-inhibitor complex. Disc diffusion assay method was used for in vitro antimicrobial screening. Results and Discussion: Investigation of possible interaction between test ligands and target lumazine synthase of Mycobacterium tuberculosis suggested 1i and 2f as best fit candidates showing hydrogen bonding, hydrophobic, aromatic and Vanderwaal's forces. Among all derivatives 1g, 1j, 1k, 1l, 2a, 2c, 2d, 2e, 2h, and 2j exhibited potent activities against bacteria and fungi compared to the standard Ciprofloxacin and Fluconazole, respectively. The superiority of 1H imidazo [4,5-b] pyridine compounds having R’ = Cl >No2 > NH2 at the phenyl/aliphatic moiety resident on the imidazopyridine, whereas leading 3H imidazo[4,5-b] pyridine compounds containing R/Ar = Cl > No2 > NH2> OCH3 substituents on the 2nd position of imidazole. PMID:25400412

  13. Aldosterone alters the participation of endothelial factors in noradrenaline vasoconstriction differently in resistance arteries from normotensive and hypertensive rats.

    PubMed

    Xavier, Fabiano E; Blanco-Rivero, Javier; Avendaño, María Soledad; Sastre, Esther; Yela, Rubén; Velázquez, Kyra; Salaíces, Mercedes; Balfagón, Gloria

    2011-03-11

    This study analyzed the effect of aldosterone (0.05mg/kg per day, 3 weeks) on vasoconstriction induced by noradrenaline in mesenteric resistance arteries from WKY rats and SHR. Contraction to noradrenaline was measured in mesenteric resistance arteries from untreated and aldosterone-treatedrats from both strains. Participation of nitric oxide (NO), superoxide anions, thromboxane A(2) (TxA(2)) and prostacyclin in this response was determined. 6-keto-prostaglandin (PG)F1alpha and thromboxane B(2) (TxB(2)) releases were determined by enzyme immunoassay. NO and superoxide anion release were also determined by fluorescence and chemiluminiscence, respectively. Aldosterone did not modify noradrenaline-induced contraction in either strain. In mesenteric resistance arteries from both aldosterone-treated groups, endothelium removal or preincubation with NO synthesis inhibitor L-NAME increased the noradrenaline-induced contraction, while incubation with the superoxide anion scavenger tempol decreased it. Preincubation with either the COX-1/2 or COX-2 inhibitor (indomethacin and NS-398, respectively) decreased the noradrenaline contraction in aldosterone-treated animals, while this response was not modified by COX-1 inhibitor SC-560. TxA(2) synthesis inhibitor (furegrelate), or TxA2 receptor antagonist (SQ 29 548) also decreased the noradrenaline contraction in aldosterone-treated animals. In untreated SHR, but not WKY rats, this response was increased by L-NAME, and reduced by tempol, indomethacin, NS-398 or SQ 29 548. Aldosterone treatment did not modify NO or TxB(2) release, but it did increase superoxide anion and 6-keto-PGF(1alpha) release in mesenteric resistance arteries from both strains. In conclusion, chronic aldosterone treatment reduces smooth muscle contraction to alpha-adrenergic stimuli, producing a new balance in the release of endothelium-derived prostanoids and NO.

  14. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    SciTech Connect

    Petri, Marcelo H.; Tellier, Céline; Michiels, Carine; Ellertsen, Ingvill; Dogné, Jean-Michel; Bäck, Magnus

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  15. Discovery of a highly selective glycogen synthase kinase-3 inhibitor (PF-04802367) that modulates tau phosphorylation in brain: Translation for PET neuroimaging

    PubMed Central

    Liang, Steven H.; Chen, Jinshan Michael; Normandin, Marc D.; Chang, Jeanne S.; Chang, George C.; Taylor, Christine K.; Trapa, Patrick; Plummer, Mark S.; Para, Kimberly S.; Conn, Edward L.; Lopresti-Morrow, Lori; Lanyon, Lorraine F.; Cook, James M.; Richter, Karl E. G.; Nolan, Charlie E.; Schachter, Joel B.; Janat, Fouad; Che, Ye; Shanmugasundaram, Veerabahu; Lefker, Bruce A.; Enerson, Bradley E.; Livni, Elijahu; Wang, Lu; Guehl, Nicolas; Patnaik, Debasis; Wagner, Florence F.; Perlis, Roy; Holson, Edward B.; Haggarty, Stephen J.; Fakhri, Georges El

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology and neurology. We have identified N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. We demonstrated its efficacy in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A 11C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding. PMID:27355874

  16. 1-(Fluoroalkylidene)-1,1-bisphosphonic Acids are Potent and Selective Inhibitors of the Enzymatic Activity of Toxoplasma gondii Farnesyl Pyrophosphate Synthase

    PubMed Central

    Szajnman, Sergio H.; Rosso, Valeria S.; Malayil, Leena; Smith, Alyssa; Moreno, Silvia N. J.; Docampo, Roberto

    2012-01-01

    α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease and against Toxoplasma gondii, the responsible agent of toxoplasmosis and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) has proven to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range exhibiting an IC50 of 30 nM. This compound was two-fold more potent than risedronate (IC50 = 74 nM) taken as a positive control. This enzymatic activity was associated to a strong cell growth inhibition against tachyzoites of T. gondii having an IC50 value of 2.7 μM. PMID:22215028

  17. Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress.

    PubMed

    Moujalled, Diane; James, Janine L; Parker, Sarah J; Lidgerwood, Grace E; Duncan, Clare; Meyerowitz, Jodi; Nonaka, Takashi; Hasegawa, Masato; Kanninen, Katja M; Grubman, Alexandra; Liddell, Jeffrey R; Crouch, Peter J; White, Anthony R

    2013-01-01

    Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219-414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities

  18. Effects of selective and non-selective inhibitors of nitric oxide synthase on morphine- and endomorphin-1-induced analgesia in acute and neuropathic pain in rats.

    PubMed

    Makuch, Wioletta; Mika, Joanna; Rojewska, Ewelina; Zychowska, Magdalena; Przewlocka, Barbara

    2013-12-01

    Nitric oxide (NO) has been reported to be involved in the mechanisms of pain generation throughout the nervous system. We examined the effects of intrathecally (i.t.) administered nitric oxide synthase (NOS) inhibitors on the antinociceptive effects of morphine and endomorphin-1 during acute pain and in chronic constriction injury (CCI)-exposed rats. We used N(G)-nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor; 7-nitroindazole (7-NI) or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM), selective inhibitors of neuronal NOS (NOS1); and 1400W dihydrochloride, a selective inhibitor of inducible NOS (NOS2). Morphine (0.5-2.5 μg) and endomorphin-1 (2.5-20 μg) in acute pain and morphine (10-40 μg) and endomorphin-1 (5-20 μg) after CCI-injury were combined with NOS inhibitors. For acute pain, the ED50 for endomorphin-1 (7.1 μg) was higher than that of morphine (1.3 μg) in the tail-flick test. For neuropathic pain, the ED50 value for morphine was much higher (43.2 μg) than that of endomorphin-1 (9.2 μg) in von Frey test. NOS inhibitors slightly influenced pain thresholds in both pain models. Moreover, in neuropathic pain, the effects of morphine were more potentiated by L-NAME, TRIM, 7-NI and 1400W (12×, 8.6×, 4.1× and 5.3×, respectively) than were the effects of endomorphin-1 (2.7×, 4.3×, 3.4× and 2.1×, respectively) in the von Frey test. Minocycline which is known to enhance the efficiency of morphine in neuropathic pain, decreased the mRNA expression of NOS1 in the DRG and NOS2 and C1q in the spinal cord after CCI. Both NOS2 and IBA-1 protein levels in the spinal cord and NOS1, NOS2 and IBA1 protein levels in DRG decreased after minocycline administration. In conclusion, our results provide evidence that both neuronal and non-neuronal NOS/NO pathways contribute to the behavioural pain responses evoked by nerve injury. The NOS inhibitors regardless of the type of pain enhanced morphine antinociception and, to a lesser extent, altered the

  19. Suppression of Aldosterone Secretion After Recumbent Saline Infusion Does Not Exclude Lateralized Primary Aldosteronism.

    PubMed

    Cornu, Erika; Steichen, Olivier; Nogueira-Silva, Luis; Küpers, Elselien; Pagny, Jean-Yves; Grataloup, Christine; Baron, Stéphanie; Zinzindohoue, Franck; Plouin, Pierre-François; Amar, Laurence

    2016-10-01

    Guidelines recommend suppression tests such as the saline infusion test (SIT) to ascertain the diagnosis of primary aldosteronism (PA) in patients with a high aldosterone:renin ratio. However, suppression tests have only been evaluated in small retrospective series, and some experts consider that they are not helpful for the diagnosis of PA. In this study, we evaluated whether low post-SIT aldosterone concentrations do exclude lateralized PA. Between February 2009 and December 2013, 199 patients diagnosed with PA on the basis of 2 elevated aldosterone:renin ratio results and a high basal plasma or urinary aldosterone level or high post-SIT aldosterone level had a selective adrenal venous sampling. We used a selectivity index of 2 and a lateralization index of 4 to interpret the adrenal venous sampling results. Baseline characteristics of the patients were the following (percent or median): men 63%, 48 years old, office blood pressure 142/88 mm Hg, serum potassium 3.4 mmol/L, aldosterone:renin ratio 113 pmol/mU, plasma aldosterone concentration 588 pmol/L. The proportion of patients with lateralized adrenal venous sampling was 12 of 41 (29%) among those with post-SIT aldosterone <139 pmol/L (5 ng/dL) and 38 of 104 (37%) among those with post-SIT aldosterone <277 pmol/L (10 ng/dL). Post-SIT aldosterone levels were not associated with the blood pressure outcome of adrenalectomy. A low post-SIT aldosterone level cannot rule out lateralized PA, even with a low threshold (139 pmol/L). Adrenal venous sampling should be considered for patients who are eligible for surgery with elevated basal aldosterone levels even if they have low aldosterone concentrations after recumbent saline suppression testing.

  20. Minimal Pharmacophoric Elements and Fragment Hopping, an Approach Directed at Molecular Diversity and Isozyme Selectivity. Design of Selective Neuronal Nitric Oxide Synthase Inhibitors

    PubMed Central

    Ji, Haitao; Stanton, Benjamin Z.; Igarashi, Jotaro; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the toxicophore library, and the side chain library. These libraries are employed to generate focused fragment libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase (nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore, a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de novo design, has been established as a new type of fragment-based inhibitor design. As an open system, the newly established approach efficiently incorporates the concept of early “ADME/Tox” considerations and provides a basic platform for medicinal chemistry-driven efforts. PMID:18321097

  1. Interactions between inducible isoforms of nitric oxide synthase and cyclo-oxygenase in vivo: investigations using the selective inhibitors, 1400W and celecoxib

    PubMed Central

    Hamilton, Lorna C; Warner, Timothy D

    1998-01-01

    Exposure of tissues to endotoxin (LPS) and/or cytokines leads to the induction of both inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). It has previously been reported that there is `cross-talk' between these two systems. However, such previous studies have been limited by the availability of highly selective inhibitors. Here we have investigated the interactions between iNOS and COX-2 in vivo using 1400W, an iNOS-selective inhibitor, and celecoxib, a COX-2-selective inhibitor.Infusion of LPS to rats for 6 h caused a time-dependent increase in the plasma concentrations of 6 keto-prostaglandin F1α (6 keto-PGF1α) and nitrite/nitrate (NO2/NO3), consistent with the induction of iNOS and COX-2. Bolus injection of arachidonic acid (AA) at t=6 h resulted in a further increase of circulating levels of 6 keto-PGF1α in LPS-treated animals.Treatment of rats with 1400W or the non-selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) inhibited the increase in plasma NO2/NO3 but were both without effect on the plasma concentration of 6 keto-PGF1α before or after AA.Treatment with the non-steroidal anti-inflammatory drugs (NSAIDs), A771726 or diclofenac, or with celecoxib significantly reduced the increase in circulating 6 keto-PGF1α caused by LPS, and the large increase in 6 keto-PGF1α following injection of AA. None of the COX inhibitors affected the increase in plasma NO2/NO3. Dexamethasone, however, significantly inhibited both the increase in 6 keto-PGF1α and the increase in NO2/NO3.In conclusion, the use of selective inhibitors does not support the concept of cross talk in vivo between iNOS and COX-2. PMID:9786506

  2. Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells.

    PubMed

    Wagner, Renate; Stübiger, Gerald; Veigel, Daniel; Wuczkowski, Michael; Lanzerstorfer, Peter; Weghuber, Julian; Karteris, Emmanouil; Nowikovsky, Karin; Wilfinger-Lutz, Nastasia; Singer, Christian F; Colomer, Ramón; Benhamú, Bellinda; López-Rodríguez, María Luz; Valent, Peter; Grunt, Thomas W

    2017-01-10

    Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

  3. Structure-Based Design of Novel Pyrimido[4,5-c]pyridazine Derivatives as Dihydropteroate Synthase Inhibitors with Increased Affinity

    SciTech Connect

    Zhao, Ying; Hammoudeh, Dalia; Yun, Mi-Kyung; Qi, Jianjun; White, Stephen W.; Lee, Richard E.

    2012-05-29

    Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS is highly conserved and is distinct from the sulfonamide binding site. It therefore represents an attractive alternative target for the design of novel antibacterial agents. We previously carried out the structural characterization of a known pyridazine inhibitor in the Bacillus anthracis DHPS pterin site and identified a number of unfavorable interactions that appear to compromise binding. With this structural information, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazines were designed to improve binding affinity. Most importantly, the N-methyl ring substitution was removed to improve binding within the pterin pocket, and the length of the side chain carboxylic acid was optimized to fully engage the pyrophosphate binding site. These inhibitors were synthesized and evaluated by an enzyme activity assay, X-ray crystallography, isothermal calorimetry, and surface plasmon resonance to obtain a comprehensive understanding of the binding interactions from structural, kinetic, and thermodynamic perspectives. This study clearly demonstrates that compounds lacking the N-methyl substitution exhibit increased inhibition of DHPS, but the beneficial effects of optimizing the side chain length are less apparent.

  4. A nitric oxide synthase inhibitor, L-NAME, attenuates saccharin drinking in a two-choice test in water-deprived rats.

    PubMed

    Czech, D A

    1999-08-01

    The nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) [0 (veh)], 10, 25, and 50 mg/kg s.c.) was administered to water-deprived, saccharin-preferring, rats in a 30-min two-bottle choice test of 0.1% sodium saccharin and tap water in a within subjects design. Saccharin intake was selectively attenuated in a dose-related manner with increasing dose of L-NAME, reaching statistical significance at 25 and 50 mg/kg L-NAME when compared to vehicle control condition (p < 0.01). In contrast, water intake was not appreciably affected. Total fluid intake was attenuated as well. Neither saccharin nor water intake in a second group of animals was significantly affected by the inactive isomer, D-NAME, suggesting a stereospecific action. These data suggest that a taste factor might contribute to the well-documented hypophagic action of NOS inhibitors in a number of animal species. The possibility that such effect might be mediated through a serotonergic mechanism is considered.

  5. The role of S-methylisothiourea hemisulfate as inducible nitric oxide synthase inhibitor against kidney iron deposition in iron overload rats

    PubMed Central

    Maleki, Maryam; Samadi, Melika; Khanmoradi, Mehrangiz; Nematbakhsh, Mehdi; Talebi, Ardeshir; Nasri, Hamid

    2016-01-01

    Background: Iron dextran is in common use to maintain iron stores. However, it is potentially toxic and may lead to iron deposition (ID) and impair functions of organs. Iron overload can regulate the expression of inducible nitric oxide synthase (iNOS) in some cells that has an important role in tissue destruction. S-methylisothiourea hemisulfate (SMT) is a direct inhibitor of iNOS, and this study was designed to investigate the effect of SMT against kidney ID in iron overload rats. Materials and Methods: 24 Wistar rats (male and female) were randomly assigned to two groups. Iron overloading was performed by iron dextran 100 mg/kg/day every other day for 2 weeks. In addition, during the study, groups 1 and 2 received vehicle and SMT (10 mg/kg, ip), respectively. Finally, blood samples were obtained, and the kidneys were prepared for histopathological procedures. Results: SMT significantly reduced the serum levels of creatinine and blood urea nitrogen. However, SMT did not alter the serum levels of iron and nitrite, and the kidney tissue level of nitrite. Co-administration of SMT with iron dextran did not attenuate the ID in the kidney. Conclusion: SMT, as a specific iNOS inhibitor, could not protect the kidney from ID while it attenuated the serum levels of kidney function biomarkers. PMID:27308268

  6. High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

    PubMed Central

    Demaeght, Peter; Osborne, Edward J.; Odman-Naresh, Jothini; Grbić, Miodrag; Nauen, Ralf; Merzendorfer, Hans

    2014-01-01

    The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as ‘mite growth inhibitors’, and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a T. urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

  7. The marine natural-derived inhibitors of glycogen synthase kinase-3β phenylmethylene hydantoins: In vitro and in vivo activities and pharmacophore modeling

    PubMed Central

    Khanfar, Mohammad A.; Asal, Bilal Abu; Mudit, Mudit; Kaddoumi, Amal; El Sayed, Khalid A.

    2009-01-01

    The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1). This natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (2) showed potent in vitro and in vivo anti-growth and anti-invasive properties against PC-3M prostate cancer cells in MTT, spheroid disaggregation, and in mice models. To explore a possible molecular target of PMHs, the most potent synthetic analogue 2 has been virtually screened against various protein kinases. Molecular modeling study has shown that 2 can be successfully docked within the binding pocket of glycogen synthase kinase-3beta (GSK-3β) similar to the well-known GSK-3β inhibitor I-5. Several PMHs showed potent in vitro GSK-3β inhibitory activity with an IC50 range of 4–20 µM. The most potent analogue 3 showed a significant increase in liver glycogen level at the 5, 15, and 25 mg/kg dose levels, in vivo. Pharmacophore model was built and validated using in-house database of active and inactive GSK-3β inhibitors. The GSK-3β inhibitory activity of PMHs entitles them to be potential leads for the treatment of cancer, Alzheimer’s disease, bipolar disorders, stroke, different tau pathologies, and type-2 diabetes. PMID:19616957

  8. HMG-CoA reductase inhibitor improves endothelial dysfunction in spontaneous hypertensive rats via down-regulation of caveolin-1 and activation of endothelial nitric oxide synthase.

    PubMed

    Suh, Jung-Won; Choi, Dong-Ju; Chang, Hyuk-Jae; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Kim, Kwang-Il; Kim, Cheol-Ho; Kim, Hyo-Soo; Oh, Buyng-Hee; Park, Young-Bae

    2010-01-01

    Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10(-9)-10(-4) M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status.

  9. Homo-timeric structural model of human microsomal prostaglandin E synthase-1 and characterization of its substrate/inhibitor binding interactions

    NASA Astrophysics Data System (ADS)

    Xing, Li; Kurumbail, Ravi G.; Frazier, Ronald B.; Davies, Michael S.; Fujiwara, Hideji; Weinberg, Robin A.; Gierse, James K.; Caspers, Nicole; Carter, Jeffrey S.; McDonald, Joseph J.; Moore, William M.; Vazquez, Michael L.

    2009-01-01

    Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic π-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H2 (PGH2), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH2 in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.

  10. Effects of nitric oxide synthase inhibitors 1-(2-trifluoromethylphenyl)--imidazole (TRIM) and 7-nitroindazole (7-NI) on learning and memory in mice.

    PubMed

    Mutlu, Oguz; Ulak, Güner; Belzung, Catherine

    2011-06-01

    Nitric oxide (NO) plays an important role in hippocampal long-term potentiation (LTP), which is involved in memory processes. This led to the hypothesis that nitric oxide synthase (NOS) inhibitors will have disturbing effects on learning and memory. The aim of our study was to investigate the effects of the new selective neuronal and inducible NOS inhibitor 1- (2-trifluoromethylphenyl) imidazole (TRIM) (10-50 mg/kg) on learning and memory and compare it to the nonselective NOS inhibitor 7-NI (15-45 mg/kg) using different behavioral tests in Swiss mice, thus clarifying the role of neuronal NOS (nNOS) and endothelial NOS (eNOS) in cognitive processes. TRIM had no specific effect on either learning or memory parameters, while 7-NI (30 mg/kg) disturbed spatial memory in the probe trial of the Morris water maze test, which was performed on the last day of the test. No differences between TRIM and the control groups were observed, while 7-NI (30 and 45 mg/kg) significantly disturbed memory in the novel object recognition test. In the social transmission of food preference test, both TRIM (50 mg/kg) and 7-NI (45 mg/kg) impaired hippocampal olfactory memory, but the total food consumption was also significantly decreased at these doses. In the passive avoidance test, TRIM did not disturb the performance, while memory impairment was observed, even with lower doses of 7-NI. All of these results suggest that TRIM has no clear effect on cognitive impairment compared to 7-NI and that inhibition of both nNOS and eNOS are necessary for the deterioration of memory processes.

  11. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    SciTech Connect

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  12. Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon1[W

    PubMed Central

    Iwakami, Satoshi; Endo, Masaki; Saika, Hiroaki; Okuno, Junichi; Nakamura, Naoki; Yokoyama, Masao; Watanabe, Hiroaki; Toki, Seiichi; Uchino, Akira; Inamura, Tatsuya

    2014-01-01

    Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon. PMID:24760819

  13. Alkaloids as inhibitors of malate synthase from Paracoccidioides spp.: receptor-ligand interaction-based virtual screening and molecular docking studies, antifungal activity, and the adhesion process.

    PubMed

    Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonçalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Cecília Maria Alves; Kato, Lucília; Freitas, Carla Dos Santos; Giannini, Maria José Soares Mendes; da Silva, Julhiany de Fátima; Soares, Célia Maria de Almeida; Pereira, Maristela

    2015-09-01

    Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated.

  14. Alkaloids as Inhibitors of Malate Synthase from Paracoccidioides spp.: Receptor-Ligand Interaction-Based Virtual Screening and Molecular Docking Studies, Antifungal Activity, and the Adhesion Process

    PubMed Central

    Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonçalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Cecília Maria Alves; Kato, Lucília; Freitas, Carla dos Santos; Giannini, Maria José Soares Mendes; da Silva, Julhiany de Fátima; Soares, Célia Maria de Almeida

    2015-01-01

    Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated. PMID:26124176

  15. A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines

    PubMed Central

    2011-01-01

    Introduction Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. Methods In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. Results In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. Conclusions G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development. PMID:22177475

  16. 16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana.

    PubMed

    Roy, Arnab; Roberts, Frank G; Wilderman, P Ross; Zhou, Ke; Peters, Reuben J; Coates, Robert M

    2007-10-17

    The secondary ent-beyeran-16-yl carbocation (7) is a key branch point intermediate in mechanistic schemes to rationalize the cyclic structures of many tetra- and pentacyclic diterpenes, including ent-beyerene, ent-kaurene, ent-trachylobane, and ent-atiserene, presumed precursors to >1000 known diterpenes. To evaluate these mechanistic hypotheses, we synthesized the heterocyclic analogues 16-aza-ent-beyerane (12) and 16-aza-ent-trachylobane (13) by means of Hg(II)- and Pb(IV)-induced cyclizations onto the Delta12 double bonds of tricyclic intermediates bearing carbamoylmethyl and aminomethyl groups at C-8. The 13,16-seco-16-norcarbamate (20a) was obtained from ent-beyeran-16-one oxime (17) by Beckmann fragmentation, hydrolysis, and Curtius rearrangement. The aza analogues inhibited recombinant ent-kaurene synthase from Arabidopsis thaliana (GST-rAtKS) with inhibition constants (IC50 = 1 x 10-7 and 1 x 10-6 M) similar in magnitude to the pseudo-binding constant of the bicyclic ent-copalyl diphosphate substrate (Km = 3 x 10-7 M). Large enhancements of binding affinities (IC50 = 4 x 10-9 and 2 x 10-8 M) were observed in the presence of 1 mM pyrophosphate, which is consistent with a tightly bound ent-beyeranyl+/pyrophosphate- ion pair intermediate in the cyclization-rearrangement catalyzed by this diterpene synthase. The weak inhibition (IC50 = 1 x 10-5 M) exhibited by ent-beyeran-16-exo-yl diphosphate (11) and its failure to undergo bridge rearrangement to kaurene appear to rule out the covalent diphosphate as a free intermediate. 16-Aza-ent-beyerane is proposed as an effective mimic for the ent-beyeran-16-yl carbocation with potential applications as an active site probe for the various ent-diterpene cyclases and as a novel, selective inhibitor of gibberellin biosynthesis in plants.

  17. A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

    PubMed

    Liu, Weitang; Yuan, Guohui; Du, Long; Guo, Wenlei; Li, Lingxu; Bi, Yaling; Wang, Jinxin

    2015-01-01

    Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation.

  18. Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Fikrika, H.; Ambarsari, L.; Sumaryada, T.

    2016-01-01

    Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

  19. Nitric oxide synthase inhibitor, aminoguanidine reduces intracerebroventricular colchicine induced neurodegeneration, memory impairments and changes of systemic immune responses in rats.

    PubMed

    Sil, Susmita; Ghosh, Tusharkanti; Ghosh, Rupsa; Gupta, Pritha

    2017-02-15

    Intracerebroventricular (i.c.v.) injection of colchicine induces neurodegeneration, memory impairments and changes of some systemic immune responses in rats. Though the role of cox 2 in these colchicine induced changes have been evaluated, the influence of nitric oxide synthase (NOS) remains to be studied. The present study was designed to assess the role of NOS on the i.c.v. colchicine induced neurodegeneration, memory impairments and changes of some systemic immune responses by inhibiting its activity with aminoguanidine. In the present study the impairments of working and reference memories, neurodegeneration (chromatolysis and plaque formation) and changes of neuroinflammatory markers in the hippocampus (increased TNF α, IL 1β, ROS and nitrite) along with changes of serum inflammatory markers (TNF α, IL 1β, ROS and nitrite) and alteration of systemic immune responses (higher phagocytic activity of blood WBC and splenic PMN, higher cytotoxicity and lower leukocyte adhesion inhibition index of splenic MNC) were measured in the intracerebroventricular colchicine injected rats (ICIR). Administration of aminoguanidine (p.o. 30/50mg/kg body weight) to ICIR resulted in recovery of neuroinflammation and partial prevention of neurodegeneration which could be corroborated with the partial recovery of memory impairments in this model. The recovery of serum inflammatory markers and the systemic immune responses in ICIR was also observed after administration of aminoguanidine. Therefore, the present study shows that aminoguanidine can protect the colchicine induced neurodegeneration, memory impairments, and changes of systemic immune systemic responses in ICIR by inhibiting the iNOS.

  20. Serum aldosterone and death, end-stage renal disease, and cardiovascular events in blacks and whites: findings from the Chronic Renal Insufficiency Cohort (CRIC) Study.

    PubMed

    Deo, Rajat; Yang, Wei; Khan, Abigail M; Bansal, Nisha; Zhang, Xiaoming; Leonard, Mary B; Keane, Martin G; Soliman, Elsayed Z; Steigerwalt, Susan; Townsend, Raymond R; Shlipak, Michael G; Feldman, Harold I

    2014-07-01

    Prior studies have demonstrated that elevated aldosterone concentrations are an independent risk factor for death in patients with cardiovascular disease. Limited studies, however, have evaluated systematically the association between serum aldosterone and adverse events in the setting of chronic kidney disease. We investigated the association between serum aldosterone and death and end-stage renal disease in 3866 participants from the Chronic Renal Insufficiency Cohort. We also evaluated the association between aldosterone and incident congestive heart failure and atherosclerotic events in participants without baseline cardiovascular disease. Cox proportional hazards models were used to evaluate independent associations between elevated aldosterone concentrations and each outcome. Interactions were hypothesized and explored between aldosterone and sex, race, and the use of loop diuretics and renin-angiotensin-aldosterone system inhibitors. During a median follow-up period of 5.4 years, 587 participants died, 743 developed end-stage renal disease, 187 developed congestive heart failure, and 177 experienced an atherosclerotic event. Aldosterone concentrations (per SD of the log-transformed aldosterone) were not an independent risk factor for death (adjusted hazard ratio, 1.00; 95% confidence interval, 0.93-1.12), end-stage renal disease (adjusted hazard ratio, 1.07; 95% confidence interval, 0.99-1.17), or atherosclerotic events (adjusted hazard ratio, 1.04; 95% confidence interval, 0.85-1.18). Aldosterone was associated with congestive heart failure (adjusted hazard ratio, 1.21; 95% confidence interval, 1.02-1.35). Among participants with chronic kidney disease, higher aldosterone concentrations were independently associated with the development of congestive heart failure but not for death, end-stage renal disease, or atherosclerotic events. Further studies should evaluate whether mineralocorticoid receptor antagonists may reduce adverse events in individuals with

  1. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    SciTech Connect

    Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  2. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    SciTech Connect

    Malaviya, Rama; Venosa, Alessandro; Hall, LeRoy; Gow, Andrew J.; Sinko, Patrick J.; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-12-15

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS{sup +} and cyclooxygenase-2{sup +}) and alternatively activated profibrotic (YM-1{sup +} and galectin-3{sup +}) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ► Nitrogen mustard (NM) induces acute lung injury and fibrosis. ► Pulmonary toxicity is associated with increased expression of iNOS. ► Transient inhibition of iNOS attenuates acute

  3. Aldosterone Inactivates the Endothelin-B Receptor via a Cysteinyl Thiol Redox Switch to Decrease Pulmonary Endothelial Nitric Oxide Levels and Modulate Pulmonary Arterial Hypertension

    PubMed Central

    Maron, Bradley A.; Zhang, Ying-Yi; White, Kevin; Chan, Stephen Y.; Handy, Diane E.; Mahoney, Christopher E.; Loscalzo, Joseph; Leopold, Jane A.

    2012-01-01

    Background Pulmonary arterial hypertension (PAH) is characterized, in part, by decreased endothelial nitric oxide (NO•) production and elevated levels of endothelin-1. Endothelin-1 is known to stimulate endothelial nitric oxide synthase (eNOS) via the endothelin-B receptor (ETB), suggesting that this signaling pathway is perturbed in PAH. Endothelin-1 also stimulates adrenal aldosterone synthesis; in systemic blood vessels, hyperaldosteronism induces vascular dysfunction by increasing endothelial reactive oxygen species (ROS) generation and decreasing NO• levels. We hypothesized that aldosterone modulates PAH by disrupting ETB-eNOS signaling through a mechanism involving increased pulmonary endothelial oxidant stress. Methods and Results In rats with PAH, elevated endothelin-1 levels were associated with elevated aldosterone levels in plasma and lung tissue and decreased lung NO• metabolites in the absence of left heart failure. In human pulmonary artery endothelial cells (HPAECs), endothelin-1 increased aldosterone levels via PGC-1α/steroidogenesis factor-1-dependent upregulation of aldosterone synthase. Aldosterone also increased ROS production, which oxidatively modified cysteinyl thiols in the eNOS-activating region of ETB to decrease endothelin-1-stimulated eNOS activity. Substitution of ETB-Cys405 with alanine improved ETB-dependent NO• synthesis under conditions of oxidant stress, confirming that Cys405 is a redox sensitive thiol that is necessary for ETB-eNOS signaling. In HPAECs, mineralocorticoid receptor antagonism with spironolactone decreased aldosterone-mediated ROS generation and restored ETB-dependent NO• production. Spironolactone or eplerenone prevented or reversed pulmonary vascular remodeling and improved cardiopulmonary hemodynamics in two animal models of PAH in vivo. Conclusions Our findings demonstrate that aldosterone modulates an ETB cysteinyl thiol redox switch to decrease pulmonary endothelium-derived NO• and promote PAH

  4. Protection from inorganic mercury effects on the in vivo dopamine release by ionotropic glutamate receptor antagonists and nitric oxide synthase inhibitors.

    PubMed

    Vidal, Lucía; Durán, Rafael; Faro, Lilian F; Campos, Francisco; Cervantes, Rosa C; Alfonso, Miguel

    2007-09-05

    The possible role of ionotropics glutamate receptors on the HgCl(2)-induced dopamine (DA) release from rat striatum was investigated by using in vivo brain microdialysis technique after administration of selective NMDA and AMPA/Kainate receptors antagonists dizocilpine (MK-801), D (-)-2-amino-5-phoshonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Moreover, we have also studied the effects of nitric oxide synthase (NOS) inhibitors L-nitro-arginine methyl ester (L-NAME) and 7-nitro-indazol (7-NI) on HgCl(2)-induced DA release. Intraestriatal infusion of 1mM HgCl(2) increased striatal DA to 1717.2+/-375.4% respect to basal levels. Infusion of 1mM HgCl(2) in 400 microM MK-801 pre-treated animals produced an increase on striatal DA levels 61% smaller than that induced in non-pre-treated animals. In the case of AP5, this treatment reduced 92% the increase produced by HgCl(2) as compared to non-pre-treated rats. Nevertheless, the administration of CNQX did not produce any effect on HgCl(2)-induced dopamine release. Intrastriatal infusion of 1mM HgCl(2) in 100 microM L-NAME pre-treated animals produced an increase on extracellular DA levels 82% smaller than produced by HgCl(2) alone. In addition, the pre-treatment with 7-NI reduced 90% the increase produced by infusion of HgCl(2) alone in rats. Thus, HgCl(2)-induced DA release could be produced at last in part, by overstimulation of NMDA receptors with NO production, since administration of NMDA receptor antagonists and NOS inhibitors protected against HgCl(2) effects on DA release.

  5. Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3β inhibitors through ligand and structure based drug design.

    PubMed

    Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

    2014-09-01

    Glycogen synthase kinase-3β elicits multi-functional effects on intracellular signaling pathways, thereby making the kinase a therapeutic target in multiple pathologies. Hence, it is important to selectively inhibit GSK-3β over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3β inhibitors. The study was designed to identify new leads by ligand based drug design, structure based drug design and in vitro evaluation. The best validated pharmacophore model (AADRRR) identified using LBDD was derived from a dataset of 135 molecules. There were 357 primary hits within the SPECS database using this pharmacophore model. A SBDD approach to the GSK-3β and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3β. GSK-3β and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3β. The molecules showed IC50 values ranging from 0.825μM to 1.116μM and were 23- to 57-fold selective for GSK-3β. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825μM. Our research identified molecules possessing benzothiophene, isoquinoline, thiazolidinedione imidazo-isoquinoline and quinazolinone scaffolds. Potency of these molecules may be due to H-bond interaction with backbone residues of Val135, Asp133 and side chain interaction with Tyr134. Selectivity over CDK5 may be due to side chain interactions with Asp200, backbone of Val61, ionic interaction with Lys60 and π-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule.

  6. Activation of the Wnt pathway through use of AR79, a glycogen synthase kinase 3β inhibitor, promotes prostate cancer growth in soft tissue and bone

    PubMed Central

    Jiang, Yuan; Dai, Jinlu; Zhang, Honglai; Sottnik, Joe L.; Keller, Jill M.; Escott, Katherine J.; Sanganee, Hitesh J.; Yao, Zhi; McCauley, Laurie K.; Keller, Evan T.

    2013-01-01

    Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling through inducing β-catenin degradation. Therefore, AR79, an inhibitor of GSK3β, is being evaluated as a bone anabolic agent. However, Wnt activation has potential to promote tumor growth. The goal of this study was to determine if AR79 impacted progression of prostate cancer (PCa). PCa tumors were established in subcutaneous and bone sites of mice followed by AR79 administration. Tumor growth, β-catenin activation, proliferation (Ki67 expression) and apoptosis (caspase 3 activity) were measured. Additionally, PCa and osteoblast cell lines were treated with AR79 and β-catenin status, proliferation (with β-catenin knocked down in some cases) and proportion of the ALDH+CD133+ stem-like cells was determined. AR79 promoted PCa growth, decreased phospho-β-catenin expression and increased total and nuclear β-catenin expression in tumors and increased tumor-induced bone remodeling. Additionally, it decreased caspase 3 and increased Ki67 expression. In addition, AR79 increased bone formation in normal mouse tibiae. AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in PCa and osteoblast cell lines and increased proliferation of PCa cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell-like proportion of the PCa cell lines. We conclude that AR79, while being bone anabolic, promotes PCa cell growth through Wnt pathway activation. PMID:24088787

  7. Transforming growth factor beta1 and aldosterone

    PubMed Central

    Matsuki, Kota; Hathaway, Catherine K.; Chang, Albert S.; Smithies, Oliver; Kakoki, Masao

    2016-01-01

    Purpose of review It is well established that blocking renin-angiotensin II-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. Recent findings TGFbeta1 suppresses adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps from 10% to 300% normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10 % hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. Summary These results identify TGFbeta signaling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism as well as assist in the development of novel therapeutic strategies for hypertension. PMID:25587902

  8. Primary Aldosteronism and ARMC5 Variants

    PubMed Central

    Zilbermint, Mihail; Xekouki, Paraskevi; Faucz, Fabio R.; Berthon, Annabel; Gkourogianni, Alexandra; Schernthaner-Reiter, Marie Helene; Batsis, Maria; Sinaii, Ninet; Quezado, Martha M.; Merino, Maria; Hodes, Aaron; Abraham, Smita B.; Libé, Rossella; Assié, Guillaume; Espiard, Stéphanie; Drougat, Ludivine; Ragazzon, Bruno; Davis, Adam; Gebreab, Samson Y.; Neff, Ryan; Kebebew, Electron; Bertherat, Jérôme; Lodish, Maya B.

    2015-01-01

    Context: Primary aldosteronism is one of the leading causes of secondary hypertension, causing significant morbidity and mortality. A number of genetic defects have recently been identified in primary aldosteronism, whereas we identified mutations in ARMC5, a tumor-suppressor gene, in cortisol-producing primary macronodular adrenal hyperplasia. Objective: We investigated a cohort of 56 patients who were referred to the National Institutes of Health for evaluation of primary aldosteronism for ARMC5 defects. Methods: Patients underwent step-wise diagnosis, with measurement of serum aldosterone and plasma renin activity followed by imaging, saline suppression and/or oral salt loading tests, plus adrenal venous sampling. Cortisol secretion was also evaluated; unilateral or bilateral adrenalectomy was performed, if indicated. DNA, protein, and transfection studies in H295R cells were conducted by standard methods. Results: We identified 12 germline ARMC5 genetic alterations in 20 unrelated and two related individuals in our cohort (39.3%). ARMC5 sequence changes in 6 patients (10.7%) were predicted to be damaging by in silico analysis. All affected patients carrying a variant predicted to be damaging were African Americans (P = .0023). Conclusions: Germline ARMC5 variants may be associated with primary aldosteronism. Additional cohorts of patients with primary aldosteronism and metabolic syndrome, particularly African Americans, should be screened for ARMC5 sequence variants because these may underlie part of the known increased predisposition of African Americans to low renin hypertension. PMID:25822102

  9. Increased anticancer activity of the thymidylate synthase inhibitor BGC9331 combined with the topoisomerase I inhibitor SN-38 in human colorectal and breast cancer cells: induction of apoptosis and ROCK cleavage through caspase-3-dependent and -independent mechanisms.

    PubMed

    Coudray, Anne-Marie; Louvet, Christophe; Kornprobst, Michel; Raymond, Eric; André, Thierry; Tournigand, Christophe; Faivre, Sandrine; De Gramont, Aimery; Larsen, Annette K; Gespach, Christian

    2005-08-01

    The folate analogue BGC9331 is a new thymidylate synthase (TS) inhibitor showing a broad spectrum of cyto-toxic activity against several human solid tumors, including colorectal cancer. In this study, we investigated the anticancer activity of BGC9331 either alone or combined with 5-fluorouracil (5-FU), MTA (multi-target antifolate), oxali-platin and SN-38, the active metabolite of the topoisomerase I inhibitor CPT-11. The antiproliferative activity of each drug and BGC9331-based combinations was investigated in the HT-29 human colorectal cancer cell line and its HT-29/5-FU counterparts selected for resistance to 5-FU. BGC9331 combined with MTA or SN-38 induced synergistic responses in HT-29 cells. Treatment of HT-29 cells with either BGC9331 or SN-38 increased caspase-3 activity and the percentage of apoptotic cells from 3 to 13%. Both drugs also augmented the proteolytic cleavage of the Rho-kinase ROCK-1 that was attenuated by the caspase-3 pathway inhibitor z-DEVD-fmk. BGC9331 combined with SN-38 further increased the percentage of apoptotic cells to 25%, and inhibited cell cycle progression and cell proliferation by 65%. This was accompanied by proteolytic activation of ROCK-1, through both caspase-3-dependent and -independent mechanisms, as shown in caspase-3-deficient MCF-7 breast cancer cells. These encouraging results warrant further preclinical investigations and clinical trials on the use of BGC9331 combined with SN-38/CPT-11 in treatment of patients with advanced colorectal or gastric cancers.

  10. Sulfhydryl angiotensin-converting enzyme inhibitor promotes endothelial cell survival through nitric-oxide synthase, fibroblast growth factor-2, and telomerase cross-talk.

    PubMed

    Donnini, Sandra; Terzuoli, Erika; Ziche, Marina; Morbidelli, Lucia

    2010-03-01

    The protective effect exerted by angiotensin-converting enzyme inhibitors (ACEI) in cardiovascular diseases caused by endothelial injury and aging has been attributed to the restoration of endothelial cell functions. Recently, we demonstrated a central role of the fibroblast growth factor-2 (FGF-2)/FGF receptor-1 system in mediating the acquisition of an angiogenic phenotype in coronary microvascular endothelium exposed to ACEI. Here, we report on the rescuing effect of ACEI on impaired endothelium and the intracellular signaling mechanisms that lead endothelial cells to enter apoptosis and to senesce. Conditions mimicking pathological cell damage (serum deprivation) lead to endothelial apoptosis as evidenced by increased caspase-3 activity. ACEI enhanced cell survival through activation of prosurvival and antiaging signals involving Akt phosphorylation, endothelial nitric-oxide synthase (eNOS) expression and activation, FGF-2 and telomerase catalytic subunit (TERT) up-regulation, and delayed senescence. In microvascular endothelial cells exposed to ACEI, Akt/eNOS pathway-dependent FGF-2 was necessary for gene transcription of TERT. These protective effects were particularly evident for sulfhydryl-containing ACEI (zofenoprilat), which were reported to exhibit potent antioxidant effects. In conclusion, ACEI with antioxidant properties up-regulate eNOS, FGF-2, and TERT mRNA, which favor endothelial cell survival and prolong their lifespan, thus restoring endothelial cell functions after vascular damage. These effects could explain the beneficial effects of these drugs in various cardiovascular diseases associated with endothelial injury and aging.

  11. RNA silencing targeting PIN (protein inhibitor of neuronal nitric oxide synthase) attenuates the development of hypertension in young spontaneously hypertensive rats.

    PubMed

    Wang, Su-Chen; Lin, Kuan-Miao; Chien, Shao-Ju; Huang, Li-Tung; Hsu, Chien-Ning; Tain, You-Lin

    2014-01-01

    Nitric oxide (NO) deficiency contributes to hypertension. We previously showed that neuronal nitric oxide synthase (nNOS) was involved in hypertension and kidney damage in spontaneously hypertensive rats (SHRs). The protein inhibitor of nNOS (PIN) has been reported to inhibit activity of nNOS.Thus, we tested whether increased PIN in the kidney results in hypertension and whether small interfering RNA (siRNA) targeting PIN attenuates hypertension in SHRs. Four-week-old male SHRs were assigned into three groups (n = 6-7/group): SHR; SHR + PIN, SHR that received siRNA targeting PIN; and SHR + NC, SHR treated with random negative control siRNA. Rats were sacrificed at 12 weeks of age. PIN protein expression was inhibited considerably when PIN siRNA was transfected into NRK52E cells (90% siRNA at 1 nM). The increases of BP were attenuated by siRNA targeting PIN in 12-week-old SHRs. Immunostaining of nNOS-α and total nNOS was greater in SHR + PIN group than SHR. Moreover, renal superoxide production and 8-hydroxydeoxyguanosine (8-OHdG) staining were more decreased in the SHR + PIN group than SHRs. We conclude that PIN siRNA reduced PIN expression in vitro and in vivo. PIN siRNA therapy attenuates hypertension in SHRs at 12 weeks of age. Our results suggest that PIN is involved in the development of hypertension.

  12. Effect of the nitric oxide donor and the nitric oxide synthase inhibitor on the liver of rats with chronic hepatitis induced by dimethylnitrosamine.

    PubMed

    Lukivskaya, O; Lis, R; Zwierz, K; Buko, V

    2004-01-01

    The present study was designed to examine the effects of the donor of nitric oxide (NO), NaNO(2) and the inhibitor of NO synthase, N(omega)-nitro-L-arginine (L-NNA), on the development of dimethylnitrosamine (DMNA)-induced chronic hepatitis in rats. L-NNA decreased rat survival and enhanced the severity of hepatic encephalopathy in the DMNA-treated animals. The aggravation of the morphological signs of hepatitis, the activation of serum alanine aminotransferase and cytosolic superoxide dismutase activities and the increase in the liver malondialdehyde content were observed in this group. The treatment with NaNO(2) improved liver morphology, decreased serum marker enzyme activities, lowered the activities of alpha-D-mannosidase and N-acetyl-beta-D-glucosaminidase compared to the DMNA-treated group. The results of the morphological and biochemical studies suggest that L-NNA increased DMNA-induced liver damage, whereas NaNO(2) partially prevented the development of chronic hepatitis. It is proposed that the opposite effects of L-NNA and NaNO(2) are partially explained by a modulation of the free radical-dependent processes in the liver.

  13. Long-term neuroprotection with 2-iminobiotin, an inhibitor of neuronal and inducible nitric oxide synthase, after cerebral hypoxia-ischemia in neonatal rats.

    PubMed

    van den Tweel, Evelyn R W; van Bel, Frank; Kavelaars, Annemieke; Peeters-Scholte, Cacha M P C D; Haumann, Johan; Nijboer, Cora H A; Heijnen, Cobi J; Groenendaal, Floris

    2005-01-01

    The short- and long-term neuroprotective effects of 2-iminobiotin, a selective inhibitor of neuronal and inducible nitric oxide synthase, were studied in 12-day-old rats following hypoxia-ischemia. Hypoxia-ischemia was induced by occlusion of the right carotid artery followed by 90 minutes of hypoxia (FiO2 0.08). Immediately on reoxygenation, 12 and 24 hours later the rats were treated with vehicle or 2-iminobiotin at a dose of 5.5, 10, 30, or 60 mg/kg per day. Histologic analysis of brain damage was performed at 6 weeks after hypoxia-ischemia. To assess early changes of cerebral tissue, levels of HSP70, nitrotyrosine, and cytochrome c were determined 24 hours after reoxygenation. Significant neuroprotection was obtained using a dose of 30 mg/kg per day of 2-iminobiotin. Levels of HSP70 were increased in the ipsilateral hemisphere in both groups (P<0.05), but the increase was significantly (P<0.05) less in the rats receiving the optimal dose of 2-iminobiotin (30 mg/kg per day). Hypoxia-ischemia did not lead to increased levels of nitrotyrosine, nor did 2-iminobiotin influence levels of nitrotyrosine. In contrast, hypoxia-ischemia induced an increase in cytochrome c level that was prevented by 2-iminobiotin. In conclusion, 2-iminobiotin administered after hypoxia-ischemia provides long-term neuroprotection. This neuroprotection is obtained by mechanisms other than a reduction of nitrotyrosine formation in proteins.

  14. Radioisotope assay for 1-aminocyclopropane-1-carboxylic acid synthase: s-adenosylhomocysteine analogs as inhibitors of the enzyme involved in plant senescence

    SciTech Connect

    Miura, G.A.; Chiang, P.K.

    1985-01-01

    A simple and rapid radioisotopic assay for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase was developed, an enzyme involved in the biosynthesis of the plant hormone ethylene. The assay utilizes an AG50-X4(NH4 (+)) column which separates S-adenosyl-L-(carboxyl-/sup 14/C)methionine (AdoMet) from the product (/sup 14/C)acc, since the latter is not bound to the resin while (/sup 14/C)adoMet is. As opposed to other assays, this procedure measures ACC directly and does not require further conversion to ethylene. When an enzyme preparation from ripe-tomato fruits (Lycopersicon esculentum Mill) was assayed, an I/sub 50/ of 2.5 + or - 0.8 micrometers for sinefungin and a K/sub m/ of 27 + or - 2 micrometers for AdoMet were obtained; these values were in good agreement with previous previous determinations made with a gas-chromatographic assay. When other nucleosides were tested as inhibitors the following order of decreasing activity was found: sinefungin, S-adenosylhomocysteine (AdoHcy), AdoHcy sulfoxide, S-n-butyladenosine, 3-deaza-adenosylhomocysteine, S-isobutyladenosine, S-isobutyladenosine, S-isobutyl-l-deazaadenosine. In contrast, S-isobutyl-3-deazaadenosine, S-isobutyl-7-deazaadenosine, 3-deazaadenosine, and adenodine were not inhibitory.

  15. Different effects of dexamethasone and the nitric oxide synthase inhibitor L-NAME on caerulein-induced rat acute pancreatitis, depending on the severity.

    PubMed

    Sugiyama, Yusuke; Kato, Shinichi; Abe, Mitsumasa; Mitsufuji, Shoji; Takeuchi, Koji

    2005-01-01

    Effects of dexamethasone and N(G)-nitro-L-arginine methyl ester (L-NAME), the nitric oxide (NO) synthase inhibitor, on caerulein-induced acute pancreatitis were examined in rats. Acute pancreatitis was induced by caerulein (20 mug/kg, s.c.) given repeatedly 2 or 4 times every hour, and serum amylase levels, pancreas weight and myeloperoxidase (MPO) activity were measured 6 h after the first injection of caerulein. Dexamethasone (3 mg/kg) and L-NAME (30 mg/kg) were administered p.o. 30 min before the first injection of caerulein. Caerulein caused moderate or severe pancreatitis, depending on the times of injections, resulting in different degrees of increase in serum amylase levels and pancreas weight, and the marked elevation of MPO activity was observed only after injections of caerulein given 4 times per hour. Both dexamethasone and L-NAME suppressed the severity of pancreatits, yet the effect of L-NAME as compared with dexamethasone was more potent against mild pancreatitis but less potent against severe pancreatitis. These results suggest that caerulein-induced acute pancreatitis shows different responsiveness to L-NAME and dexamethasone, depending on the severity; the former is more effective against pancreatitis with less inflammation, while the latter is more effective against pancreatitis with severe inflammation. It is assumed that endogenous NO may be involved in oedema formation as the early event in the development of acute pancreatitis.

  16. A quantitative structure-activity relationship (QSAR) study on a few series of potent, highly selective inhibitors of nitric oxide synthase.

    PubMed

    Bharti, Vishwa Deepak; Gupta, Satya P; Kumar, Harish

    2014-02-01

    QSAR study was performed on a series of 1,2-dihydro-4-quinazolinamines, 4,5-dialkylsubstituted-2-imino-1,3-thiazolidine derivatives and 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives studied by Tinker et al. [J Med Chem (2003), 46, 913-916], Ueda et al. [Bioorg Med Chem (2004) 12, 4101-4116] and Ueda et al. [Bioorg Med Chem Lett (2004) 14, 313-316], respectively, as potent, highly selective inhibitors of inducible nitric oxide synthase (iNOS). The iNOS inhibition activity of the whole series of compounds was analyzed in relation to the physicochemical and molecular properties of the compounds. The QSAR analysis revealed that the inhibition potency of the compounds was controlled by a topological parameter 1chi(v) (Kier's first order valence molecular connectivity index), density (D), surface tension (St) and length (steric parameter) of a substituent. This suggested that the drug-receptor interaction predominantly involved the dispersion interaction, but the bulky molecule would face steric problem because of which the molecule may not completely fit in active sites of the receptor and thus may not have the optimum interaction.

  17. Elevated levels of the serum endogenous inhibitor of nitric oxide synthase and metabolic control in rats with streptozotocin-induced diabetes.

    PubMed

    Xiong, Yan; Fu, Yun-feng; Fu, Si-hai; Zhou, Hong-hao

    2003-08-01

    This study was designed to determine the relationship between elevated levels of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), and metabolic control in rats with streptozotocin-induced diabetes. Serum levels of ADMA were measured by high-performance liquid chromatography at 8 weeks after diabetes was induced. Endothelium-dependent relaxation to acetylcholine was tested in aortic rings from nondiabetic age-matched control, untreated diabetic, and insulin-treated diabetic rats to evaluate endothelial function. Serum concentrations of glucose, glycosylated serum protein, and malondialdehyde were examined to estimate metabolic control. Serum levels of ADMA increased dramatically in untreated diabetic rats compared with control rats. This elevation in ADMA levels was accompanied by impairment of the endothelium-dependent relaxation response to acetylcholine in aortic rings. Long-term insulin treatment not only prevented the elevation of serum ADMA levels, but also improved the impairment of endothelium-dependent relaxation in diabetic rats. Serum levels of glucose, glycosylated serum protein, and malondialdehyde were significantly increased in parallel with the elevation of ADMA in untreated diabetic rats compared with control rats. These parameters were normalized after diabetic rats received insulin treatment for 8 weeks. These results provide the first evidence that an elevation in the concentration of ADMA in rats with streptozotocin-induced diabetes is closely related to metabolic control of the disease.

  18. Design, Synthesis, Calorimetry and Crystallographic analysis of 2-Alkylaminoethyl-1,1-Bisphosphonates as inhibitors of Trypanosoma cruzi Farnesyl Diphosphate Synthase

    PubMed Central

    Aripirala, Srinivas; Szajnman, Sergio H.; Jakoncic, Jean; Rodriguez, Juan B.; Docampo, Roberto; Gabelli, Sandra B.; Amzel, L. Mario

    2016-01-01

    Linear 2-alkylaminoethyl-1,1-bisphosphonates are effective agents against proliferation of Trypanosoma cruzi--the etiologic agent of American trypanosomiasis (Chagas disease)--exhibiting IC50 values in the nanomolar range against the parasites. This activity is associated with inhibition at the low nanomolar level of the T. cruzi farnesyl diphosphate synthase (TcFPPS). X-ray structures and thermodynamic data of the complexes TcFPPS with five compounds of this family show that the inhibitors bind to the allylic site of the enzyme with their alkyl chain occupying the cavity that binds the isoprenoid chain of the substrate. The compounds bind to TcFPPS with unfavorable enthalpy compensated by a favorable entropy that results from a delicate balance between two opposing effects: the loss of conformational entropy due to freezing of single bond rotations, and the favorable burial of the hydrophobic alkyl chains. The data suggest that introduction of strategically placed double bonds and methyl branches should increase affinity substantially. PMID:22715997

  19. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

  20. Synthesis and evaluation of 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives as glycogen synthase kinase-3 (GSK-3) inhibitors.

    PubMed

    Chun, Kwangwoo; Park, Ji-Seon; Lee, Han-Chang; Kim, Young-Ha; Ye, In-Hea; Kim, Kang-Jeon; Ku, Il-Whea; Noh, Min-Young; Cho, Goang-Won; Kim, Heejaung; Kim, Seung Hyun; Kim, Jeongmin

    2013-07-01

    New potent glycogen synthase kinase-3 (GSK-3) inhibitors, 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives, were designed by modeling, synthesized and evaluated in vitro. Compound 17c showed good potency in enzyme and cell-based assays (IC50=111 nM, EC50=1.78 μM). Moreover, it has demonstrated desirable water solubility, PK profile, and moderate brain penetration.

  1. Circadian Rhythm of Plasma Aldosterone Concentration in Patients with Primary Aldosteronism

    PubMed Central

    Kem, David C.; Weinberger, Myron H.; Gomez-Sanchez, Celso; Kramer, Norman J.; Lerman, Robert; Furuyama, Shunsuke; Nugent, Charles A.

    1973-01-01

    Plasma aldosterone, cortisol, and renin activity were measured in nine recumbent patients with hyperaldosteronism, including seven with adenomas, one with idiopathic hyperplasia, and one with glucocorticoid suppressible hyperplasia. All had peak values of plasma aldosterone concentration from 3 a.m. to noon and lowest values at 6 p.m. or midnight. This rhythm was similar to the circadian pattern of plasma cortisol in the same patients. When these data were normalized to eliminate the wide variation in ranges of plasma aldosterone and cortisol between individuals, there was an excellent correlation (r = + 0.87, P < 0.005) between the two hormones. In contrast, plasma aldosterone concentrations did not correlate with plasma renin activity before or after normalization of data. Short term suppression of ACTH by administration of dexamethasone eliminated the circadian variation of plasma aldosterone in both patients with hyperplasia and in four of five patients with adenomas, while it markedly altered the rhythm in the fifth. Similar doses of dexamethasone were administered to four normal subjects and did not flatten the circadian rhythm of plasma aldosterone. These data suggest that patients with primary aldosteronism have a circadian rhythm of plasma aldosterone mediated by changes in ACTH. PMID:4353776

  2. GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo

    PubMed Central

    Alderton, Wendy K; Angell, Anthony D R; Craig, Caroline; Dawson, John; Garvey, Edward; Moncada, Salvador; Monkhouse, Jayne; Rees, Daryl; Russell, Linda J; Russell, Rachel J; Schwartz, Sheila; Waslidge, Neil; Knowles, Richard G

    2005-01-01

    GW274150 ([2-[(1-iminoethyl) amino]ethyl]-L-homocysteine) and GW273629 (3-[[2-[(1-iminoethyl)amino]ethyl]sulphonyl]-L-alanine) are potent, time-dependent, highly selective inhibitors of human inducible nitric oxide synthase (iNOS) vs endothelial NOS (eNOS) (>100-fold) or neuronal NOS (nNOS) (>80-fold). GW274150 and GW273629 are arginine competitive, NADPH-dependent inhibitors of human iNOS with steady state Kd values of <40 and <90 nM, respectively.GW274150 and GW273629 inhibited intracellular iNOS in J774 cells in a time-dependent manner, reaching IC50 values of 0.2±0.04 and 1.3±0.16 μM, respectively. They were also acutely selective in intact rat tissues: GW274150 was >260-fold and 219-fold selective for iNOS against eNOS and nNOS, respectively, while GW273629 was >150-fold and 365-fold selective for iNOS against eNOS and nNOS, respectively.The pharmacokinetic profile of GW274150 was biphasic in healthy rats and mice with a terminal half-life of ∼6 h. That of GW273629 was also biphasic in rats, producing a terminal half-life of ∼3 h. In mice however, elimination of GW273629 appeared monophasic and more rapid (∼10 min). Both compounds show a high oral bioavailability (>90%) in rats and mice.GW274150 was effective in inhibiting LPS-induced plasma NOx levels in mice with an ED50 of 3.2±0.7 mg kg−1 after 14 h intraperitoneally (i.p.) and 3.8±1.5 mg kg−1 after 14 h when administered orally. GW273629 showed shorter-lived effects on plasma NOx and an ED50 of 9±2 mg kg−1 after 2 h when administered i.p.The effects of GW274150 and GW273629 in vivo were consistent with high selectivity for iNOS, as these inhibitors were of low potency against nNOS in the rat cerebellum and did not cause significant effects on blood pressure in instrumented mice. PMID:15778742

  3. Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase.

    PubMed Central

    Szabó, C; Southan, G J; Thiemermann, C

    1994-01-01

    Enhanced formation of nitric oxide (NO) by both the constitutive and the inducible isoforms of NO synthase (NOS) has been implicated in the pathophysiology of a variety of diseases, including circulatory shock. Non-isoform-selective inhibition of NO formation, however, may lead to side effects by inhibiting the constitutive isoform of NOS and, thus, the various physiological actions of NO. S-Methylisothiourea sulfate (SMT) is at least 10- to 30-fold more potent as an inhibitor of inducible NOS (iNOS) in immunostimulated cultured macrophages (EC50, 6 microM) and vascular smooth muscle cells (EC50, 2 microM) than NG-methyl-L-arginine (MeArg) or any other NOS inhibitor yet known. The effect of SMT on iNOS activity can be reversed by excess L-arginine in a concentration-dependent manner. SMT (up to 1 mM) does not inhibit the activity of xanthine oxidase, diaphorase, lactate dehydrogenase, monoamine oxidase, catalase, cytochrome P450, or superoxide dismutase. SMT is equipotent with MeArg in inhibiting the endothelial, constitutive isoform of NOS in vitro and causes increases in blood pressure similar to those produced by MeArg in normal rats. SMT, however, dose-dependently reverses (0.01-3 mg/kg) the hypotension and the vascular hyporeactivity to vasoconstrictor agents caused by endotoxin [bacterial lipopolysaccharide (LPS), 10 mg/kg, i.v.] in anesthetized rats. Moreover, therapeutic administration of SMT (5 mg/kg, i.p., given 2 hr after LPS, 10 mg/kg, i.p.) attenuates the rises in plasma alanine and aspartate aminotransferases, bilirubin, and creatinine and also prevents hypocalcaemia when measured 6 hr after administration of LPS. SMT (1 mg/kg, i.p.) improves 24-hr survival of mice treated with a high dose of LPS (60 mg/kg, i.p.). Thus, SMT is a potent and selective inhibitor of iNOS and exerts beneficial effects in rodent models of septic shock. SMT, therefore, may have considerable value in the therapy of circulatory shock of various etiologies and other

  4. Mechanisms of renal control of potassium homeostasis in complete aldosterone deficiency.

    PubMed

    Todkar, Abhijeet; Picard, Nicolas; Loffing-Cueni, Dominique; Sorensen, Mads V; Mihailova, Marija; Nesterov, Viatcheslav; Makhanova, Natalia; Korbmacher, Christoph; Wagner, Carsten A; Loffing, Johannes

    2015-02-01

    Aldosterone-independent mechanisms may contribute to K(+) homeostasis. We studied aldosterone synthase knockout (AS(-/-)) mice to define renal control mechanisms of K(+) homeostasis in complete aldosterone deficiency. AS(-/-) mice were normokalemic and tolerated a physiologic dietary K(+) load (2% K(+), 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K(+) intake (5% K(+)), AS(-/-) mice decompensated and became hyperkalemic. High-K(+) diets induced upregulation of the renal outer medullary K(+) channel in AS(-/-) mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K(+) excretion was detected only with a 2% K(+) diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS(-/-) mice than in AS(+/+) mice and was downregulated in mice of both genotypes in response to increased K(+) intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS(-/-) mice. In contrast with the kidney, the distal colon of AS(-/-) mice did not respond to dietary K(+) loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K(+) load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K(+) channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K(+) secretion and increased intratubular availability of Na(+) that can be reabsorbed in exchange for K(+) secreted.

  5. Mechanisms of Renal Control of Potassium Homeostasis in Complete Aldosterone Deficiency

    PubMed Central

    Todkar, Abhijeet; Picard, Nicolas; Loffing-Cueni, Dominique; Sorensen, Mads V.; Mihailova, Marija; Nesterov, Viatcheslav; Makhanova, Natalia; Korbmacher, Christoph; Wagner, Carsten A.

    2015-01-01

    Aldosterone-independent mechanisms may contribute to K+ homeostasis. We studied aldosterone synthase knockout (AS−/−) mice to define renal control mechanisms of K+ homeostasis in complete aldosterone deficiency. AS−/− mice were normokalemic and tolerated a physiologic dietary K+ load (2% K+, 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K+ intake (5% K+), AS−/− mice decompensated and became hyperkalemic. High-K+ diets induced upregulation of the renal outer medullary K+ channel in AS−/− mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K+ excretion was detected only with a 2% K+ diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS−/− mice than in AS+/+ mice and was downregulated in mice of both genotypes in response to increased K+ intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS−/− mice. In contrast with the kidney, the distal colon of AS−/− mice did not respond to dietary K+ loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K+ load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K+ channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K+ secretion and increased intratubular availability of Na+ that can be reabsorbed in exchange for K+ secreted. PMID:25071088

  6. SFE/SFHTA/AFCE consensus on primary aldosteronism, part 7: Medical treatment of primary aldosteronism.

    PubMed

    Pechère-Bertschi, Antoinette; Herpin, Daniel; Lefebvre, Hervé

    2016-07-01

    Spironolactone, which is a potent mineralocorticoid receptor antagonist, represents the first line medical treatment of primary aldosteronism (PA). As spironolactone is also an antagonist of the androgen and progesterone receptor, it may present side effects, especially in male patients. In case of intolerance to spironolactone, amiloride may be used to control hypokaliemia and we suggest that eplerenone, which is a more selective but less powerful antagonist of the mineralocorticoid receptor, be used in case of intolerance to spironolactone and insufficient control of hypertension by amiloride. Specific calcic inhibitors and thiazide diuretics may be used as second or third line therapy. Medical treatment of bilateral forms of PA seem to be as efficient as surgical treatment of lateralized PA for the control of hypertension and the prevention of cardiovascular and renal morbidities. This allows to propose medical treatment of PA to patients with lateralized forms of PA who refuse surgery or to patients with PA who do not want to be explored by adrenal venous sampling to determine whether they have a bilateral or lateralized form.

  7. Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression

    PubMed Central

    Kunnimalaiyaan, Selvi; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

    2015-01-01

    Background Glycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation. Methods The pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0–20 μM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1. Results AR-A014418 treatment had a significant dose-dependent growth reduction (P < 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1. Conclusions This study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics. PMID:26147011

  8. Constitutive activation of glycogen synthase kinase-3β correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer

    PubMed Central

    2010-01-01

    Background Aberrant regulation of glycogen synthase kinase-3β (GSK-3β) has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3β phosphorylated at Tyr216 (pGSK-3β) and its relationship with other tumor-associated proteins in human gastric cancers. Methods Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3β inhibitor lithium chloride (LiCl) for immunoblot analysis. Results We found that pGSK-3β was expressed in 129 (46%) of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P < 0.001). The expression of pGSK-3β inversely correlated with lymphatic invasion (P < 0.001) and lymph node metastasis (P < 0.001) and correlated with a longer patient survival (P < 0.001). In addition, pGSK-3β expression positively correlated with that of p16, p21, p27, p53, APC, PTEN, MGMT, SMAD4, or KAI1 (P < 0.05), but not with that of cyclin D1. This was confirmed by immunoblot analysis using SNU-668 gastric cancer cells treated with LiCl. Conclusions GSK-3β activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3β activation is a useful prognostic marker for the early-stage gastric cancer. PMID:20704706

  9. The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Methyl Ester Diminishes the Immunomodulatory Effects of Parental Arginine in Rats with Subacute Peritonitis

    PubMed Central

    Lo, Hui-Chen; Hung, Ching-Yi; Huang, Fu-Huan; Su, Tzu-Cheng; Lee, Chien-Hsing

    2016-01-01

    The combined treatment of parenteral arginine and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) have been shown to improve liver function and systemic inflammation in subacute peritonitic rats. Here, we investigated the effects of single and combined parenteral arginine and L-NAME treatments on leukocyte and splenocyte immunity. Male Wistar rats were subjected to cecal punctures and were intravenously given total parenteral nutrition solutions with or without arginine and/or L-NAME supplementations for 7 days. Non-surgical and sham-operated rats with no cecal puncture were given a chow diet and parenteral nutrition, respectively. Parenteral feeding elevated the white blood cell numbers and subacute peritonitis augmented the parenteral nutrition-induced alterations in the loss of body weight gain, splenomegaly, and splenocyte decreases. Parenteral arginine significantly increased the B-leukocyte level, decreased the natural killer T (NKT)-leukocyte and splenocyte levels, alleviated the loss in body weight gain and total and cytotoxic T-splenocyte levels, and attenuated the increases in plasma nitrate/nitrite and interferon-gamma production by T-splenocytes. L-NAME infusion significantly decreased NKT-leukocyte level, tumor-necrosis factor (TNF)-alpha production by T-splenocytes and macrophages, and interferon-gamma production by T-leukocytes, monocytes, and T-splenocytes, as well as increased interleukin-6 production by T-leukocytes and monocytes and nitrate/nitrite production by T-leukocytes. Combined treatment significantly decreased plasma nitrate/nitrite, the NKT-leukocyte level, and TNF-alpha production by T-splenocytes. Parenteral arginine may attenuate immune impairment and L-NAME infusion may augment leukocyte proinflammatory response, eliminate splenocyte proinflammatory and T-helper 1 responses, and diminish arginine-induced immunomodulation in combined treatment in subacute peritonitic rats. PMID:27007815

  10. Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

    PubMed Central

    Muregi, Francis W.; Ohta, Isao; Masato, Uchijima; Kino, Hideto; Ishih, Akira

    2011-01-01

    Background The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. Methodology/Principal Findings To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. Conclusions/Significance The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may

  11. A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP

    PubMed Central

    Woodard, Crystal; Liao, Gangling; Goodwin, C. Rory; Hu, Jianfei; Xie, Zhi; dos Reis, Thaila F.; Newman, Rob; Rho, Heesool; Qian, Jiang

    2015-01-01

    ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCE KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human

  12. Aldosterone increases the apical Na sup + permeability of toad bladder by two different mechanisms

    SciTech Connect

    Asher, C.; Garty, H. )

    1988-10-01

    The aldosterone-induced augmentation of Na{sup +} transport in toad bladder was analyzed by comparing the hormonal actions on the transepithelial short-circuit current and on the amiloride-sensitive {sup 22}Na{sup +} uptake in isolated membrane vesicles. Incubating bladders with 0.5 {mu}M aldosterone for 3 hr evoked more than a 2-fold increase of the short-circuit current but had no effect on the amiloride-sensitive Na{sup +} transport in apical vesicles derived from the treated tissue. A longer incubation produced an additional augmentation of the short-circuit current, which was accompanied by about a 3-fold increase of the channel activity in isolated membranes. The stimulatory effect of aldosterone sustained in vesicles was inhibited by the antagonist spironolactone and the protein synthesis inhibitor cycloheximide. It is suggested that aldosterone elevates the apical Na{sup +} permeability of target epithelia by two different mechanisms: a relatively fast effect which is insensitive to triiodothyronine or butyrate and is not sustained by the isolated membrane, and a slower or later response blocked by these reagents, which is preserved by the isolated membrane. The data also indicate that these processes are mediated by different nuclear receptors.

  13. Characteristics of aldosterone binding in rat and human serum.

    PubMed

    Coirini, H; White, A; Marusic, E T; De Nicola, A F

    1982-01-01

    Binding of cortisol and corticosterone by serum proteins is well established, but discrepancies exist regarding aldosterone. We have observed that approximately 1% of 3H-aldosterone incubated with rat serum was bound in a time-dependent process, although it was not competed by a large excess of non-radioactive aldosterone, assessed by Florisil separation or gel filtration on Sephadex G-50 columns. After electrophoresis on cellulose acetate of rat serum incubated with 3H-aldosterone, specific or non-specific binding to protein fractions was not obtained. Further, a 10 000-fold molar excess of aldosterone (10 microM) displaced only 34% of the bound 3H-aldosterone to rat serum, preventing the calculation of the IC50 value. Increasing concentrations of aldosterone (3-83 nM) did not displace 3H-corticosterone bound in rat serum to presumably corticosterone binding globulin (CBG). In contrast, inhibition of this binding by 3-83 nM corticosterone was concentration dependent, showing an IC50 value of 10(-8) M. In normal human serum, binding of 3H-aldosterone demonstrated competition by a 100 and 1 000-fold excess of aldosterone. Displacement curves of 3H corticosterone bound to human serum by 1.7-75 nM corticosterone or 0.05-8.8 microM aldosterone yielded IC50 values in the range of 10(-8) M for corticosterone and 10(-6) M for aldosterone. With horse serum, aldosterone's binding affinity was three orders of magnitude lower than that of corticosterone. These studies suggest that in the rat aldosterone was loosely and weakly bound to a high capacity binder, possibly albumin. In agreement with the work of others, in humans aldosterone may be bound to both CBG and albumin. The current data do not substantiate for the presence of specific aldosterone binding proteins in serum.

  14. Phosphanilic Acid Inhibits Dihydropteroate Synthase

    DTIC Science & Technology

    1989-11-01

    dihydropteroate synthases of P. aeruginosa and E . coli were about equally susceptible to inhibition by PA. These results suggest that cells of P. aeruginosa...are more permeable to PA than cells of E . coli . Although a weak inhibitor, PA acted on dihydropteroate synthase in the same manner as the sulfonamides...with which PA is structurally related. Inhibition of E . coli by PA in a basal salts-glucose medium was prevented by p-aminobenzoic acid (pABA). However

  15. The ratios of aldosterone / plasma renin activity (ARR) versus aldosterone / direct renin concentration (ADRR).

    PubMed

    Glinicki, Piotr; Jeske, Wojciech; Bednarek-Papierska, Lucyna; Kruszyńska, Aleksandra; Gietka-Czernel, Małgorzata; Rosłonowska, Elżbieta; Słowińska-Srzednicka, Jadwiga; Kasperlik-Załuska, Anna; Zgliczyński, Wojciech

    2015-12-01

    Primary aldosteronism (PA) is estimated to occur in 5-12% of patients with hypertension. Assessment of aldosterone / plasma renin activity (PRA) ratio (ARR) has been used as a screening test in patients suspected of PA. Direct determination of renin (DRC) and calculation of aldosterone / direct renin concentration ratio (ADRR) could be similarly useful for screening patients suspected of PA. The study included 62 patients with indication for evaluation of the renin-angiotensin-aldosterone system and 35 healthy volunteers. In all participants we measured concentrations of serum aldosterone, plasma direct renin, and PRA after a night's rest and again after walking for two hours. The concentrations of aldosterone, direct renin, and PRA were measured by isotopic methods (radioimmunoassay (RIA) / immunoradiometric assay (IRMA)). Correlations of ARR with ADRR in the supine position were r = 0.9162, r(2) = 0.8165 (p < 0.01); and in the up-right position were r = 0.7765, r(2) = 0.9153 (p < 0.01). The cut-off values of ARR and ADRR ≥ 100 presented highest specificity (99%) for the diagnosis of PA; however, quite acceptable specificity and sensitivity (> 80% and 100%, respectively) appeared for the ratios ≥ 30. We suggest that for practical and economic reasons ARR can be replaced by ADRR.

  16. Angiotensin receptor blocker drugs and inhibition of adrenal beta-arrestin-1-dependent aldosterone production: Implications for heart failure therapy

    PubMed Central

    Lymperopoulos, Anastasios; Aukszi, Beatrix

    2017-01-01

    Aldosterone mediates many of the physiological and pathophysiological/cardio-toxic effects of angiotensin II (AngII). Its synthesis and secretion from the zona glomerulosa cells of the adrenal cortex, elevated in chronic heart failure (HF), is induced by AngII type 1 receptors (AT1Rs). The AT1R is a G protein-coupled receptor, mainly coupling to Gq/11 proteins. However, it can also signal through β-arrestin-1 (βarr1) or -2 (βarr2), both of which mediate G protein-independent signaling. Over the past decade, a second, Gq/11 protein-independent but βarr1-dependent signaling pathway emanating from the adrenocortical AT1R and leading to aldosterone production has become appreciated. Thus, it became apparent that AT1R antagonists that block both pathways equally well are warranted for fully effective aldosterone suppression in HF. This spurred the comparison of all of the currently marketed angiotensin receptor blockers (ARBs, AT1R antagonists or sartans) at blocking activation of the two signaling modes (G protein-, and βarr1-dependent) at the AngII-activated AT1R and hence, at suppression of aldosterone in vitro and in vivo. Although all agents are very potent inhibitors of G protein activation at the AT1R, candesartan and valsartan were uncovered to be the most potent ARBs at blocking βarr activation by AngII and at suppressing aldosterone in vitro and in vivo in post-myocardial infarction HF animals. In contrast, irbesartan and losartan are virtually G protein-“biased” blockers at the human AT1R, with very low efficacy for βarr inhibition and aldosterone suppression. Therefore, candesartan and valsartan (and other, structurally similar compounds) may be the most preferred ARB agents for HF pharmacotherapy, as well as for treatment of other conditions characterized by elevated aldosterone.

  17. Effects of the nitric oxide synthase inhibitor L-NMMA on cerebrovascular and cardiovascular responses to hypoxia and hypercapnia in humans

    PubMed Central

    Ide, Kojiro; Worthley, Matthew; Anderson, Todd; Poulin, Marc J

    2007-01-01

    Cerebral blood flow is highly sensitive to alterations in the partial pressures of O2 and CO2 (PO2 and PCO2, respectively) in the arterial blood. In humans, the extent to which nitric oxide (NO) is involved in this regulation is unclear. We hypothesized that the NO synthase (NOS) inhibitor NG-monomethyl-l-arginine (l-NMMA), attenuates the sensitivity of middle cerebral artery blood velocity () to isocapnic hypoxia (end-tidal PO2 = 50 Torr) and euoxic hypercapnia (end-tidal PCO2 =+9 Torr above resting values) in 10 volunteers (age, 28.7 ± 1.3 years; height, 179.2 ± 2.4 cm; weight, 78.0 ± 3.7 kg; mean ±s.e.m.). The techniques of transcranial Doppler ultrasound and dynamic end-tidal forcing were used to measure , and control end-tidal PO2 and end-tidal PCO2, respectively. At baseline (isocapnic euoxia), following intravenous administration of l-NMMA, mean arterial blood pressure (MAP) increased (76.3 ± 7.3 to 86.2 ± 9.4 mmHg) and heart rate (HR) decreased (59.5 ± 9.0 to 55.2 ± 9.5 beats min−1) but was unchanged. Hypoxia-induced increases in MAP, HR and were similar with and without l-NMMA (5.0 ± 0.7 versus 7.1 ± 1.0 mmHg, 11.5 ± 1.4 versus 12.4 ± 1.5 beats min−1, 6.5 ± 0.8 versus 6.6 ± 0.8 cm s−1 for ΔMAP, ΔHR and Δ, respectively). Hypercapnia-induced increases in MAP, HR and were similar with and without l-NMMA (7.4 ± 3.1 versus 8.1 ± 2.2 mmHg, 10.4 ± 4.6 versus 10.0 ± 4.2 beats min−1, 16.5 ± 1.5 versus 17.6 ± 1.5 cm s−1 for ΔMAP, ΔHR and Δ, respectively) but the sensitivity of the response at the removal of hypercapnia was attenuated with l-NMMA. In young healthy humans, pharmacological blockade of nitric oxide synthesis does not affect the increases in cerebral blood flow with hypoxia and hypercapnia, suggesting that nitric oxide is not required for the cerbrovascular responses to hypoxia and hypercapnia. PMID:17673507

  18. Pulmonary hypertension triggered by lipopolysaccharide in ascites-susceptible and -resistant broilers is not amplified by aminoguanidine, a specific inhibitor of inducible nitric oxide synthase.

    PubMed

    Bowen, O T; Erf, G F; Anthony, N B; Wideman, R F

    2006-03-01

    Nitric oxide (NO) is a potent pulmonary vasodilator that modulates the pulmonary vasoconstriction and pulmonary hypertension (PH) triggered by bacterial lipopolysaccharide (LPS) in broilers. The amplitude and duration of the LPS-induced PH are markedly enhanced following pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME), which inhibits NO synthesis by both the constitutive (endothelial) and inducible (inflammatory) forms of nitric oxide synthase (eNOS and iNOS, respectively). In the present study L-NAME and the selective iNOS inhibitor aminoguanidine (AG) were administered to differentiate between iNOS and eNOS as the primary source of NO that attenuates the pulmonary vascular response to LPS. Clinically healthy male progeny from ascites-susceptible and ascites-resistant lines were anesthetized, and their pulmonary artery was cannulated. The initial pulmonary arterial pressure (PAP) was recorded, then the broilers either remained untreated (control group) or were injected i.v. with AG. Ten minutes later all birds received an i.v. injection of LPS, followed 40 min later by an i.v. injection of L-NAME. When compared with untreated controls, AG neither increased the baseline PAP nor did it increase or prolong the PH response to LPS. The ascites-susceptible broilers maintained a higher PAP than the ascites-resistant broilers throughout the experiment, and the ascites-resistant broilers exhibited greater relative increases in PAP in response to LPS than did the ascites-susceptible broilers. Within 40 min after the LPS injection, PAP subsided to a level that did not differ from the respective preinjection value for each line. Injecting L-NAME reversed the decline in PAP, and within 5 min PAP returned to hypertensive levels approaching the maximum peak PH response to LPS. The absence of any impact of AG coupled with the profound response to L-NAME indicates that NO synthesized by eNOS rather than iNOS likely modulated the acute (within 1 h) PH elicited by

  19. Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

    SciTech Connect

    Wu Defeng; Cederbaum, Arthur . E-mail: arthur.cederbaum@mssm.edu

    2006-10-15

    Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N {sup G}-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48 {+-} 6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23 {+-} 5%, while, SNAP or DETA-NONO increased viability to 66 {+-} 8 or 71 {+-} 6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA

  20. Hypertension induced by nitric oxide synthase inhibitor increases responsiveness of ventricular myocardium and aorta of rat tissue to adrenomedullin stimulation in vitro.

    PubMed

    Pan, Chun Shui; Jiang, Wei; Zhong, Guang Zhen; Zhao, Jing; Pang, Yong Zheng; Tang, Chao Shu; Qi, Yong Fen

    2005-12-12

    In this work, we aimed to observe the changes in adrenomedullin (ADM) and its receptor-calcitonin receptor-like receptor (CL), receptor activity-modifying protein (RAMP) 1, RAMP2 and RAMP3-in cardiac ventricles and aortas of hypertensive rats, and the responsiveness of injured cardiovascular tissue to ADM, then to illustrate the protective mechanism of ADM on the cardiovascular system. Male SD rats were subjected to treatment with chronic N(G)-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase. The ADM contents and cAMP production in myocardia and aortas were measured by RIA. The mRNA levels of ADM, CL, and RAMP1-3 were determined by RT-PCR. L-NNA induced severe hypertension and cardiomegaly. The ir-ADM content in plasma, ventricles and aortas in L-NNA-treated animals increased by 80%, 72% and 57% (all p<0.01), respectively. Furthermore, mRNA levels of ADM, CL, RAMP2 and RAMP3 were elevated by 91%, 33%, 50% and 72.5% (all p<0.01), respectively, in ventricles and by 95%, 177%, 74.7% and 85% (all p<0.01), respectively, in aortas. mRNA level of RAMP1 was elevated by 129% (p<0.01) in aortas but no significant difference in ventricles. The elevated mRNA levels of RAMP2 and RAMP3 were positively correlated with that of ADM in hypertrophic ventricles (r=0.633 and 0.828, p<0.01, respectively) and the elevated mRNA levels of CL, RAMP2 and RAMP3 were positively correlated with that of ADM in aortas (r=0.941, 0.943 and 0.736, all p<0.01, respectively). The response of ventricular myocardia and aortas to ADM administration potentiated, and the production of cAMP was increased by 41% and 68% (both p<0.01), respectively. ADM-stimulated cAMP generation in ventricular myocardia and aortas was blocked by administration of both ADM22-52, the specific antagonist of ADM receptor, and CGRP8-37, the antagonist of the CGRP1 receptor. The results showed an increased in cardiovascular ADM generation and an up-regulation of the gene expression of ADM and its receptor-CL, RAMP1

  1. Role of Genetic Polymorphism of Angiotensin-Converting Enzyme, Plasminogen Activator Inhibitor-1 and Endothelial Nitric Oxide Synthase in the Prognosis of Coronary Artery Disease

    PubMed Central

    Zhang, Ai Yuan; Ji, Xiang Wu; Zhang, Ai Juan; Guan, Li Xue; Huang, Jing; Wang, Jing Xian

    2010-01-01

    Background This study was to investigate the effects of multiple genetic polymorphisms and conventional risk factors in the prognosis of coronary artery disease (CAD). Methods One hundred and fifty five patients with CAD were prospectively recruited, they were subgrouped as single vessel disease (SVD) and multiple vessel disease (MVD). All patients were detected I/D polymorphism of angiotensin-converting enzyme (ACE) gene, 4G/5G polymorphism of plasminogen activator inhibitor-1 (PAI-1) gene, and G894→T mutation of endothelial nitric oxide synthase (eNOS) gene. The patients were followed up for 10-65 months, mean 35 months. End points were major adverse cardiovascular events (MACE), including angina, myocardial infarction, and cardiac sudden death. Results During the follow-up period, MACE developed in 81 patients, 73 patients with angina, seven with myocardial infarction, and one with cardiac sudden death. CAD patients with MVD were more probable of developing MACE during follow-up. Distribution of PAI-1 gene polymorphism was significantly different between SVD and MVD patients, p < 0.001. The frequency of DD genotype of ACE and 4G/4G genotype of PAI-1 in patients with MACE were significantly higher than those in patients without MACE, p < 0.001 and p = 0.002, respectively. Incidence of diabetes mellitus was significantly higher in patients with MACE than in patients without MACE, P = 0.03. Cox regression analysis showed that diabetes mellitus (HR 2.36, 95% CI 1.33-4.46, p = 0.003), 4G/4G polymorphism of PAI-1 gene (HR 3.45, 95% CI 1.71-6.56, p = 0.009), and D/D polymorphism of ACE gene (HR 2.99, 95% CI 1.84-5.76, p = 0.005), were independent predictors of the MACE. Conclusions Our results showed that the conventional risk factors and genetic polymorphisms have significant influence on prognosis of CAD patients. CAD patients with diabetes mellitus, DD genotype of ACE, and 4G/4G genotype of PAI-1 suggested poor prognosis.

  2. Upregulation of Steroidogenic Acute Regulatory Protein by Hypoxia Stimulates Aldosterone Synthesis in Pulmonary Artery Endothelial Cells to Promote Pulmonary Vascular Fibrosis

    PubMed Central

    Maron, Bradley A.; Oldham, William M.; Chan, Stephen Y.; Vargas, Sara O.; Arons, Elena; Zhang, Ying-Yi; Loscalzo, Joseph; Leopold, Jane A.

    2014-01-01

    Background The molecular mechanism(s) regulating hypoxia-induced vascular fibrosis are unresolved. Hyperaldosteronism correlates positively with vascular remodeling in pulmonary arterial hypertension (PAH), suggesting that aldosterone may contribute to the pulmonary vasculopathy of hypoxia. The hypoxia-sensitive transcription factors c-Fos/c-Jun regulate steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step of aldosterone steroidogenesis. We hypothesized that c-Fos/c-Jun upregulation by hypoxia activates StAR-dependent aldosterone synthesis in human pulmonary artery endothelial cells (HPAECs) to promote vascular fibrosis in PAH. Methods and Results Patients with PAH, rats with Sugen/hypoxia-PAH, and mice exposed to chronic hypoxia expressed increased StAR in remodeled pulmonary arterioles, providing a basis for investigating hypoxia-StAR signaling in HPAECs. Hypoxia (2.0% FiO2) increased aldosterone levels selectively in HPAECs, which was confirmed by liquid chromatography-mass spectrometry. Increased aldosterone by hypoxia resulted from enhanced c-Fos/c-Jun binding to the proximal activator protein (AP-1) site of the StAR promoter in HPAECs, which increased StAR expression and activity. In HPAECs transfected with StAR-siRNA or treated with the AP-1 inhibitor, SR-11302, hypoxia failed to increase aldosterone, confirming that aldosterone biosynthesis required StAR activation by c-Fos/c-Jun. The functional consequences of aldosterone were confirmed by pharmacological inhibition of the mineralocorticoid receptor with spironolactone or eplerenone, which attenuated hypoxia-induced upregulation of the fibrogenic protein connective tissue growth factor and collagen III in vitro, and decreased pulmonary vascular fibrosis to improve pulmonary hypertension in Conclusions Our findings identify autonomous aldosterone synthesis in HPAECs due to hypoxia-mediated upregulation of StAR as a novel molecular mechanism that promotes pulmonary vascular

  3. Effect of ions of potassium and lithium on NO synthase expression in the human adrenal cortex.

    PubMed

    Kovzun, E I; Lukashenya, O S; Pushkarev, V M; Mikosha, A S; Tron'ko, N D

    2014-01-01

    The expression of endothelial and inducible NO synthase in the human adrenal glands was studied under a change in the concentration of K(+), which plays a regulatory role in aldosterone secretion. K(+) ions stimulated the expression of both isoforms of NO synthase in the human adrenal cortex. A stimulatory effect of K(+) on NO synthase is probably related to activation of the calmodulin system and potassium-induced translocation of protein kinase C. Lithium produced n inhibitory effect on both isoforms of NO synthase, which suggests that protein kinase C serves a major regulator of expression in the human adrenal glands.

  4. From a natural product lead to the identification of potent and selective benzofuran-3-yl-(indol-3-yl)maleimides as glycogen synthase kinase 3beta inhibitors that suppress proliferation and survival of pancreatic cancer cells.

    PubMed

    Gaisina, Irina N; Gallier, Franck; Ougolkov, Andrei V; Kim, Ki H; Kurome, Toru; Guo, Songpo; Holzle, Denise; Luchini, Doris N; Blond, Sylvie Y; Billadeau, Daniel D; Kozikowski, Alan P

    2009-04-09

    Recent studies have demonstrated that glycogen synthase kinase 3beta (GSK-3beta) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3beta inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3beta and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3beta inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3beta inhibitors 5 and 26 resulted in suppression of GSK-3beta activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3beta inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders.

  5. Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting duct.

    PubMed

    Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

    2013-10-01

    Aldosterone increases tubular Na(+) absorption largely by increasing α-epithelial Na(+) channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

  6. Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS.

    PubMed

    Koda, Noriko; Tsutsui, Yasutaka; Niwa, Haruki; Ito, Seiji; Woodward, David F; Watanabe, Kikuko

    2004-04-15

    Prostaglandin (PG) D(2) ethanolamide (prostamide D(2)) was reduced to 9alpha,11beta-PGF(2) ethanolamide (9alpha,11beta-prostamide F(2)) by PGF synthase, which also catalyzes the reduction of PGH(2) and PGD(2) to PGF(2alpha) and 9alpha,11beta-PGF(2), respectively. These enzyme activities were measured by a new method, the liquid chromatographic-electrospray ionization-mass spectrometry (LC/ESI/MS) technique, which could simultaneously detect the substrate and all products. PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2) were separated on a TSKgel ODS 80Ts column, ionized by electrospray, and detected in the negative mode. Selected ion monitoring (SIM) of m/z 353 ([M-H](-)), 353 ([M-H](-)), 351 ([M-H](-)), 333 ([M-H-H(2)O](-)), 456 ([M+59](-)), and m/z 358 ([M-37](-)) was used for quantifying PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2), respectively. The detection limit for PGF(2alpha) and 9alpha,11beta-PGF(2) was 0.01pmol; that for PGH(2) and PGD(2), 0.1pmol; and that for prostamide D(2) and 9alpha,11beta-prostamide F(2), 0.5 and 0.03pmol, respectively. The LC/ESI/MS technique for measuring PGF synthase activity showed higher sensitivity than other methods. Using this method, we found that Bimatoprost, the ethyl amide analog of 17-phenyl-trinor PGF(2alpha) and an anti-glaucoma agent, inhibited all three reductase activities of PGF synthase when used at a low concentration. These results suggest that Bimatoprost also behaves as a potent PGF synthase inhibitor in addition to having prostamide-like activity.

  7. In vivo regulation of gene expression of enzymes controlling aldosterone synthesis in rat adrenal.

    PubMed

    LeHoux, J G; Tremblay, A

    1992-12-01

    We studied the effect of alterations in the intake of sodium and potassium as well as changes in circulating adrenocorticotropin (ACTH) on the expression of the two rate-limiting systems of aldosterone formation in the rat. Low sodium and high potassium intake promoted time-dependent increases in the zona glomerulosa cytochrome P450scc (P450scc) and cytochrome P450c11 (P450c11) protein and mRNA levels, but no changes were found in the zona fasciculata-reticularis. In addition, these responses were associated with markedly elevated transcriptional activities. To further define the contribution of P450c11 and P450c18 (aldosterone synthase) in response to these differing intakes, we evaluated their mRNA levels using gene-specific oligonucleotide probes. P450c18 mRNA was restricted to the zona glomerulosa, whereas P450c11 mRNA was detected in both zona glomerulosa and zona fasciculata-reticularis. Furthermore, only P450c18 mRNA was induced by both low sodium or high potassium intake, as P450c11 mRNA levels remained unchanged. Captopril, an inhibitor of angiotensin-I converting enzyme, abolished the enhancing effects of the low sodium regimen on P450scc and P450c18 mRNA levels. Captopril also suppressed the augmentation of P450c18 mRNA observed with potassium supplementation but had no effect on P450scc mRNA levels. When the hypocholesterolemic drug 4-aminopyrazolopyrimidine (4-APP) was administered to rats for 3 consecurive days, both the level of plasma ACTH and the adrenal content of mRNA encoding P450scc increased 24 h post final injection. The coadministration of dexamethasone with 4-APP prevented these increases. In contrast, the mRNA content of P450c11 remained at control levels. In conclusion, this work demonstrates that variations in the intake of sodium and potassium act on the expression of the CYP11B2 gene, but not on that of the CYP11B1 gene. Moreover angiotensin-II (A-II) is an important factor in this mechanism of action. Both ions also enhance the

  8. Identification of a Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitor, BIP-135, that Prolongs the Median Survival Time of Δ7 SMA KO Mouse Model of Spinal Muscular Atrophy.

    PubMed

    Chen, Po C; Gaisina, Irina N; El-Khodor, Bassem F; Ramboz, Sylvie; Makhortova, Nina R; Rubin, Lee L; Kozikowski, Alan P

    2012-01-18

    The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA, and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration.

  9. Albuminuria is associated with an increased prostasin in urine while aldosterone has no direct effect on urine and kidney tissue abundance of prostasin.

    PubMed

    Oxlund, Christina; Kurt, Birgül; Schwarzensteiner, Ilona; Hansen, Mie R; Stæhr, Mette; Svenningsen, Per; Jacobsen, Ib A; Hansen, Pernille B; Thuesen, Anne D; Toft, Anja; Hinrichs, Gitte R; Bistrup, Claus; Jensen, Boye L

    2017-02-24

    The proteinase prostasin is a candidate mediator for aldosterone-driven proteolytic activation of the epithelial sodium channel (ENaC). It was hypothesized that the aldosterone-mineralocorticoid receptor (MR) pathway stimulates prostasin abundance in kidney and urine. Prostasin was measured in plasma and urine from type 2 diabetic patients with resistant hypertension (n = 112) randomized to spironolactone/placebo in a clinical trial. Prostasin protein level was assessed by immunoblotting in (1) human and rat urines with/without nephrotic syndrome, (2) human nephrectomy tissue, (3) urine and kidney from aldosterone synthase-deficient (AS(-/-)) mice and ANGII- and aldosterone-infused mice, and in (4) kidney from adrenalectomized rats. Serum aldosterone concentration related to prostasin concentration in urine but not in plasma. Plasma prostasin concentration increased significantly after spironolactone compared to control. Urinary prostasin and albumin related directly and were reduced by spironolactone. In patients with nephrotic syndrome, urinary prostasin protein was elevated compared to controls. In rat nephrosis, proteinuria coincided with increased urinary prostasin, unchanged kidney tissue prostasin, and decreased plasma prostasin while plasma aldosterone was suppressed. Prostasin protein abundance in human nephrectomy tissue was similar across gender and ANGII inhibition regimens. Prostasin urine abundance was not different in AS(-/-) and aldosterone-infused mice. Prostasin kidney level was not different from control in adrenalectomized rats and AS(-/-) mice. We found no evidence for a direct relationship between mineralocorticoid receptor signaling and kidney and urine prostasin abundance. The reduction of urinary prostasin in spironolactone-treated patients is most likely the result of an improved glomerular filtration barrier function and generally reduced proteinuria.

  10. The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase.

    PubMed Central

    Thiemermann, C.; Ruetten, H.; Wu, C. C.; Vane, J. R.

    1995-01-01

    1. We have investigated whether (i) endotoxaemia caused by E. coli lipopolysaccharide in the anaesthetized rat causes a multiple organ dysfunction syndrome (MODS; e.g. circulatory failure, renal failure, liver failure), and (ii) an enhanced formation of nitric oxide (NO) due to induction of inducible NO synthase (iNOS) contributes to the MODS. In addition, this study elucidates the beneficial and adverse effects of aminoethyl-isothiourea (AE-ITU), a relatively selective inhibitor of iNOS activity, and NG-methyl-L-arginine (L-NMMA), a non-selective inhibitor of NOS activity on the MODS caused by endotoxaemia. 2. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 117 +/- 3 mmHg (time 0) to 97 +/- 4 mmHg at 2 h (P < 0.05, n = 15) and 84 +/- 4 mmHg at 6 h (P < 0.05, n = 15). The pressor effect of noradrenaline (NA, 1 micrograms kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with AE-ITU (1 mg kg-1, i.v. plus 1 mg kg-1 h-1 starting at 2 h after LPS) caused only a transient rise in MAP, but significantly attenuated the delayed vascular hyporeactivity seen in LPS-rats. Infusion of L-NMMA (3 mg kg-1, i.v. plus 3 mg kg-1 h-1) caused a rapid and sustained rise in MAP and attenuated the delayed vascular hyporeactivity to NA. Neither AE-ITU nor L-NMMA had any effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 3. Endotoxaemia for 6 h was associated with a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT), gamma-glutamyl-transferase (gamma GT), and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with AE-ITU significantly attenuated this liver dysfunction (rise in GOT, GPT, gamma GT and bilirubin) (P < 0.05, n = 10). In contrast, L-NMMA reduced the increase in the serum levels of gamma GT and bilirubin, but not in GOT and GPT (n = 5). Injection of LPS also caused a

  11. Attenuation of endotoxin-induced multiple organ dysfunction by 1-amino-2-hydroxy-guanidine, a potent inhibitor of inducible nitric oxide synthase.

    PubMed Central

    Ruetten, H.; Southan, G. J.; Abate, A.; Thiemermann, C.

    1996-01-01

    1. We have investigated the effects of (i) several guanidines on the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) in murine cultured macrophages and rat aortic vascular smooth muscle cells (RASM); and (ii) 1-amino-2-hydroxy-guanidine, the most potent inhibitor of iNOS activity discovered, on haemodynamics, multiple organ (liver, renal, and pancreas) dysfunction and iNOS activity in rats with endotoxic shock. 2. The synthesized guanidine analogues caused concentration-dependent inhibitions of the increase in nitrite formation caused by lipopolysaccaride (LPS, 1 microgram ml-1) in J774.2 macrophages and RASM cells with the following rank order of potency: 1-amino-2-hydroxy-guanidine > 1-amino-2-methyl-guanidine > 1-amino-1-methyl-guanidine > 1-amino-1,2-dimethyl-guanidine. Interestingly, 1-amino-2-hydroxy-guanidine (IC50: J774.2, 68 microM; RASM, 114 microM) was more potent in inhibiting nitrite formation caused by LPS than NG-methyl-L-arginine, but less potent than aminoethyl-isothiourea. 3. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 115 +/- 4 mmHg (time 0) to 98 +/- 5 mmHg at 2 h (P < 0.05, n = 10) and 69 +/- 5 mmHg at 6 h (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 mg kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine (10 mg kg-1, i.v. plus 10 mg kg-1 h-1 starting at 2 h after LPS) prevented the delayed hypotension and vascular hyporeactivity seen in LPS-rats. However, 1-amino-2-hydroxy-guanidine had no effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 4. Endotoxaemia for 6 h caused a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT) and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine significantly attenuated the liver dysfunction caused

  12. JS ISH-ECCR-4 THE PLASMA ALDOSTERONE / ANGIOTENSIN II RATIO FOR THE SCREENING OF SECONDARY HYPERTENSION.

    PubMed

    Poglitsch, Marko

    2016-09-01

    the portion of physiologically active angiotensin II in a patient sample. The performance of the AA2-Ratio is superior to the ARR in terms of the diagnostic window, which can be explained by the much closer functional link between aldosterone and angiotensin II comparing to the molecular distant relationship between aldosterone and renin. It has been shown that the correlation between renin concentration or activity and angiotensin II is poor especially in patients treated with anti-hypertensive drugs. In sharp contrast, the AA2-Ratio does not interfere with first line anti-hypertensive drugs including ACE inhibitors.The AA2-Ratio has the potential to be superior to the ARR in terms of its ability to better reflecting the activity of the true direct circulating regulator of aldosterone (i.e. angiotensin II rather than renin), and being unaffected by antihypertensive medications including ACE inhibitors. Data obtained in proof-of-concept studies proved the AA2-Ratio to be a powerful and cost-effective diagnostic tool for the diagnosis of PA among hypertensive patients. The stability of the AA2-Ratio in the presence of ACE-inhibition points to a potential use of the AA2-Ratio PA screening in hypertensive patients without ACE-inhibitor discontinuation.

  13. The Regulation of Aldosterone Secretion in Anephric Man

    PubMed Central

    Bayard, Francis; Cooke, C. Robert; Tiller, David J.; Beitins, Inese Z.; Kowarski, Avinoam; Walker, W. Gordon; Migeon, Claude J.

    1971-01-01

    The regulation of aldosterone secretion in anephric man was investigated in studies on nephrectomized patients who were being intermittently hemodialyzed while awaiting renal transplantation. The effects of supine and upright posture on the concentration of plasma aldosterone on the 1st day postdialysis and on a 3rd or 4th day postdialysis were compared to the effects of postural variation in normal subjects who were on a low sodium intake and on a high sodium intake. In contrast with the normal subjects who exhibited higher concentrations of plasma aldosterone after 2 hr of upright posture than in the supine position and low concentrations of plasma aldosterone on a high sodium intake, the anephric patients showed less consistent variations in plasma aldosterone due to changes in posture and exhibited higher concentrations of plasma aldosterone on the 3rd or 4th day postdialysis, despite an increase in body weight, than on the 1st day postdialysis. The increase in the concentration of plasma aldosterone in the anephric patients between the 1st day postdialysis and the 3rd or 4th day postdialysis indicates that aldosterone secretion is not responding primarily, in this situation, to volume-related stimuli. There was a high degree of correlation between the concentration of plasma aldosterone and the corresponding levels of serum potassium concentration, which also rose significantly between the 1st day postdialysis and the 3rd or 4th day postdialysis. Furthermore, when potassium accumulation between dialyses was prevented in three of these patients, the concentration of plasma aldosterone fell to minimally detectable levels. The results of these studies suggest that the primary regulator of aldosterone secretion in the absence of the kidneys is potassium. PMID:4329001

  14. Aldosterone Inhibits the Fetal Program and Increases Hypertrophy in the Heart of Hypertensive Mice

    PubMed Central

    Azibani, Feriel; Devaux, Yvan; Coutance, Guillaume; Schlossarek, Saskia; Polidano, Evelyne; Fazal, Loubina; Merval, Regine; Carrier, Lucie; Solal, Alain Cohen; Chatziantoniou, Christos; Launay, Jean-Marie; Samuel, Jane-Lise; Delcayre, Claude

    2012-01-01

    Background Arterial hypertension (AH) induces cardiac hypertrophy and reactivation of “fetal” gene expression. In rodent heart, alpha-Myosin Heavy Chain (MyHC) and its micro-RNA miR-208a regulate the expression of beta-MyHC and of its intronic miR-208b. However, the role of aldosterone in these processes remains unclear. Methodology/Principal Findings RT-PCR and western-blot were used to investigate the genes modulated by arterial hypertension and cardiac hyperaldosteronism. We developed a model of double-transgenic mice (AS-Ren) with cardiac hyperaldosteronism (AS mice) and systemic hypertension (Ren). AS-Ren mice had increased (x2) angiotensin II in plasma and increased (x2) aldosterone in heart. Ren and AS-Ren mice had a robust and similar hypertension (+70%) versus their controls. Anatomical data and echocardiography showed a worsening of cardiac hypertrophy (+41%) in AS-Ren mice (P<0.05 vs Ren). The increase of ANP (x 2.5; P<0.01) mRNA observed in Ren mice was blunted in AS-Ren mice. This non-induction of antitrophic natriuretic peptides may be involved in the higher trophic cardiac response in AS-Ren mice, as indicated by the markedly reduced cardiac hypertrophy in ANP-infused AS-Ren mice for one month. Besides, the AH-induced increase of ßMyHC and its intronic miRNA-208b was prevented in AS-Ren. The inhibition of miR 208a (−75%, p<0.001) in AS-Ren mice compared to AS was associated with increased Sox 6 mRNA (x 1.34; p<0.05), an inhibitor of ßMyHC transcription. Eplerenone prevented all aldosterone-dependent effects. Conclusions/Significance Our results indicate that increased aldosterone in heart inhibits the induction of atrial natriuretic peptide expression, via the mineralocorticoid receptor. This worsens cardiac hypertrophy without changing blood pressure. Moreover, this work reveals an original aldosterone-dependent inhibition of miR-208a in hypertension, resulting in the inhibition of β-myosin heavy chain expression through the induction of

  15. Polyphosphoester-based cationic nanoparticles serendipitously release integral biologically-active components to serve as novel degradable inducible nitric oxide synthase inhibitors.

    PubMed

    Shen, Yuefei; Zhang, Shiyi; Zhang, Fuwu; Loftis, Alexander; Pavía-Sanders, Adriana; Zou, Jiong; Fan, Jingwei; Taylor, John-Stephen A; Wooley, Karen L

    2013-10-18

    A degradable polyphosphoester (PPE)-based cationic nanoparticle (cSCK), which is integrated constructed as a novel degradable drug device, demonstrates surprisingly efficient inhibition of inducible nitric oxide synthase (iNOS) transcription, and eventually inhibits nitric oxide (NO) over-production, without loading of any specific therapeutic drugs. This system may serve as a promising anti-inflammatory agent toward the treatment of acute lung injury.

  16. Central interactions of aldosterone and angiotensin II in aldosterone- and angiotensin II-induced hypertension.

    PubMed

    Xue, Baojian; Beltz, Terry G; Yu, Yang; Guo, Fang; Gomez-Sanchez, Celso E; Hay, Meredith; Johnson, Alan Kim

    2011-02-01

    Many studies have implicated both angiotensin II (ANG II) and aldosterone (Aldo) in the pathogenesis of hypertension, the progression of renal injury, and cardiac remodeling after myocardial infarction. In several cases, ANG II and Aldo have been shown to have synergistic interactions in the periphery. In the present studies, we tested the hypothesis that ANG II and Aldo interact centrally in Aldo- and ANG II-induced hypertension in male rats. In rats with blood pressure (BP) and heart rate (HR) measured by DSI telemetry, intracerebroventricular (icv) infusions of the mineralocorticoid receptor (MR) antagonists spironolactone and RU28318 or the angiotensin type 1 receptor (AT1R) antagonist irbesartan significantly inhibited Aldo-induced hypertension. In ANG II-induced hypertension, icv infusion of RU28318 significantly reduced the increase in BP. Moreover, icv infusions of the reactive oxygen species (ROS) scavenger tempol or the NADPH oxidase inhibitor apocynin attenuated Aldo-induced hypertension. To confirm these effects of pharmacological antagonists, icv injections of either recombinant adeno-associated virus carrying siRNA silencers of AT1aR (AT1aR-siRNA) or MR (MR-siRNA) significantly attenuated the development of Aldo-induced hypertension. The immunohistochemical and Western blot analyses of AT1aR-siRNA- or MR-siRNA-injected rats showed a marked reduction in the expression of AT1R or MR in the paraventricular nucleus compared with scrambled siRNA rats. When animals from all studies underwent ganglionic blockade with hexamethonium, there was a smaller reduction in the fall of BP in animals receiving icv AT1R or MR antagonists. These results suggest that ANG II and Aldo interact in the brain in a mutually cooperative manner such that the functional integrity of both brain AT1R and MR are necessary for hypertension to be induced by either systemic ANG II or Aldo. The pressor effects produced by systemic ANG II or Aldo involve increased central ROS and

  17. Renin-Angiotensin-aldosterone system in autosomal dominant polycystic kidney disease.

    PubMed

    Tkachenko, Oleksandra; Helal, Imed; Shchekochikhin, Dmitry; Schrier, Robert W

    2013-02-01

    Autosomal dominant polycystic kidney disease is the most frequent life-threatening hereditary disease. Prognostic factors for progressive renal impairment have been identified such as gender, race, age, proteinuria, hematuria, hypertension. Hypertension is the only risk factor for renal dysfunction in autosomal dominant polycystic kidney disease, which is presently treatable. Better understanding of the pathophysiology of hypertension will help in defining appropriate interventions. The renin-angiotensin-aldosterone-system is the pivotal factor in the pathogenesis of hypertension in autosomal dominant polycystic kidney disease. Basic research and clinical studies in autosomal dominant polycystic kidney disease implicated activation of the renin-angiotensin-aldosterone-system. Therapy of hypertension in autosomal dominant polycystic kidney disease with angiotensin-converting enzyme inhibitors or angiotensin receptor blocker has the potential to prevent cardiovascular complications and slow the progression of renal disease. The results of two large multicenter double-blind placebo controlled randomized clinical trials (the HALT-PKD trials) possibly will elucidate the beneficial effects of the renin-angiotensin-aldosterone-system inhibition in autosomal dominant polycystic kidney disease.

  18. Azilsartan, aliskiren, and combination antihypertensives utilizing renin-angiotensin-aldosterone system antagonists.

    PubMed

    Lanier, Gregg; Sankholkar, Kedar; Aronow, Wilbert S

    2014-01-01

    Health care providers managing hypertension (HTN) have a large selection of pharmacologic agents to choose from, including several different classes of drugs and many similar drugs within each class. Antagonism of the renin-angiotensin-aldosterone system has been shown to be very effective for HTN, especially in patients with cardiovascular disease, diabetes, and heart failure. Within this group, there have been 2 new agents recently introduced to the US market and approved by the Food and Drug Administration. It is important for the HTN specialist to be familiar with the merits of these 2 drugs: the angiotensin receptor blocker Edarbi (azilsartan) and the renin inhibitor Tekturna (aliskiren). Additionally, there have been several new, fixed-dose combination antihypertensives introduced to the market since 2006 that use a renin-angiotensin-aldosterone antagonist. Seven of these combine 2 drugs together in a single pill: Edarbyclor (azilsartan/chlorthalidone), Exforge (amlodipine/valsartan), Azor (olmesartan/amlodipine), Twynsta (amlodipine/telmisartan), Tekturna HCT [aliskiren/hydrochlorothiazide (HCTZ)], Valturna (aliskiren/valsartan), Tekamlo (aliskiren/amlodipine). Three triple-drug combination medications have also been introduced recently: Exforge HCT (amlodipine/valsartan/HCTZ), Tribenzor (olmesartan/amlodipine/HCTZ), and Amturnide (aliskiren/amlodipine/hydrocholorothiazide). This review will summarize the trial data and important pharmacologic merits of these 2 new renin-angiotensin-aldosterone antagonists and the advantages of initiating treatment with one of the new fixed-dose, combination drugs approved over the last 5 years.

  19. Synthesis and evaluation of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines as receptor tyrosine kinase and thymidylate synthase inhibitors and as antitumor agents.

    PubMed

    Zaware, Nilesh; Kisliuk, Roy; Bastian, Anja; Ihnat, Michael A; Gangjee, Aleem

    2017-04-01

    In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3-7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed.

  20. Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase - The importance of accommodating the active site water.

    PubMed

    Heyes, Logan C; Reichau, Sebastian; Cross, Penelope J; Jameson, Geoffrey B; Parker, Emily J

    2014-12-01

    3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9±0.4μM. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated.

  1. LH, progesterone, and TSH can stimulate aldosterone in vitro: a study on normal adrenal cortex and aldosterone producing adenoma.

    PubMed

    Nicolini, G; Balzan, S; Morelli, L; Iacconi, P; Sabatino, L; Ripoli, A; Fommei, E

    2014-05-01

    Endocrine factors different from ACTH or angiotensin II can stimulate aldosterone secretion and have a role in the pathophysiology of hyperaldosteronism. Aldosterone may increase in luteotropic/progestogenic and in hypothyroid states; LH and, occasionally, TSH receptors have been detected in normal adrenal cortex and aldosterone-producing adenoma. The aim of the study was to compare adrenal contents of LH and TSH receptors between normal cortex and aldosterone-producing adenoma and to evaluate the ability of LH, its product progesterone, and TSH to stimulate aldosterone secretion in vitro from primary adrenocortical cells. Surgical aldosterone-producing adenoma fragments from 19 patients and adrenal cortex fragments from 10 kidney donors were used for Western blotting and cell cultures. LH (n=26), TSH (n=19) and progesterone (n=8) receptor proteins were investigated; LH receptor-mRNA was also tested in 8 samples. Aldosterone responses in vitro to LH, progesterone, and TSH stimulation were assayed. LH and TSH receptors were more expressed in adenoma than normal cortex (p<0.01, p<0.05, respectively); progesterone receptor was observed in 6/8 samples. Aldosterone increased after in vitro stimulation with LH (5/12 adenoma, 1/7 normal cells), progesterone (4/5 adenoma, 5/6 normal cells), and TSH (3/5 adenoma and 3/5 normal cells). LH and TSH receptors were more expressed in aldosterone producing adenoma than normal adrenal cortex. LH, progesterone, and TSH can stimulate aldosterone in vitro. Similar mechanisms could participate in vivo in the aldosterone increase in lutheotropic, progestogenic, or hypothyroid states and may exist in both normal adrenal cortex and adenoma in responsive individuals.

  2. Aldosterone does not modify gene expression in human endothelial cells.

    PubMed

    Verhovez, A; Williams, T A; Morello, F; Monticone, S; Brizzi, M F; Dentelli, P; Fallo, F; Fabris, B; Amenta, F; Gomez-Sanchez, C; Veglio, F; Mulatero, P

    2012-03-01

    The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

  3. Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

    SciTech Connect

    Rajendran, V.M.; Kashgarian, M.; Binder, H.J. )

    1989-11-05

    Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.

  4. Hyperkalaemia in the age of aldosterone antagonism.

    PubMed

    Chapagain, A; Ashman, N

    2012-11-01

    Hyperkalaemia is well recognized as a medical emergency. However, with the publication of trials showing benefit with renin-aldosterone axis suppression in heart failure, the epidemiology of patients presenting with hyperkalaemia has changed. The reported incidence of rate of serious hyperkalaemia (>6.0 mEq/l of potassium) ranges from 6 to 12% in patients on spironolactone with congestive cardiac failure (CCF). A rational choice of therapy based on present evidence is different from the traditionally used algorithm, given our understanding of the physiology relevant to this patient group. This article discusses the changing face of hyperkalaemia and the present evidence and discusses options in treatment of hyperkalaemia.

  5. Aldosterone and mineralocorticoid receptors in the cardiovascular system.

    PubMed

    Funder, John W

    2010-01-01

    Aldosterone is currently thought to exert its physiologic effects by activating epithelial mineralocorticoid receptors, and its pathologic effects on the cardiovascular system via mineralocorticoid receptors in the heart and blood vessels. Recent studies have extended this understanding to include a reevaluation of the roles of aldosterone and mineralocorticoid receptor activation in blood pressure control; the rapid, nongenomic effects of aldosterone; the role of cortisol as a mineralocorticoid receptor agonist under conditions of redox change/tissue damage/reactive oxygen species generation; the growing consensus that primary aldosteronism accounts for approximately 10% of all essential hypertension; recent new insights into the cardioprotective role of spironolactone; and the development of third- and fourth-generation mineralocorticoid receptor antagonists for use in cardiovascular and other inflammatory disease. These findings on aldosterone action and mineralocorticoid receptor blockade are analyzed in the context of the prevention and treatment of cardiovascular disease.

  6. Effects of ACTH on the last step of aldosterone biosynthesis.

    PubMed

    Cozza, E N; Ceballos, N R; Lantos, C P

    1989-12-01

    The production of tritiated aldosterone and tritiated SM (a saponifiable 18-hydroxycorticosterone derivative) by rat adrenals were studied at various incubation times in absence or presence of two concentrations of ACTH. Tritiated 18-hydroxycorticosterone or 18-deoxyaldosterone served as precursors. The lower ACTH concentration (150 pM) increased the production of tritiated aldosterone. Whereas, the higher ACTH concentration (1.5 microM) stimulated tritiated aldosterone production at shorter incubation time (30 min), while after 60 min it inhibited. This time dependency would reflect variations in the levels of endogenous steroids. On the other hand, the effects of ACTH on tritiated SM production were opposite to those on tritiated aldosterone. In effect, while 150 pM ACTH inhibited SM production, 1.5 microM ACTH stimulated it. These results suggest that ACTH promotes opposite effects on the productions of aldosterone and SM and therefore both productions would be coordinated under the regulation of ACTH.

  7. Triphenylethanamine Derivatives as Cholesteryl Ester Transfer Protein Inhibitors: Discovery of N-[(1R)-1-(3-Cyclopropoxy-4-fluorophenyl)-1-[3-fluoro-5-(1,1,2,2-tetrafluoroethoxy)phenyl]-2-phenylethyl]-4-fluoro-3-(trifluoromethyl)benzamide (BMS-795311).

    PubMed

    Qiao, Jennifer X; Wang, Tammy C; Adam, Leonard P; Chen, Alice Ye A; Taylor, David S; Yang, Richard Z; Zhuang, Shaobin; Sleph, Paul G; Li, Julia P; Li, Danshi; Yin, Xiaohong; Chang, Ming; Chen, Xue-Qing; Shen, Hong; Li, Jianqing; Smith, Daniel; Wu, Dauh-Rurng; Leith, Leslie; Harikrishnan, Lalgudi S; Kamau, Muthoni G; Miller, Michael M; Bilder, Donna; Rampulla, Richard; Li, Yi-Xin; Xu, Carrie; Lawrence, R Michael; Poss, Michael A; Levesque, Paul; Gordon, David A; Huang, Christine S; Finlay, Heather J; Wexler, Ruth R; Salvati, Mark E

    2015-11-25

    Cholesteryl ester transfer protein (CETP) inhibitors raise HDL-C in animals and humans and may be antiatherosclerotic by enhancing reverse cholesterol transport (RCT). In this article, we describe the lead optimization efforts resulting in the discovery of a series of triphenylethanamine (TPE) ureas and amides as potent and orally available CETP inhibitors. Compound 10g is a potent CETP inhibitor that maximally inhibited cholesteryl ester (CE) transfer activity at an oral dose of 1 mg/kg in human CETP/apoB-100 dual transgenic mice and increased HDL cholesterol content and size comparable to torcetrapib (1) in moderately-fat fed hamsters. In contrast to the off-target liabilities with 1, no blood pressure increase was observed with 10g in rat telemetry studies and no increase of aldosterone synthase (CYP11B2) was detected in H295R cells. On the basis of its preclinical profile, compound 10g was advanced into preclinical safety studies.

  8. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

  9. Structure-based design and synthesis of N(omega)-nitro-L-arginine-containing peptidomimetics as selective inhibitors of neuronal nitric oxide synthase. Displacement of the heme structural water.

    PubMed

    Seo, Jiwon; Igarashi, Jotato; Li, Huiying; Martasek, Pavel; Roman, Linda J; Poulos, Thomas L; Silverman, Richard B

    2007-05-03

    The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N'-nitroguanidine (1) and 4-N-(Nomega-nitro-l-argininyl)-trans-4-amino-l-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). On the basis of this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3-5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 5 and 6). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures.

  10. Purification, kinetics, inhibitors and CD for recombinant β-amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases.

    PubMed

    Ito, Ryousuke; Masukawa, Yukari; Hoshino, Tsutomu

    2013-03-01

    β-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant β-amyrin synthase. The β-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 μm and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The β-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the

  11. Human Adrenocortical Remodeling Leading to Aldosterone-Producing Cell Cluster Generation

    PubMed Central

    Hayashi, Yuichiro; Al-Eyd, Ghaith; Nakagawa, Ken; Morita, Shinya; Kosaka, Takeo; Oya, Mototsugu; Mitani, Fumiko; Suematsu, Makoto; Kabe, Yasuaki

    2016-01-01

    Background. The immunohistochemical detection of aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1) has enabled the identification of aldosterone-producing cell clusters (APCCs) in the subcapsular portion of the human adult adrenal cortex. We hypothesized that adrenals have layered zonation in early postnatal stages and are remodeled to possess APCCs over time. Purposes. To investigate changes in human adrenocortical zonation with age. Methods. We retrospectively analyzed adrenal tissues prepared from 33 autopsied patients aged between 0 and 50 years. They were immunostained for CYP11B2 and CYP11B1. The percentage of APCC areas over the whole adrenal area (AA/WAA, %) and the number of APCCs (NOA, APCCs/mm2) were calculated by four examiners. Average values were used in statistical analyses. Results. Adrenals under 11 years old had layered zona glomerulosa (ZG) and zona fasciculata (ZF) without apparent APCCs. Some adrenals had an unstained (CYP11B2/CYP11B1-negative) layer between ZG and ZF, resembling the rat undifferentiated cell zone. Average AA/WAA and NOA correlated with age, suggesting that APCC development is associated with aging. Possible APCC-to-APA transitional lesions were incidentally identified in two adult adrenals. Conclusions. The adrenal cortex with layered zonation remodels to possess APCCs over time. APCC generation may be associated with hypertension in adults. PMID:27721827

  12. Aldosterone and the cardiovascular system: a dangerous association.

    PubMed

    Cachofeiro, Victoria; López-Andrés, Natalia; Miana, Maria; Martín-Fernández, Beatriz; de Las Heras, Natalia; Martínez, Ernesto; Lahera, Vicente; Fortuño, María Antonia

    2010-12-01

    Initial studies have focussed on the actions of aldosterone in renal electrolyte handling and, as a consequence, blood pressure control. More recently, attention has primarily been focussed on its actions on the heart and vascular system, where it is locally produced. Aldosterone by binding mineralocorticoid receptors causes oxidative stress, fibrosis and triggers an inflammatory response in the cardiovascular system. All these effects could be underlying the role of aldo-sterone on cardiac and vascular remodelling associated with different pathological situations. At the vascular level, aldo-sterone affects endothelial function because administration of aldosterone to rats impaired endothelium-dependent relaxations. In addition, the administration of mineralocorticoid receptor antagonists ameliorates endothelium-dependent relaxation in models of both hypertension and atherosclerosis, and in patients with heart failure. Several mechanisms can participate in this effect, including production of vasoconstrictor factors and a reduction in nitric oxide levels. This reduction can involve both a decrease in its production as well as an increase in its degradation by reactive oxygen species. Aldosterone can produce oxidative stress by the activation of transcription factors such as the NF-κB system, which can also trigger an inflammatory process through the production of different cytokines. At cardiac level, high levels of aldosterone can also adversely impact heart function by producing cardiac hypertrophy, diastolic dysfunction and electrical remodelling through changes in ionic channels. All these effects can explain the beneficial effect of mineralocorticoid blockade in the cardiovascular system.

  13. Nocturnal oscillations of plasma aldosterone in relation to sleep stages.

    PubMed

    Krauth, M O; Saini, J; Follenius, M; Brandenberger, G

    1990-10-01

    This study examined the relationship between aldosterone secretion and sleep stages in conjunction with two aldosterone regulating hormone systems, the renin-angiotensin system (RAS) and adrenocorticotropin (ACTH), and also K+. Nocturnal plasma patterns of aldosterone, plasma renin activity (PRA), ACTH and K+ were established in blood collected at 10-min intervals in two groups of 6 subjects. Both groups underwent two 9 hour overnight-studies, consisting of one control night and one experimental night. The first group was maintained on a low Na diet and the other was given a beta-blocker, atenolol. Polygraphic recordings of sleep were scored according to established criteria. For the control night, REM sleep usually began at peak level or in the descending phase of aldosterone oscillations. As previously described, PRA reflected REM-NREM sleep alteration, levels increased in NREM and decreased during REM sleep. ACTH fluctuations did not oscillate with sleep stages, but levels were very seldom in the ascending phase at REM sleep onset. Plasma K+ remained almost constant throughout the night. The relative importance of the ACTH and the RAS on nocturnal aldosterone secretion and the relationship between aldosterone oscillations and sleep stages remained unclear. Modulating renin levels by either consuming a low Na diet or administration of a beta-blocker enabled this relationship to be clarified. The RAS dominated aldosterone secretion when stimulated by a low sodium diet. Aldosterone oscillations then reflected PRA oscillations with a delay of about 20 min and the relationship of aldosterone to sleep stages was dependent on the relationship of PRA with sleep stages.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Treatment with the nitric oxide synthase inhibitor L-NAME provides a survival advantage in a mouse model of Kras mutation-positive, non-small cell lung cancer

    PubMed Central

    Xu, MengMeng; Counter, Christopher M.

    2016-01-01

    Oncogenic mutations in the gene KRAS are commonly detected in non-small cell lung cancer (NSCLC). This disease is inherently difficult to treat, and combinations involving platinum-based drugs remain the therapeutic mainstay. In terms of novel, pharmacologically actionable targets, nitric oxide synthases (NOS) have been implicated in the etiology of KRAS-driven cancers, including lung cancer, and small molecular weight NOS inhibitors have been developed for the treatment of other diseases. Thus, we evaluated the anti-neoplastic activity of the oral NOS inhibitor L-NAME in a randomized preclinical trial using a genetically engineered mouse model of Kras and p53 mutation-positive NSCLC. We report here that L-NAME decreased lung tumor growth in vivo, as assessed by sequential radiological imaging, and provided a survival advantage, perhaps the most difficult clinical parameter to improve upon. Moreover, L-NAME enhanced the therapeutic benefit afforded by carboplatin chemotherapy, provided it was administered as maintenance therapy after carboplatin. Collectively, these results support the clinical evaluation of L-NAME for the treatment of KRAS mutation-positive NSCLC. PMID:27285753

  15. Role of polymorphisms in factor V (FV Leiden), prothrombin, plasminogen activator inhibitor type-1 (PAI-1), methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) genes as risk factors for thrombophilias.

    PubMed

    Miranda-Vilela, A L

    2012-09-01

    Thrombophilias are defined as a predisposition to thrombosis due to hematological changes which induce blood hypercoagulability; they can be inherited or acquired. They are individually characterized by a large phenotypic variability, even when they occur within the same family. Hereditary thrombophilias are, in most cases, due to changes related to physiological coagulation inhibitors or mutations in the genes of coagulation factors. High levels of plasma homocysteine may also be responsible for vaso-occlusive episodes and may have acquired (nutritional deficiencies of folate and vitamins B6 and B12) and/or genetic causes (mutations in the genes responsible for expression of enzymes involved in the intracellular metabolism of homocysteine). Considering that: (1) thromboses are events of multigenic and multifactorial etiopathology; (2) the presence of mutations in several genes significantly increases the risk of their occurrence; (3) the vascular territory (venous and/or arterial) affected involves different pathophysiological mechanisms and treatments, knowledge of genetic variants that may contribute to the risk and variability of the phenotypic manifestations of these diseases is extremely important. This understanding may provide support for a more individualized and therefore more effective treatment for thrombophilia carriers. Thus, this mini-review aims to address a comprehensive summary of thrombophilias and thrombosis, and discuss the role of polymorphisms in Factor V (FV Leiden), Prothrombin, Plasminogen activator inhibitor type-1 (PAI-1), Methylenetetrahydrofolate reductase (MTHFR) and Cystathionine β-synthase (CBS) genes as risk factors for thrombophilias.

  16. A head-to-head comparison of eneamide and epoxyamide inhibitors of glucosamine-6-phosphate synthase from the dapdiamide biosynthetic pathway.

    PubMed

    Hollenhorst, Marie A; Ntai, Ioanna; Badet, Bernard; Kelleher, Neil L; Walsh, Christopher T

    2011-05-17

    The dapdiamides make up a family of antibiotics that have been presumed to be cleaved in the target cell to enzyme-inhibitory N-acyl-2,3-diaminopropionate (DAP) warheads containing two alternative electrophilic moieties. Our prior biosynthetic studies revealed that an eneamide warhead is made first and converted to an epoxyamide via a three-enzyme branch pathway. Here we provide a rationale for this logic. We report that the R,R-epoxyamide warhead is a more efficient covalent inactivator of glucosamine-6-phosphate synthase by 1 order of magnitude versus the eneamide, and this difference correlates with a >10-fold difference in antibiotic activity for the corresponding acyl-DAP dipeptides.

  17. Structural, functional, and molecular alterations produced by aldosterone plus salt in rat heart: association with enhanced serum and glucocorticoid-regulated kinase-1 expression.

    PubMed

    Martín-Fernández, Beatriz; de las Heras, Natalia; Miana, María; Ballesteros, Sandra; Delgado, Carmen; Song, Su; Hintze, Thomas; Cachofeiro, Victoria; Lahera, Vicente

    2011-01-01

    We aimed to evaluate the structural, functional, inflammatory, and oxidative alterations, as well as serum and glucocorticoid-regulated kinase-1 (SGK-1) expression, produced in rat heart by aldosterone + salt administration. Fibrosis mediators such as connective tissue growth factor, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2 were also evaluated. Treatment with spironolactone was evaluated to prove mineralocorticoid mediation. Male Wistar rats received aldosterone (1 mg[middle dot]kg-1[middle dot]d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg[middle dot]kg-1[middle dot]d-1). Systolic and diastolic blood pressures, left ventricle (LV) systolic pressure, and LV end-diastolic pressure were elevated (P < 0.05) in aldosterone + salt-treated rats. In aldosterone + salt-treated rats, -dP/dt decreased (P < 0.05), but +dP/dt was similar in all groups. Spironolactone normalized (P < 0.05) systolic blood pressure, diastolic blood pressure, LV systolic pressure, LV end-diastolic pressure, and -dP/dt. Relative heart weight, collagen content, messenger RNA expression of transforming growth factor beta, connective tissue growth factor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases 2, tumor necrosis factor alpha, interleukin-1[beta], p22phox, endothelial nitric oxide synhtase, and SGK-1 were increased (P < 0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (P < 0.05). SGK-1 might be a key mediator in the structural, functional, and molecular cardiac alterations induced by aldosterone + salt in rats. All the observed changes and mediators are related with the activation of mineralocorticoid receptors.

  18. Capsaicin- and mustard oil-induced extracellular signal-regulated protein kinase phosphorylation in sensory neurons in vivo: effects of neurokinins 1 and 2 receptor antagonists and of a nitric oxide synthase inhibitor.

    PubMed

    Donnerer, Josef; Liebmann, Ingrid; Schuligoi, Rufina

    2009-01-01

    Stimulation of primary sensory neurons with capsaicin or mustard oil leads to phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) via activation of transient receptor potential V1 (TRPV1) or TRPA1, respectively. p-ERK1/2 was determined by Western immunoblotting in the dorsal root ganglia and in the sciatic nerve of rats following either systemic or perineural capsaicin treatment, or mustard oil application to the hind paw skin. To investigate the possible involvement of neurokinin 1 (NK(1)) and NK(2) receptors as well as of nitric oxide, the selective antagonists, SR140333 for NK(1) and SR48968 for NK(2), and the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), were employed. The increase of p-ERK1/2 after systemic capsaicin treatment was markedly attenuated by SR140333, while only the increase in the dorsal root ganglia was impaired by SR48968; in contrast, inhibition of nitric oxide synthase had no effect. Perineural capsaicin induced an increase in p-ERK1/2 in the ipsilateral sciatic nerve and in the dorsal root ganglia. This effect was not influenced by SR140333 or L-NAME. We found for the first time that mustard oil application to the hind paw skin caused an increase in p-ERK1/2 in the sciatic nerve and in the dorsal root ganglia and only the phosphorylation in the latter was attenuated by SR140333 while L-NAME showed no effect. From the present results, it may be assumed that capsaicin- or mustard oil-induced p-ERK1/2 in sensory neurons is not solely directly linked to TRPV1 or TRPA1 channels, but under certain conditions NK(1)- and NK(2)-mediated mechanisms are involved.

  19. A Case of Primary Aldosteronism with End Stage Renal Disease

    PubMed Central

    Na, Hyun Hee; Park, Kyung Jun; Kim, Sun Young

    2006-01-01

    A 52-year-old woman was referred to our hospital due to chronic renal failure with a 10-year history of hypertension. We found polycystic kidney disease, pulmonary tuberculosis and an aldosterone-producing adrenocortical mass. At this time, her serum potassium level and blood pressure were within the normal range. She refused hemodialysis and then was hospitalized because of uremic encephalopathy. On admission, her serum potassium level was normal without treatment and plasma aldosterone concentration highly elevated. She received hemodialysis, and thereafter hypokalemia developed. We then administered spironolactone, whereupon serum potassium level returned to the normal range. In this case, we thought that normokalemia was balanced hypokalemia of primary aldosteronism with hyperkalemia of chronic renal failure, and that hypokalemia developed after hemodialysis was due to an imbalanced primary aldosteronism with end stage renal disease. PMID:24459492

  20. Effect of Eplerenone, a Selective Aldosterone Blocker, on the Development of Diabetic Nephropathy in Type 2 Diabetic Rats

    PubMed Central

    Ahn, Jae Hee; Hong, Ho Cheol; Cho, Myong Jin; Kim, Yoon Jung; Choi, Hae Yoon; Eun, Chai Ryoung; Yang, Sae Jeong; Yoo, Hye Jin; Kim, Hee Young; Seo, Ji A; Kim, Sin Gon; Choi, Kyung Mook; Baik, Sei Hyun; Choi, Dong Seop

    2012-01-01

    Background Aldosterone antagonists are reported to have beneficial effects on diabetic nephropathy by effective blocking of the renin-angiotensin-aldosterone system. We investigated the renoprotective effect of the selective aldosterone receptor blocker eplerenone, the angiotensin converting enzyme inhibitor lisinopril, and combined eplerenone and lisinopril treatment in type 2 diabetic rats. Methods Animals were divided into six groups as follows: Otsuka Long-Evans Tokushima Fatty (OLETF) rat control, OLETF rats treated with a low dose of eplerenone (50 mg/kg/day), OLETF rats treated with a high dose of eplerenone (200 mg/kg/day), OLETF rats treated with lisinopril (10 mg/kg/day), OLETF rats treated with a combination of both drugs (eplerenone 200 mg/kg/day and lisinopril 10 mg/kg/day), and obese non-diabetic Long-Evans Tokushima Otsuka rats for 26 weeks. Results Urinary albumin excretion was significantly lower in the lisinopril group, but not in the eplerenone group. Urinary albumin excretion was decreased in the combination group than in the lisinopril group. Glomerulosclerosis and renal expression of type I and type IV collagen, plasminogen activator inhibitor-1, transforming growth factor-β1, connective tissue growth factor, and fibronectin mRNA were markedly decreased in the lisinopril, eplerenone, and combination groups. Conclusion Eplerenone and lisinopril combination showed additional benefits on type 2 diabetic nephropathy compared to monotherapy of each drug. PMID:22540049

  1. Global- and renal-specific sympathoinhibition in aldosterone hypertension.

    PubMed

    Lohmeier, Thomas E; Liu, Boshen; Hildebrandt, Drew A; Cates, Adam W; Georgakopoulos, Dimitrios; Irwin, Eric D

    2015-06-01

    Recent technology for chronic electric activation of the carotid baroreflex and renal nerve ablation provide global and renal-specific suppression of sympathetic activity, respectively, but the conditions for favorable antihypertensive responses in resistant hypertension are unclear. Because inappropriately high plasma levels of aldosterone are prevalent in these patients, we investigated the effects of baroreflex activation and surgical renal denervation in dogs with hypertension induced by chronic infusion of aldosterone (12 μg/kg per day). Under control conditions, basal values for mean arterial pressure and plasma norepinephrine concentration were 100±3 mm Hg and 134±26 pg/mL, respectively. By day 7 of baroreflex activation, plasma norepinephrine was reduced by ≈40% and arterial pressure by 16±2 mm Hg. All values returned to control levels during the recovery period. Arterial pressure increased to 122±5 mm Hg concomitant with a rise in plasma aldosterone concentration from 4.3±0.4 to 70.0±6.4 ng/dL after 14 days of aldosterone infusion, with no significant effect on plasma norepinephrine. After 7 days of baroreflex activation at control stimulation parameters, the reduction in plasma norepinephrine was similar but the fall in arterial pressure (7±1 mm Hg) was diminished (≈55%) during aldosterone hypertension when compared with control conditions. Despite sustained suppression of sympathetic activity, baroreflex activation did not have central actions to inhibit either the stimulation of vasopressin secretion or drinking induced by increased plasma osmolality during chronic aldosterone infusion. Finally, renal denervation did not attenuate aldosterone hypertension. These findings suggest that aldosterone excess may portend diminished blood pressure lowering to global and especially renal-specific sympathoinhibition during device-based therapy.

  2. SY 03-3 OVERVIEW OF SOMATIC MUTATIONS AND EPIGENETIC REGULATION OF ALDOSTERONE PRODUCING ADENOMA (APA).

    PubMed

    Umemura, Satoshi

    2016-09-01

    clear cells with abundant microvesicular cytoplasm with numerous lipid droplets ('fasciculata-like'). The histological pattern of tumors with CACNA1D and ATPase mutations was still controversial (Clin Endo 2015; 83: 779, JCEM 2016;101:494)(3) Epigenetics in APAIntegrated analysis of genome-wide methylation and gene expression shows epigenetic regulation of CYP11B2 in aldosteronomas. APA were hypomethylated compared with normal, nonfunctioning adrenocortical tumors and APA-adjacent adrenal gland samples from the same patient. CYP11B2 was upregulated and hypomethylated, suggesting that dysregulated methylation preferentially upregulates CYP11B2 with commensurate inhibition of other key genes involved in steroid biosynthesis. Comparing the methylation status of CYP11B2 CpG islands in paired DNA samples from aldosteronomas and blood from the same patients showed CpG hypomethylation was found in aldosteronomas but not in peripheral blood DNA in all cases. These results may suggest the involvement of these DNA hypomethylation in aldosterone production. (J Clin Endocrinol Metab 2014; 99: E536. Eur J Endocrinol 2015; 173: 185)(4) Aldosterone-producing cell clusters (APCCs) with high expression of CYP11B2 in both normal and PA adrenal tissueRecently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. Known aldosterone driver mutations were identified in some of APCCs, including CACNA1D and ATP1A1, which were not observed in the adjacent normal adrenal tissue. These studies suggest that APCCs are common in normal adrenals, and APCCs harbor somatic mutations known to cause excess aldosterone production. Furthermore, the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. The mutations in KCNJ5 were not detected in those APCCs. The frequency of somatic mutations in CACNA1D in APCCs from normal adrenals increases with age. These results may

  3. Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.

    PubMed

    Hopcroft, Philip J; Fisher, David I

    2016-02-01

    The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values.

  4. Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants.

    PubMed Central

    Chang, A K; Duggleby, R G

    1998-01-01

    Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold. PMID:9677339

  5. Adrenal Venous Sampling: Where Is the Aldosterone Disappearing to?

    SciTech Connect

    Solar, Miroslav; Ceral, Jiri; Krajina, Antonin; Ballon, Marek; Malirova, Eva; Brodak, Milos; Cap, Jan

    2010-08-15

    Adrenal venous sampling (AVS) is generally considered to be the gold standard in distinguishing unilateral and bilateral aldosterone hypersecretion in primary hyperaldosteronism. However, during AVS, we noticed a considerable variability in aldosterone concentrations among samples thought to have come from the right adrenal glands. Some aldosterone concentrations in these samples were even lower than in samples from the inferior vena cava. We hypothesized that the samples with low aldosterone levels were unintentionally taken not from the right adrenal gland, but from hepatic veins. Therefore, we sought to analyze the impact of unintentional cannulation of hepatic veins on AVS. Thirty consecutive patients referred for AVS were enrolled. Hepatic vein sampling was implemented in our standardized AVS protocol. The data were collected and analyzed prospectively. AVS was successful in 27 patients (90%), and hepatic vein cannulation was successful in all procedures performed. Cortisol concentrations were not significantly different between the hepatic vein and inferior vena cava samples, but aldosterone concentrations from hepatic venous blood (median, 17 pmol/l; range, 40-860 pmol/l) were markedly lower than in samples from the inferior vena cava (median, 860 pmol/l; range, 460-4510 pmol/l). The observed difference was statistically significant (P < 0.001). Aldosterone concentrations in the hepatic veins are significantly lower than in venous blood taken from the inferior vena cava. This finding is important for AVS because hepatic veins can easily be mistaken for adrenal veins as a result of their close anatomic proximity.

  6. Effects of aldosterone on insulin sensitivity and secretion.

    PubMed

    Luther, James M

    2014-12-01

    Dr. Conn originally reported an increased risk of diabetes in patients with hyperaldosteronism in the 1950s, although the mechanism remains unclear. Aldosterone-induced hypokalemia was initially described to impair glucose tolerance by impairing insulin secretion. Correction of hypokalemia by potassium supplementation only partially restored insulin secretion and glucose tolerance, however. Aldosterone also impairs glucose-stimulated insulin secretion in isolated pancreatic islets via reactive oxygen species in a mineralocorticoid receptor-independent manner. Aldosterone-induced mineralocorticoid receptor activation also impairs insulin sensitivity in adipocytes and skeletal muscle. Aldosterone may produce insulin resistance secondarily by altering potassium, increasing inflammatory cytokines, and reducing beneficial adipokines such as adiponectin. Renin-angiotensin system antagonists reduce circulating aldosterone concentrations and also the risk of type 2 diabetes in clinical trials. These data suggest that primary and secondary hyperaldosteronism may contribute to worsening glucose tolerance by impairing insulin sensitivity or insulin secretion in humans. Future studies should define the effects of MR antagonists and aldosterone on insulin secretion and sensitivity in humans.

  7. Mechanisms of acetohydroxyacid synthases.

    PubMed

    Chipman, David M; Duggleby, Ronald G; Tittmann, Kai

    2005-10-01

    Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

  8. Activation of the Endogenous Renin-Angiotensin-Aldosterone System or Aldosterone Administration Increases Urinary Exosomal Sodium Channel Excretion

    PubMed Central

    Qi, Ying; Wang, Xiaojing; Rose, Kristie L.; MacDonald, W. Hayes; Zhang, Bing; Schey, Kevin L.

    2016-01-01

    Urinary exosomes secreted by multiple cell types in the kidney may participate in intercellular signaling and provide an enriched source of kidney-specific proteins for biomarker discovery. Factors that alter the exosomal protein content remain unknown. To determine whether endogenous and exogenous hormones modify urinary exosomal protein content, we analyzed samples from 14 mildly hypertensive patients in a crossover study during a high-sodium (HS, 160 mmol/d) diet and low-sodium (LS, 20 mmol/d) diet to activate the endogenous renin-angiotensin-aldosterone system. We further analyzed selected exosomal protein content in a separate cohort of healthy persons receiving intravenous aldosterone (0.7 μg/kg per hour for 10 hours) versus vehicle infusion. The LS diet increased plasma renin activity and aldosterone concentration, whereas aldosterone infusion increased only aldosterone concentration. Protein analysis of paired urine exosome samples by liquid chromatography-tandem mass spectrometry–based multidimensional protein identification technology detected 2775 unique proteins, of which 316 exhibited significantly altered abundance during LS diet. Sodium chloride cotransporter (NCC) and α- and γ-epithelial sodium channel (ENaC) subunits from the discovery set were verified using targeted multiple reaction monitoring mass spectrometry quantified with isotope-labeled peptide standards. Dietary sodium restriction or acute aldosterone infusion similarly increased urine exosomal γENaC[112–122] peptide concentrations nearly 20-fold, which correlated with plasma aldosterone concentration and urinary Na/K ratio. Urine exosomal NCC and αENaC concentrations were relatively unchanged during these interventions. We conclude that urinary exosome content is altered by renin-angiotensin-aldosterone system activation. Urinary measurement of exosomal γENaC[112–122] concentration may provide a useful biomarker of ENaC activation in future clinical studies. PMID:26113616

  9. Activation of the Endogenous Renin-Angiotensin-Aldosterone System or Aldosterone Administration Increases Urinary Exosomal Sodium Channel Excretion.

    PubMed

    Qi, Ying; Wang, Xiaojing; Rose, Kristie L; MacDonald, W Hayes; Zhang, Bing; Schey, Kevin L; Luther, James M

    2016-02-01

    Urinary exosomes secreted by multiple cell types in the kidney may participate in intercellular signaling and provide an enriched source of kidney-specific proteins for biomarker discovery. Factors that alter the exosomal protein content remain unknown. To determine whether endogenous and exogenous hormones modify urinary exosomal protein content, we analyzed samples from 14 mildly hypertensive patients in a crossover study during a high-sodium (HS, 160 mmol/d) diet and low-sodium (LS, 20 mmol/d) diet to activate the endogenous renin-angiotensin-aldosterone system. We further analyzed selected exosomal protein content in a separate cohort of healthy persons receiving intravenous aldosterone (0.7 μg/kg per hour for 10 hours) versus vehicle infusion. The LS diet increased plasma renin activity and aldosterone concentration, whereas aldosterone infusion increased only aldosterone concentration. Protein analysis of paired urine exosome samples by liquid chromatography-tandem mass spectrometry-based multidimensional protein identification technology detected 2775 unique proteins, of which 316 exhibited significantly altered abundance during LS diet. Sodium chloride cotransporter (NCC) and α- and γ-epithelial sodium channel (ENaC) subunits from the discovery set were verified using targeted multiple reaction monitoring mass spectrometry quantified with isotope-labeled peptide standards. Dietary sodium restriction or acute aldosterone infusion similarly increased urine exosomal γENaC[112-122] peptide concentrations nearly 20-fold, which correlated with plasma aldosterone concentration and urinary Na/K ratio. Urine exosomal NCC and αENaC concentrations were relatively unchanged during these interventions. We conclude that urinary exosome content is altered by renin-angiotensin-aldosterone system activation. Urinary measurement of exosomal γENaC[112-122] concentration may provide a useful biomarker of ENaC activation in future clinical studies.

  10. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    SciTech Connect

    Zanotti, Franco; Gnoni, Antonio; Mangiullo, Roberto; Papa, Sergio

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  11. New Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Inhibitors, Nalidixic Acid Linked to Isatin Schiff Bases via Certain l-Amino Acid Bridges.

    PubMed

    Naglah, Ahmed M; Ahmed, Atallah F; Wen, Zhi-Hong; Al-Omar, Mohamed A; Amr, Abd El-Galil E; Kalmouch, Atef

    2016-04-15

    A series of new Schiff bases were synthesized by condensation of isatins with the nalidixic acid-l-amino acid hydrazides. Prior to hydrazide formation, a peptide linkage has been prepared via coupling of nalidixic acid with appropriate l-amino acid methyl esters to yield 3a-c. The chemical structures of the new Schiff bases (5b and 5d-h) were confirmed by means of IR, NMR, mass spectroscopic, and elemental analyses. The anti-inflammatory activity of these Schiff bases was evaluated via measurement of the expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells model. The Schiff bases exhibited significant dual inhibitory effect against the induction of the pro-inflammatory iNOS and COX-2 proteins with variable potencies. However, they strongly down-regulated the iNOS expression to the level of 16.5% ± 7.4%-42.2% ± 19.6% compared to the effect on COX-2 expression (<56.4% ± 3.1% inhibition) at the same concentration (10 μM). The higher iNOS inhibition activity of the tested Schiff bases, relative to that of COX-2, seems to be a reflection of the combined suppressive effects exerted by their nalidixic acid, isatins (4a-c), and l-amino acid moieties against iNOS expression. These synthesized nalidixic acid-l-amino acid-isatin conjugates can be regarded as a novel class of anti-inflammatory antibacterial agents.

  12. CAUSES AND CONSEQUENCES OF ZINC DYSHOMEOSTASIS IN RATS WITH CHRONIC ALDOSTERONISM

    PubMed Central

    Gandhi, Malay S.; Deshmukh, Prajwal A.; Kamalov, German; Zhao, Tieqiang; Zhao, Wenyuan; Whaley, Jonathan T.; Tichy, Jill R.; Bhattacharya, Syamal K.; Ahokas, Robert A.; Sun, Yao; Gerling, Ivan C.; Weber, Karl T.

    2009-01-01

    Iterations in Ca2+ and Mg2+ balance accompany aldosteronism (inappropriate for dietary Na+ intake). Increased Zn excretion and Zn translocation to injured tissues, including the heart, also occurs. Several causes and consequences of Zn dyshomeostasis in rats receiving aldosterone/salt treatment (ALDOST) were examined: 1) the role of urinary acidification in promoting hyperzincuria, acetazolamide (75 mg/kg), a carbonic anhydrase inhibitor, was used as cotreatment to raise urinary HCO3− excretion; 2) assess Zn levels in the heart, including cardiomyocyte cytosolic free [Zn2+]i and mitochondrial Zn, the expression of metallothionein (MT-I), a Zn binding protein, and biomarkers of oxidative stress; and 3) monitor oxidative stress and cardiac pathology in response to ZnSO4 supplement (40 mg/day). Compared to controls, at 4 wks ALDOST we found: an acidification of urine and metabolic alkalosis associated with increased urinary Zn excretion and hypozincemia, each of which were prevented by acetazolamide; a rise in cardiac Zn including increased [Zn2+]i and mitochondrial Zn, associated with increased tissue MT-I, 8-isoprostane, malondialdehyde, and gp91phox, coupled with oxidative stress in plasma and urine; and ZnSO4 prevented hypozincemia, but not ionized hypocalcemia, and attenuated oxidative stress and microscopic scarring without preventing the vasculitis and perivascular fibrosis of intramural coronary arteries. Thus, the hyperzincuria seen with ALDOST is due to urinary acidification. The oxidative stress that appears in the heart is accompanied by increased tissue Zn serving as an antioxidant. Cotreatment with ZnSO4 attenuated cardiomyocyte necrosis, however, polynutrient supplement may be required to counteract the dyshomeostasis of all 3 cations that accompanies aldosteronism and contribute to cardiac pathology. PMID:18806605

  13. Effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with the nitric oxide synthase inhibitor and dexamethasone.

    PubMed

    Arutyunyan, Tamara V; Korystova, Antonina F; Kublik, Ludmila N; Levitman, Maria Kh; Shaposhnikova, Vera V; Korystov, Yuri N

    2013-12-01

    The action of taxifolin on the angiotensin-converting enzyme (ACE) and the formation of reactive oxygen and nitrogen species (ROS/RNS) in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor (N ω-nitro-L-arginine methyl ester (L-NAME)) or dexamethasone have been studied. The ACE activity in aorta sections was determined by measuring the hydrolysis of hippuryl-L-histidyl-L-leucine, and the ROS/RNS production was measured by oxidation of dichlorodihydrofluorescein. It was shown that taxifolin at a dose of 30-100 μg/kg/day decreases the ACE activity in the aorta of aging rats and of rats treated with L-NAME or dexamethasone to the level of the ACE activity in young control rats. Taxifolin (100 μg/kg/day) was found to also reduce the amount of ROS/RNS in the aorta that increased as a result of L-NAME intake. L-NAME treatment increases the contribution of 5-lipoxygenase and NADPH oxidase to ROS/RNS production in the aorta, while taxifolin (100 μg/kg/day) decreases the contribution of these enzymes to the normal level.

  14. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

    PubMed Central

    Hernández-Perera, O; Pérez-Sala, D; Navarro-Antolín, J; Sánchez-Pascuala, R; Hernández, G; Díaz, C; Lamas, S

    1998-01-01

    Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors. PMID:9637705

  15. Glycogen synthase kinase 3 (GSK3)-inhibitor SB216763 promotes the conversion of human umbilical cord mesenchymal stem cells into neural precursors in adherent culture.

    PubMed

    Gao, Liyang; Zhao, Mingyan; Li, Peng; Kong, Junchao; Liu, Zhijun; Chen, Yonghua; Huang, Rui; Chu, Jiaqi; Quan, Juanhua; Zeng, Rong

    2017-01-01

    The ability to generate neural progenitor cells from human umbilical cord mesenchymal stem cells (hUC-MSCs) has provided an option to treat neurodegenerative diseases. To establish a method for this purpose, we characterized the early neural markers of hUC-MSCs-derived cells under different conditions. We found that neither the elimination of signals for alternative fate nor N2 supplement was sufficient to differentiate hUC-MSCs into neural precursor cells, but the GSK3 inhibitor SB216763 could promote an efficient neural commitment of hUC-MSCs. The results indicated that Wnt/β-catenin might play an important role during the early neural differentiation of hUC-MSCs. Here, we report a method for hUC-MSCs to commit efficiently into a neural fate within a short period of time. This protocol provides an efficient method for hUC-MSCs-based neural regeneration.

  16. Prostaglandins, renin, aldosterone, and catecholamines in preeclampsia.

    PubMed

    Pedersen, E B; Christensen, N J; Christensen, P; Johannesen, P; Kornerup, H J; Kristensen, S; Lauritsen, J G; Leyssac, P P; Rasmussen, A B; Wohlert, M

    1983-01-01

    Urinary excretion of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha), plasma concentrations of renin (PRC), aldosterone (PAC), noradrenaline (PNA) and adrenaline (PA) were determined in the third trimester of pregnancy, 5 days and 3 months after delivery in preeclampsia and normotensive pregnant and non-pregnant control subjects. PGE2 was higher in pregnant control subjects than in non-pregnant subjects, but reduced to non-pregnant level in preeclampsia. PGF2 alpha was the same in preeclampsia and normotensive pregnancy but higher than in the non-pregnant group. PRC and PAC were increased during pregnancy, but considerably lesser in preeclampsia than during normotensive pregnancy. PNA and PA were the same in all three groups. All parameters were normal 3 months after delivery. There were no correlations between any of the hormones and blood pressure in any of the groups. PGE2 was positively correlated to PRC. The lack of renal PGE2 in preeclampsia might be responsible for the decrease in renal blood flow and sodium excretion, and the changes in PRC and PAC are supposed to be secondary to changes in PGE2. It is hypothesised that preeclampsia is a state of prostaglandin deficiency.

  17. The Role of Aldosterone in Obesity-Related Hypertension.

    PubMed

    Kawarazaki, Wakako; Fujita, Toshiro

    2016-04-01

    Obese subjects often have hypertension and related cardiovascular and renal diseases, and this has become a serious worldwide health problem. In obese subjects, impaired renal-pressure natriuresis causes sodium retention, leading to the development of salt-sensitive hypertension. Physical compression of the kidneys by visceral fat and activation of the sympathetic nervous system, renin-angiotensin systems (RAS), and aldosterone/mineralocorticoid receptor (MR) system are involved in this mechanism. Obese subjects often exhibit hyperaldosteronism, with increased salt sensitivity of blood pressure (BP). Adipose tissue excretes aldosterone-releasing factors, thereby stimulating aldosterone secretion independently of the systemic RAS, and aldosterone/MR activation plays a key role in the development of hypertension and organ damage in obesity. In obese subjects, both salt sensitivity of BP, enhanced by obesity-related metabolic disorders including aldosterone excess, and increased dietary sodium intake are closely related to the incidence of hypertension. Some salt sensitivity-related gene variants affect the risk of obesity, and together with salt intake, its combination is possibly associated with the development of hypertension in obese subjects. With high salt levels common in modern diets, salt restriction and weight control are undoubtedly important. However, not only MR blockade but also new diagnostic modalities and therapies targeting and modifying genes that are related to salt sensitivity, obesity, or RAS regulation are expected to prevent obesity and obesity-related hypertension.

  18. Bilateral Adrenocortical Masses Producing Aldosterone and Cortisol Independently

    PubMed Central

    Lee, Seung-Eun; Lee, You-Bin; Seok, Hyeri; Shin, In Seub; Eun, Yeong Hee; Kim, Jung-Han; Oh, Young Lyun

    2015-01-01

    A 31-year-old woman was referred to our hospital with symptoms of hypertension and bilateral adrenocortical masses with no feature of Cushing syndrome. The serum aldosterone/renin ratio was elevated and the saline loading test showed no suppression of the plasma aldosterone level, consistent with a diagnosis of primary hyperaldosteronism. Overnight and low-dose dexamethasone suppression tests showed no suppression of serum cortisol, indicating a secondary diagnosis of subclinical Cushing syndrome. Adrenal vein sampling during the low-dose dexamethasone suppression test demonstrated excess secretion of cortisol from the left adrenal mass. A partial right adrenalectomy was performed, resulting in normalization of blood pressure, hypokalemia, and high aldosterone level, implying that the right adrenal mass was the main cause of the hyperaldosteronism. A total adrenalectomy for the left adrenal mass was later performed, resulting in a normalization of cortisol level. The final diagnosis was bilateral adrenocortical adenomas, which were secreting aldosterone and cortisol independently. This case is the first report of a concurrent cortisol-producing left adrenal adenoma and an aldosterone-producing right adrenal adenoma in Korea, as demonstrated by adrenal vein sampling and sequential removal of adrenal masses. PMID:26248855

  19. The Role of Aldosterone in Obesity-Related Hypertension

    PubMed Central

    Kawarazaki, Wakako

    2016-01-01

    Obese subjects often have hypertension and related cardiovascular and renal diseases, and this has become a serious worldwide health problem. In obese subjects, impaired renal-pressure natriuresis causes sodium retention, leading to the development of salt-sensitive hypertension. Physical compression of the kidneys by visceral fat and activation of the sympathetic nervous system, renin–angiotensin systems (RAS), and aldosterone/mineralocorticoid receptor (MR) system are involved in this mechanism. Obese subjects often exhibit hyperaldosteronism, with increased salt sensitivity of blood pressure (BP). Adipose tissue excretes aldosterone-releasing factors, thereby stimulating aldosterone secretion independently of the systemic RAS, and aldosterone/MR activation plays a key role in the development of hypertension and organ damage in obesity. In obese subjects, both salt sensitivity of BP, enhanced by obesity-related metabolic disorders including aldosterone excess, and increased dietary sodium intake are closely related to the incidence of hypertension. Some salt sensitivity-related gene variants affect the risk of obesity, and together with salt intake, its combination is possibly associated with the development of hypertension in obese subjects. With high salt levels common in modern diets, salt restriction and weight control are undoubtedly important. However, not only MR blockade but also new diagnostic modalities and therapies targeting and modifying genes that are related to salt sensitivity, obesity, or RAS regulation are expected to prevent obesity and obesity-related hypertension. PMID:26927805

  20. Racial differences in sensitivity of blood pressure to aldosterone.

    PubMed

    Tu, Wanzhu; Eckert, George J; Hannon, Tamara S; Liu, Hai; Pratt, Linda M; Wagner, Mary Anne; Dimeglio, Linda A; Jung, Jeesun; Pratt, J Howard

    2014-06-01

    Blacks in comparison with whites are at risk for a more serious form of hypertension with high rates of complications. Greater sodium retention is thought to underlie the blood pressure (BP)-determining physiology of blacks, but specific mechanisms have not been identified. In a prospective observational study of BP, 226 black children and 314 white children (mean age, 10.6 years) were enrolled initially. Assessments were repeated in 85 blacks and 136 whites after reaching adulthood (mean age, 31 years). The relationship of BP to plasma aldosterone concentration in the context of the prevailing level of plasma renin activity was studied in blacks and whites. In a secondary interventional study, 9-α fludrocortisone was administered for 2 weeks to healthy adult blacks and whites to simulate hyperaldosteronism. BP responses in the 2 race groups were then compared. Although black children had lower levels of plasma renin activity and plasma aldosterone, their BP was positively associated with the plasma aldosterone concentration, an effect that increased as plasma renin activity decreased (P=0.004). Data from black adults yielded similar results. No similar relationship was observed in whites. In the interventional study, 9-α fludrocortisone increased BP in blacks but not in whites. In conclusion, aldosterone sensitivity is a significant determinant of BP in young blacks. Although its role in establishing the risk of hypertension is not known, it could be as relevant as the actual level of aldosterone.

  1. Purinergic inhibition of ENaC produces aldosterone escape.

    PubMed

    Stockand, James D; Mironova, Elena; Bugaj, Vladislav; Rieg, Timo; Insel, Paul A; Vallon, Volker; Peti-Peterdi, Janos; Pochynyuk, Oleh

    2010-11-01

    The mechanisms underlying "aldosterone escape," which refers to the excretion of sodium (Na(+)) during high Na(+) intake despite inappropriately increased levels of mineralocorticoids, are incompletely understood. Because local purinergic tone in the aldosterone-sensitive distal nephron downregulates epithelial Na(+) channel (ENaC) activity, we tested whether this mechanism mediates aldosterone escape. Here, urinary ATP concentration increased with dietary Na(+) intake in mice. Physiologic concentrations of ATP decreased ENaC activity in a dosage-dependent manner. P2Y(2)(-/-) mice, which lack the purinergic receptor, had significantly less increased Na(+) excretion than wild-type mice in response to high-Na(+) intake. Exogenous deoxycorticosterone acetate and deletion of the P2Y(2) receptor each modestly increased the resistance of ENaC to changes in Na(+) intake; together, they markedly increased resistance. Under the latter condition, ENaC could not respond to changes in Na(+) intake. In contrast, as a result of aldosterone escape, wild-type mice had increased Na(+) excretion in response to high-Na(+) intake regardless of the presence of high deoxycorticosterone acetate. These data suggest that control of ENaC by purinergic signaling is necessary for aldosterone escape.

  2. Purinergic Inhibition of ENaC Produces Aldosterone Escape

    PubMed Central

    Mironova, Elena; Bugaj, Vladislav; Rieg, Timo; Insel, Paul A.; Vallon, Volker; Peti-Peterdi, Janos

    2010-01-01

    The mechanisms underlying “aldosterone escape,” which refers to the excretion of sodium (Na+) during high Na+ intake despite inappropriately increased levels of mineralocorticoids, are incompletely understood. Because local purinergic tone in the aldosterone-sensitive distal nephron downregulates epithelial Na+ channel (ENaC) activity, we tested whether this mechanism mediates aldosterone escape. Here, urinary ATP concentration increased with dietary Na+ intake in mice. Physiologic concentrations of ATP decreased ENaC activity in a dosage-dependent manner. P2Y2−/− mice, which lack the purinergic receptor, had significantly less increased Na+ excretion than wild-type mice in response to high-Na+ intake. Exogenous deoxycorticosterone acetate and deletion of the P2Y2 receptor each modestly increased the resistance of ENaC to changes in Na+ intake; together, they markedly increased resistance. Under the latter condition, ENaC could not respond to changes in Na+ intake. In contrast, as a result of aldosterone escape, wild-type mice had increased Na+ excretion in response to high-Na+ intake regardless of the presence of high deoxycorticosterone acetate. These data suggest that control of ENaC by purinergic signaling is necessary for aldosterone escape. PMID:20813869

  3. Recent advances in the study of rTS proteins. rTS expression during growth and in response to thymidylate synthase inhibitors in human tumor cells.

    PubMed

    Dolnick, B J; Lu, K; Yin, M B; Rustum, Y M

    1997-01-01

    The rTS proteins have now been shown to be expressed in a variety of cell lines, with expression of rTS beta being found elevated in three cell lines which are resistant to TS inhibitors (3, 4) (Figure 1). In one of these cell lines (K562 B1A), the cells were selected for resistance to MTX, which has a primary site of action on DHFR, but was found to be cross-resistant to FUdR (4). The other two cell lines were selected for resistance to either 5-fluorouracil (H630-1) or a combination of ZD1694 and FU. In each case, elevation of rTS beta appears to be a selected response to thymidylate stress. In HCT-8 and HCT-8/DF2 cells, treatment of cells for a short period of time (2 hr) resulted in the elevation of rTS beta levels, again suggestive that expression of rTS beta is a response to thymidylate stress. rTS beta appears to be regulated with cell growth, its levels increasing at mid-log and at late-log/saturation phase in H630 and H630-1 cells (Fig. 2), and increasing with late-log in several other cell lines as well (Fig. 3). The increase in rTS beta is suggestive of a cellular function associated with a state where growth is no longer desirable, reminiscent of the starvation-sensing protein homolog RSPA in E. coli (22). While this relationship would not explain the spike in rTS beta levels in mid-log H630 and H630-1 cells, it does make sense if the rTS proteins (particularly rTS beta) are involved in down-regulating thymidylate biosynthesis. The potential mechanism of this down-regulation may be speculated to be the catabolism of some precursor for thymidylate biosynthesis or some direct effect upon TS through modulation by some other ligand, either a metabolite or another protein. Studies on the expression of rTS proteins in clinical specimens indicate that rTS beta is expressed at high levels in kidney and kidney tumor (Dolnick, unpublished results). Given the physiologic role of the kidney, high level expression of rTS in this organ is consistent with a role in a

  4. Role of the anterior region of the third ventricle in the cardiovascular responses produced by systemic injection of a nitric oxide synthase inhibitor

    NASA Technical Reports Server (NTRS)

    Lewis, S. J.; Whalen, E. J.; Beltz, T. G.; Johnson, A. K.

    1999-01-01

    This study examined whether a prior electrolytic lesion of the tissue surrounding the anteroventral third ventricle (AV3V) would affect the increase in mean arterial blood pressure (MAP) and the fall in heart rate (HR) produced by systemic injection of the nitric oxide synthesis (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 25 micromol/kg, i.v.) in conscious rats. L-NAME produced a smaller increase in MAP in AV3V-lesion than in sham-lesion rats (+19+/-3 vs. +40+/-3 mmHg, respectively; P<0.05). In contrast, L-NAME produced similar falls in HR in the AV3V-lesion and sham-lesion rats (-103+/-15 vs. -97+/-8 bpm, respectively; P<0.05). These findings demonstrate that the L-NAME-induced pressor response is dependent upon the integrity of the AV3V region, whereas the L-NAME-induced bradycardia is not. Copyright 1999 Elsevier Science B. V.

  5. A nitric oxide synthase inhibitor (L-NAME) attenuates abstinence-induced withdrawal from both cocaine and a cannabinoid agonist (WIN 55212-2) in Planaria.

    PubMed

    Rawls, Scott M; Rodriguez, Tonatiu; Baron, David A; Raffa, Robert B

    2006-07-12

    We previously reported that planarians (Dugesia dorotocephala) that have been exposed to cocaine for 1 h undergo abstinence-induced withdrawal when placed into cocaine-free, but not cocaine-containing, water. We now report that planarians also display dose-related abstinence-induced withdrawal following exposure to the synthetic cannabinoid agonist WIN 55212-2, but not its inactive enantiomer (WIN 55212-3). The withdrawal from WIN 55212-2 was manifested as a significant (P < 0.05) decrease in the rate of planarian spontaneous locomotor activity over a 5-min observation period, using a recently designed metric (pLMV). We also report that withdrawal from cocaine (80 microM) or WIN 55212-2 (10 microM) was attenuated by the selective inhibitor of nitric oxide synthesis L-NAME (L-nitro-arginine methyl ester), which had no effect of its own on pLMV. These results suggest a common NO-dependent pathway of withdrawal from cocaine and WIN 55212-2 in Planaria.

  6. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage.

    PubMed

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Komur, Baran; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  7. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    PubMed Central

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders. PMID:27382570

  8. ASP9853, an inhibitor of inducible nitric oxide synthase dimerization, in combination with docetaxel: Preclinical investigation and a Phase I study in advanced solid tumors

    PubMed Central

    Luke, Jason J.; LoRusso, Patricia; Shapiro, Geoffrey I.; Krivoshik, Andrew; Schuster, Robin; Yamazaki, Takao; Arai, Yukinori; Fakhoury, Allam; Dmuchowski, Carl; Infante, Jeffrey R.

    2016-01-01

    Purpose ASP9853 is an inhibitor of iNOS dimerization, which results in decreased NO production. Here we report preclinical pharmacology of ASP9853 and the impact of ASP9853 in combination with a taxane on tumor volume in vivo. In addition, a Phase I open-label study of ASP9853 plus docetaxel was conducted to assess this combination in patients with advanced solid tumors. Methods The preclinical efficacy of ASP9853 in combination with a taxane was studied in tumor-bearing mice. In the clinic, patients with solid tumors that had progressed or failed to respond to previous therapies were treated with once daily ASP9853 in combination with docetaxel once every 3 weeks to assess safety and tolerability and to determine the maximum tolerated dose (MTD) and the recommended Phase II dose (RP2D) of the combination. Results ASP9853 in combination with docetaxel showed greater tumor growth inhibition than docetaxel alone against non-small lung cancer xenografts. Twenty patients were treated with ASP9853 and docetaxel. Five patients experienced neutropenic dose limiting toxicities. Owing to overall toxicity that limited further dose escalation, the ASP9853 concentrations predicted for efficacy based on the preclinical data were not achieved. Due to toxicity and lack of clear efficacy, the study was terminated without determination of MTD or RP2D. Conclusions Inhibition of iNOS by ASP9853 in combination with docetaxel was not tolerable and resulted in the possible potentiation of neutropenia. Manipulation of the iNOS pathway, with or without chemotherapy, appears to be more complicated than initially expected. PMID:26811179

  9. Inhibition of endothelial nitric oxyde synthase increases capillary formation via Rac1-dependent induction of hypoxia-inducible factor-1α and plasminogen activator inhibitor-1.

    PubMed

    Petry, Andreas; BelAiba, Rachida S; Weitnauer, Michae; Görlach, Agnes

    2012-11-01

    Disruption of endothelial homeostasis results in endothelial dysfunction, characterised by a dysbalance between nitric oxide (NO) and reactive oxygen species (ROS) levels often accompanied by a prothrombotic and proproliferative state. The serine protease thrombin not only is instrumental in formation of the fibrin clot, but also exerts direct effects on the vessel wall by activating proliferative and angiogenic responses. In endothelial cells, thrombin can induce NO as well as ROS levels. However, the relative contribution of these reactive species to the angiogenic response towards thrombin is not completely clear. Since plasminogen activator inhibitor-1 (PAI-1), a direct target of the proangiogenic transcription factors hypoxia-inducible factors (HIFs), exerts prothrombotic and proangiogenic activities we investigated the role of ROS and NO in the regulation of HIF-1α, PAI-1 and capillary formation in response to thrombin. Thrombin enhanced the formation of NO as well as ROS generation involving the GTPase Rac1 in endothelial cells. Rac1-dependent ROS formation promoted induction of HIF-1α, PAI-1 and capillary formation by thrombin, while NO reduced ROS bioavailability and subsequently limited induction of HIF-1α, PAI-1 and the angiogenic response. Importantly, thrombin activation of Rac1 was diminished by NO, but enhanced by ROS. Thus, our findings show that capillary formation induced by thrombin via Rac1-dependent activation of HIF-1 and PAI-1 is limited by the concomitant release of NO which reduced ROS bioavailability. Rac1 activity is sensitive to ROS and NO, thereby playing an essential role in fine tuning the endothelial response to thrombin.

  10. Renal Aldosterone Receptors: Studies with [3H]Aldosterone and the Anti-Mineralocorticoid [3H]Spirolactone (SC-26304)

    PubMed Central

    Marver, Diana; Stewart, John; Funder, John W.; Feldman, David; Edelman, Isidore S.

    1974-01-01

    In vivo, a spirolactone (SC-26304) inhibited the effects of aldosterone on urinary K+:Na+ ratios and the binding of [3H]aldosterone to renal cytoplasmic and nuclear receptors. Cytoplasmic binding of [3H]aldosterone and [3H]spirolactone (SC-26304) was similar in magnitude and involved the same set of sites. Under three sets of conditions—(i) in the intact rat, (ii) in kidney slices, and (iii) in reconstitution studies (mixing prelabeled cytoplasm with either purified renal nuclei or chromatin), [3H]spirolactone (SC-26304) did not yield specific nuclear complexes in contrast to the reproducible generation of these complexes with [3H]aldosterone. In glycerol density gradients, cytoplasmic [3H]aldosterone receptor complexes sedimented at 8.5 S and 4 S in low concentrations of salt and at 4.5 S in high concentrations of salt. Cytoplasmic [3H]spirolactone (SC-26304) receptor complexes sedimented at 3 S in low concentrations of salt and 4 S in high concentrations of salt. These results are discussed in terms of an allosteric model of the receptor system. Images PMID:4364539

  11. Aldosterone and volume management in hypertensive heart disease.

    PubMed

    Sica, Domenic A

    2014-05-01

    Aldosterone-receptor antagonists dose-dependently reduce both the epithelial and nonepithelial actions of aldosterone. These compounds are used commonly in the treatment of hypertension, with or without aldosteronism, and in the volume-overload periods of various forms of heart failure, cirrhosis, and renal failure. In this regard, the relevant site of action for these compounds is compartmentalized to the distal nephron. The cardiac benefits of aldosterone-receptor blockade now are sufficiently well established to warrant routine use of these compounds for their survival benefits in moderate to advanced stages of heart failure. Aldosterone-receptor antagonists spironolactone and eplerenone commonly are used in the treatment of resistant forms of hypertension. Spironolactone, but not eplerenone, is a commonly used add-on diuretic that provides incremental benefit for salt-and-water excretion in excess of what may be seen with a loop diuretic given together with a thiazide-type diuretic. The dose-response relationship for natriuresis with spironolactone has not been explored completely as to its combination therapy responses. The quite high doses of spironolactone used in patients with cirrhosis and ascites would infer that the overall treatment effect with this compound exceeds simple receptor blockade and may include a nervous system effect that operationally reduces renal sympathetic nerve traffic. The adverse electrolyte and renal function side effects with aldosterone-receptor antagonists are not uncommon in at-risk patients, such as those with chronic kidney disease, and require that dosing be mindful of the tendency of these drugs to importantly increase serum potassium levels.

  12. Isolated production of aldosterone by a malignant adrenal carcinoma.

    PubMed Central

    Levine, D. S.; Fischer, D. G.; Forman, B. H.

    1984-01-01

    A 45-year-old female developed hypertension and hypokalemia. Elevated plasma aldosterone and suppressed plasma renin levels were measured with no evidence for glucocorticoid or androgen abnormalities. A left adrenal tumor was removed that showed histologic criteria for malignancy. It is commonly taught that malignant adrenal tumors are recognized by their multiple hormone production. However, isolated aldosterone production by a carcinoma can occur and requires close follow-up observation and therapy for this highly malignant tumor. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:6537692

  13. Blockade of T-type voltage-dependent Ca2+ channels by benidipine, a dihydropyridine calcium channel blocker, inhibits aldosterone production in human adrenocortical cell line NCI-H295R.

    PubMed

    Akizuki, Osamu; Inayoshi, Atsushi; Kitayama, Tetsuya; Yao, Kozo; Shirakura, Shiro; Sasaki, Katsutoshi; Kusaka, Hideaki; Matsubara, Masahiro

    2008-04-28

    Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for treatment of hypertension and angina. Benidipine exerts pleiotropic pharmacological features, such as renoprotective and cardioprotective effects. In pathophysiological conditions, the antidiuretic hormone aldosterone causes development of renal and cardiovascular diseases. In adrenal glomerulosa cells, aldosterone is produced in response to extracellular potassium, which is mainly mediated by T-type voltage-dependent Ca2+ channels. More recently, it has been demonstrated that benidipine inhibits T-type Ca2+ channels in addition to L-type Ca2+ channels. Therefore, effect of calcium channel blockers, including benidipine, on aldosterone production and T-type Ca2+ channels using human adrenocortical cell line NCI-H295R was investigated. Benidipine efficiently inhibited KCl-induced aldosterone production at low concentration (3 and 10 nM), with inhibitory activity more potent than other calcium channel blockers. Patch clamp analysis indicated that benidipine concentration-dependently inhibited T-type Ca2+ currents at 10, 100 and 1000 nM. As for examined calcium channel blockers, inhibitory activity for T-type Ca2+ currents was well correlated with aldosterone production. L-type specific calcium channel blockers calciseptine and nifedipine showed no effect in both assays. These results indicate that inhibition of T-type Ca2+ channels is responsible for inhibition of aldosterone production in NCI-H295R cells. Benidipine efficiently inhibited KCl-induced upregulation of 11-beta-hydroxylase mRNA and aldosterone synthase mRNA as well as KCl-induced Ca2+ influx, indicating it as the most likely inhibition mechanism. Benidipine partially inhibited angiotensin II-induced aldosterone production, plus showed additive effects when used in combination with the angiotensin II type I receptor blocker valsartan. Benidipine also partially inhibited angiotensin II-induced upregulation of the above m

  14. Low doses of ethanol decrease the activity of the angiotensin-converting enzyme in the aorta of aging rats and rats treated with a nitric oxide synthase inhibitor and dexamethasone.

    PubMed

    Emel'yanov, Maksim O; Korystova, Antonina F; Kublik, Ludmila N; Levitman, Maria Kh; Shaposhnikova, Vera V; Korystov, Yuri N

    2012-01-01

    In the present study, the activity of ACE (angiotensin-converting enzyme) in the aorta of senescent rats and rats treated with the NOS (NO synthase) inhibitor L-NAME (NG-nitro-L-arginine methyl ester) or dexamethasone and the effect of low doses of ethanol (0.2-1.2 g/kg of body weight, daily for 8-12 days) on this activity were studied. We found that ACE activity increased with age and in response to L-NAME and dexamethasone treatment. Ethanol at a dose of 0.4 g/kg of body weight per day decreased ACE activity in the aorta of aged rats and of rats treated with L-NAME or dexamethasone to the level of activity in young control rats. The optimal ethanol dose (the dose inducing a maximum decrease in ACE activity) increased with increasing doses of dexamethasone: 0.4 g/kg of body weight per day at 30 μg of dexamethasone/kg of body weight and 0.8 g/kg of body weight per day at 100 μg of dexamethasone/kg of body weight. It was also found that optimal doses of ethanol increased the number of cells in the thymus of rats treated with dexamethasone. The optimal dose of ethanol of 0.4 g/kg of body weight per day, which induced a maximum decrease in ACE activity in rat aorta, corresponded to a dose of 30 g of ethanol/day, which, according to epidemiological data, produces a maximum decrease in the incidence of cardiovascular disease in humans. In conclusion, the decrease in ACE activity in vessels may be one of the main mechanisms of the beneficial effects of low doses of ethanol on human health.

  15. Two squalene synthase inhibitors, E5700 and ER-119884, interfere with cellular proliferation and induce ultrastructural and lipid profile alterations in a Candida tropicalis strain resistant to fluconazole, itraconazole, and amphotericin B.

    PubMed

    Ishida, Kelly; Visbal, Gonzalo; Rodrigues, Juliany Cola Fernandes; Urbina, Julio A; de Souza, Wanderley; Rozental, Sonia

    2011-08-01

    Three quinuclidine-based squalene synthase (SQS) inhibitors (BPQ-OH, E5700, and ER-119884) were evaluated against five Candida tropicalis strains with different susceptibility profiles to fluconazole (FLC), itraconazole (ITC), terbinafine (TRB), and amphotericin B (AMB). Although the quinuclidine derivatives were inactive against most C. tropicalis strains tested at concentrations up to 16 μg/ml, E5700 and ER-119884 showed antifungal activity against C. tropicalis ATCC 28707, a strain resistant to FLC, ITC, and AMB, with IC(50) and IC(90) values (i.e., the minimum inhibitory concentrations of the drugs determined as the lowest drug concentrations leading to a 50 and 90% of reduction in turbidity at 492 nm, respectively, after 48 h of incubation) of 1 and 4 μg/ml, respectively. Analysis of free sterols showed that non-treated C. tropicalis ATCC 28707 cells contained only 14-methylated sterols and that treatment with E5700 or ER-119884 led to a marked reduction of squalene content and the complete disappearance of the endogenous sterols. The fatty acid and phospholipid profiles in C. tropicalis ATCC 28707 cells grown in the presence of E5700 and ER-119884 were also markedly altered, with a large increase in the content of linolenic acid (C18:3), associated with a reduction in the content of linoleic (C18:2) and oleic (C18:1) acids. Treatment of C. tropicalis ATCC 28707 with E5700 or ER-119884 IC(50) values induced several ultrastructural alterations, including a marked increase in the thickness of the cell wall and the appearance of a large number of electron-dense vacuoles. In conclusion, our results indicated that E5700 and ER-119884 inhibited the growth and altered the lipid prolife and the ultrastructure of a multiple drug-resistant C. tropicalis strain. Therefore, such compounds could act as leads for the development of new treatment options against multidrug resistant Candida species.

  16. Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843.

    PubMed

    Wolfe, L A; Mullin, R J; Laethem, R; Blumenkopf, T A; Cory, M; Miller, J F; Keith, B R; Humphreys, J; Smith, G K

    1999-01-01

    Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme-antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies

  17. Effect of aldosterone-producing adenoma on endothelial function and Rho-associated kinase activity in patients with primary aldosteronism.

    PubMed

    Matsumoto, Takeshi; Oki, Kenji; Kajikawa, Masato; Nakashima, Ayumu; Maruhashi, Tatsuya; Iwamoto, Yumiko; Iwamoto, Akimichi; Oda, Nozomu; Hidaka, Takayuki; Kihara, Yasuki; Kohno, Nobuoki; Chayama, Kazuaki; Goto, Chikara; Aibara, Yoshiki; Noma, Kensuke; Liao, James K; Higashi, Yukihito

    2015-04-01

    The purpose of this study was to evaluate vascular function and activity of Rho-associated kinases (ROCKs) in patients with primary aldosteronism. Vascular function, including flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation, and ROCK activity in peripheral leukocytes were evaluated in 21 patients with aldosterone-producing adenoma (APA), 23 patients with idiopathic hyperaldosteronism (IHA), and 40 age-, sex-, and blood pressure-matched patients with essential hypertension (EHT). FMD was significantly lower in the APA group than in the IHA and EHT groups (3.2±2.0% versus 4.6±2.3% and 4.4±2.2%; P<0.05, respectively), whereas there was no significant difference in FMD between the IHA and EHT groups. There was no significant difference in nitroglycerine-induced vasodilation in the 3 groups. ROCK activity was higher in the APA group than in the IHA and EHT groups (1.29±0.57 versus 1.00±0.46 and 0.81±0.36l; P<0.05, respectively), whereas there was no significant difference in ROCK activity between the IHA and EHT groups. FMD correlated with age (r=-0.31; P<0.01), plasma aldosterone concentration (r=-0.35; P<0.01), and aldosterone:renin ratio (r=-0.34; P<0.01). ROCK activity correlated with age (r=-0.24; P=0.04), plasma aldosterone concentration (r=0.33; P<0.01), and aldosterone:renin ratio (r=0.46; P<0.01). After adrenalectomy, FMD and ROCK activity were restored in patients with APA. APA was associated with both endothelial dysfunction and increased ROCK activity compared with those in IHA and EHT. APA may have a higher risk of future cardiovascular events.

  18. Dopaminergic Inhibition of Metoclopramide-induced Aldosterone Secretion in Man

    PubMed Central

    Carey, Robert M.; Thorner, Michael O.; Ortt, Elizabeth M.

    1980-01-01

    This study was designed to investigate the role of dopaminergic mechanisms in the control of aldosterone secretion in man. Five normal male subjects in metabolic balance at 150 meq sodium/d and 60 meq potassium/d constant intake received the specific dopamine antagonist, metoclopramide, 10 mg i.v. on 2 consecutive d. On the 1st d, the subjects received an infusion of 5% glucose solution (vehicle) from 60 min before to 60 min after metoclopramide administration; on the 2nd d, an infusion of dopamine 4 μg/kg per min was substituted for vehicle. Metoclopramide in the presence of vehicle increased plasma aldosterone concentrations from 2.4±1.1 to a maximum of 17.2±2.8 ng/100 ml (P < 0.01) and serum prolactin concentrations from 7.5±5.0 to a maximum of 82.2±8.7 ng/ml (P < 0.01). Dopamine 4 μg/kg per min did not alter basal plasma aldosterone concentrations, but blunted the aldosterone responses to metoclopramide significantly; in the presence of dopamine, plasma aldosterone concentrations increased from 3.1±0.5 to 6.2±1.4 ng/100 ml (P < 0.05) in response to metoclopramide. The incremental aldosterone responses to metoclopramide were significantly lower in the presence of dopamine than with vehicle. Dopamine 4 μg/kg per min suppressed basal prolactin to <3 ng/ml and inhibited the prolactin responses to metoclopramide; serum prolactin concentrations increased to a maximum of 8.5±2.3 ng/ml with metoclopramide in the presence of dopamine. The subjects were studied in the same manner except that dopamine 2 μg/kg per min was administered instead of the 4-μg/kg per min dose. Dopamine 2 μg/kg per min attenuated the aldosterone and prolactin responses to metoclopramide, but was less effective than the 4-μg/kg per min dose of dopamine. Metoclopramide 10 mg i.v. was administered to five additional subjects after pretreatment with the dopamine agonist, bromocriptine, 2.5 mg or placebo at 6 p.m., midnight, and 6 a.m. before study. Bromocriptine suppressed basal serum

  19. Synthesis and evaluation of 2,5-dihydrochorismate analogues as inhibitors of the chorismate-utilising enzymes.

    PubMed

    Payne, Richard J; Bulloch, Esther M M; Toscano, Miguel M; Jones, Michelle A; Kerbarh, Olivier; Abell, Chris

    2009-06-07

    A library of 2,5-dihydrochorismate analogues were designed as inhibitors of the chorismate-utilising enzymes including anthranilate synthase, isochorismate synthase, salicylate synthase and 4-amino-4-deoxychorismate synthase. The inhibitors were synthesised in seven or eight steps from shikimic acid, sourced from star anise. The compounds exhibited moderate but differential inhibition against the four chorismate-utilising enzymes.

  20. Inhibition of Fatty Acid Synthase in Prostate Cancer by Orlistat, a Novel Therapeutic

    DTIC Science & Technology

    2007-11-01

    C.W. Fatty acid synthase inhibitors: new directions for oncology. Expert Opinion on Investigational Drugs (2007) 16(11): 1817-29 (Invited Review...acid synthase inhibitors: new directions for oncology. Expert Opinion on Investigational Drugs (2007) 16(11): 1817-29 (Invited Review) Abstracts...Kuhajda FP: Fatty-acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 2000, 16:202-208. 3. Smith S: The animal fatty

  1. Reversible heart rhythm complexity impairment in patients with primary aldosteronism

    NASA Astrophysics Data System (ADS)

    Lin, Yen-Hung; Wu, Vin-Cent; Lo, Men-Tzung; Wu, Xue-Ming; Hung, Chi-Sheng; Wu, Kwan-Dun; Lin, Chen; Ho, Yi-Lwun; Stowasser, Michael; Peng, Chung-Kang

    2015-08-01

    Excess aldosterone secretion in patients with primary aldosteronism (PA) impairs their cardiovascular system. Heart rhythm complexity analysis, derived from heart rate variability (HRV), is a powerful tool to quantify the complex regulatory dynamics of human physiology. We prospectively analyzed 20 patients with aldosterone producing adenoma (APA) that underwent adrenalectomy and 25 patients with essential hypertension (EH). The heart rate data were analyzed by conventional HRV and heart rhythm complexity analysis including detrended fluctuation analysis (DFA) and multiscale entropy (MSE). We found APA patients had significantly decreased DFAα2 on DFA analysis and decreased area 1-5, area 6-15, and area 6-20 on MSE analysis (all p < 0.05). Area 1-5, area 6-15, area 6-20 in the MSE study correlated significantly with log-transformed renin activity and log-transformed aldosterone-renin ratio (all p < = 0.01). The conventional HRV parameters were comparable between PA and EH patients. After adrenalectomy, all the altered DFA and MSE parameters improved significantly (all p < 0.05). The conventional HRV parameters did not change. Our result suggested that heart rhythm complexity is impaired in APA patients and this is at least partially reversed by adrenalectomy.

  2. Activating mutations in CTNNB1 in aldosterone producing adenomas

    PubMed Central

    Åkerström, Tobias; Maharjan, Rajani; Sven Willenberg, Holger; Cupisti, Kenko; Ip, Julian; Moser, Ana; Stålberg, Peter; Robinson, Bruce; Alexander Iwen, K.; Dralle, Henning; Walz, Martin K.; Lehnert, Hendrik; Sidhu, Stan; Gomez-Sanchez, Celso; Hellman, Per; Björklund, Peyman

    2016-01-01

    Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation. PMID:26815163

  3. Circadian rhythm of aldosterone in dairy cattle during the summer

    NASA Astrophysics Data System (ADS)

    Aranas, T. J.; Roussel, J. D.; Seybt, S. H.

    1987-09-01

    Twelve Holstein heifers, pregnant from 120 150 days were used to study the circadian rhythm of aldosterone, cortisol, progesterone, sodium and potassium in dairy cattle during the summer in Louisiana. Cortisol was not significantly influenced by time (time 1 = 06.00 h). Aldosterone, sodium, potassium and progesterone changed significantly (P<.01) with time. Aldosterone peaked (116.5±17.2 pg/ml) at 08.00 h and then generally declined to 16.00 h (26.7±2.0 pg/ml). Sodium generally increased from 06.00 h (320.1±7.3 mg%) to 18.00 h (377.9±6.1 mg%), and then declined. Potassium generally increased from 06.00 h (20.9±0.5 mg%) to 22.00 h (23.0±0.3 mg%). Progesterone generally increased from 07.00 h (2.8±0.4 mg/ml) to 24.00 h (7.5±1.4 mg/ml). Aldosterone was significantly related to temperature associated with the time of the day samples were taken (r = 0.66, P<.02).

  4. 21 CFR 862.1045 - Aldosterone test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aldosterone test system. 862.1045 Section 862.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  5. 21 CFR 862.1045 - Aldosterone test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aldosterone test system. 862.1045 Section 862.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  6. 21 CFR 862.1045 - Aldosterone test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aldosterone test system. 862.1045 Section 862.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  7. 21 CFR 862.1045 - Aldosterone test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aldosterone test system. 862.1045 Section 862.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  8. 21 CFR 862.1045 - Aldosterone test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aldosterone test system. 862.1045 Section 862.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  9. Dopamine Inhibits Angiotensin-Stimulated Aldosterone Biosynthesis in Bovine Adrenal Cells

    PubMed Central

    Mc Kenna, Terence J.; Island, Donald P.; Nicholson, Wendell E.; Liddle, Grant W.

    1979-01-01

    The possibility that dopamine may play a role in the in vivo control of aldosterone production in man was suggested to us by reports from others; (a) that bromocriptine, a dopaminergic agonist, inhibits the aldosterone response to diuresis and to the infusion of angiotensin or ACTH; and (b) that metaclopramide, a dopamine blocking agent, causes elevations in plasma aldosterone levels. To determine whether such effects were direct or indirect, we examined the action of dopamine on aldosterone biosynthesis in isolated, bovine adrenal cells. Dopamine significantly inhibits the aldosterone response to angiotensin (P < 0.001), but does not influence basal aldosterone biosynthesis. It has previously been reported that angiotensin stimulates both the early and late phases of aldosterone biosynthesis. The present experiments demonstrated that the enhancing effect of angiotensin on the conversion of deoxycorticosterone to aldosterone (late phase of aldosterone biosynthesis) was almost completely inhibited by dopamine (P < 0.001). A significant inhibitory effect of dopamine (10 nM) was seen even when aldosterone biosynthesis was stimulated by a grossly supraphysiological concentration of angiotensin II (10 μM). However, these studies did not demonstrate any direct effect of dopamine on the early phase of aldosterone biosynthesis (cholesterol to pregnenolone) basally or when stimulated, or on the late phase of aldosterone biosynthesis under basal conditions. These in vitro studies suggest a direct inhibitory role for dopamine on the late phase of aldosterone biosynthesis, which may account for the in vivo inhibition of the aldosterone response to angiotensin in subjects treated with a dopaminergic agent. PMID:447857

  10. [Primary aldosteronism and pregnancy: report of 2 cases].

    PubMed

    Germain, Alfredo M; Kottman, Cristián; Valdés, Gloria

    2002-12-01

    Based on two patients, we discuss the difficulties in diagnosing and managing primary aldosteronism in pregnancy, which derive from changes of the renin-angiotensin-aldosterone axis, from the uncertainty regarding blood pressure control along gestation and postpartum, and from the contraindication to the use of spironolactone. The first case is a 27 years old woman with a long standing refractory hypertension, a hemorrhagic stroke with left brachial hemiplegia and crural hemiparesia, two miscarriages, one stillbirth and one offspring with intrauterine growth retardation. Due to hypokalemia, a plasma aldosterone/renin activity ratio of 91, and a negative genetic screening for glucocorticoid remediable aldosteronism (GRA), a primary hyperaldosteronism with normal adrenals in CT scan was diagnosed, and good blood pressure control was attained with spironolactone. After two and a half years of normotension, a fifth pregnancy, managed with methyldopa evolved with satisfactory blood pressures, plasma potassium, fetal growth, uterine and umbilical arterial resistance indexes, and maternal endothelial function. At 37 1/2 weeks of pregnancy the patient delivered a healthy newborn weighing 2,960 g. Blood pressure rose during the 48 hours of postpartum in the absence of proteinuria and required i.v. hydralazine. The second patient is a 37 years old woman, with known refractory hypertension for 7 years, hypokalemia, plasma aldosterone/renin activity ratio greater than 40, normal adrenals in the CAT scan, and a negative genetic screening for GRA. She had normotensive pregnancies 5 and 3 years prior to the detection of hypertension, with hypertensive crisis in both postpartum periods, retrospectively considered as expressions of primary hyperaldosteronism.

  11. Type I receptors in parotid, colon, and pituitary are aldosterone selective in vivo

    SciTech Connect

    Sheppard, K.; Funder, J.W. )

    1987-10-01

    Previous in vivo studies have demonstrated that type I receptors in the rat kidney are aldosterone selective, whereas those in the hippocampus do not appear to discriminate between aldosterone and corticosterone. The authors have injected mature rats with ({sup 3}H)aldosterone or ({sup 3}H)corticosterone plus 100-fold excess of RU 28362, with or without unlabeled aldosterone or corticosterone, and compared type I receptor occupancy in two classic mineralocorticoid target tissues (parotid and colon) and in the pituitary. Mature rats were killed 10-180 min after tracer administration; ({sup 3}H)aldosterone was well taken up and retained in all tissues, whereas ({sup 3}H)corticosterone was significantly retained only in the pituitary 10 min after tracer administration. To assess a possible role for corticosterone-binding globulin (CBG) in conferring aldosterone specificity on type I receptors, 10-day-old rats (with very low levels of CBG) were similarly injected. In the colon and parotid, ({sup 3}H)aldosterone binding was at least an order of magnitude higher than that of corticosterone; in the pituitary aldosterone binding was approximately three times that of corticosterone. They interpret these data as evidence that in the parotid and colon type I receptors are aldosterone selective by a non-CBG-requiring mechanism, whereas in the pituitary there appear to be both aldosterone-selective and nonselective type I sites.

  12. Placental growth factor mediates aldosterone-dependent vascular injury in mice.

    PubMed

    Jaffe, Iris Z; Newfell, Brenna G; Aronovitz, Mark; Mohammad, Najwa N; McGraw, Adam P; Perreault, Roger E; Carmeliet, Peter; Ehsan, Afshin; Mendelsohn, Michael E

    2010-11-01

    In clinical trials, aldosterone antagonists reduce cardiovascular ischemia and mortality by unknown mechanisms. Aldosterone is a steroid hormone that signals through renal mineralocorticoid receptors (MRs) to regulate blood pressure. MRs are expressed and regulate gene transcription in human vascular cells, suggesting that aldosterone might have direct vascular effects. Using gene expression profiling, we identify the pro-proliferative VEGF family member placental growth factor (PGF) as an aldosterone-regulated vascular MR target gene in mice and humans. Aldosterone-activated vascular MR stimulated Pgf gene transcription and increased PGF protein expression and secretion in the mouse vasculature. In mouse vessels with endothelial damage and human vessels from patients with atherosclerosis, aldosterone enhanced expression of PGF and its receptor, FMS-like tyrosine kinase 1 (Flt1). In atherosclerotic human vessels, MR antagonists inhibited PGF expression. In vivo, aldosterone infusion augmented vascular remodeling in mouse carotids following wire injury, an effect that was lost in Pgf-/- mice. In summary, we have identified PGF as what we believe to be a novel downstream target of vascular MR that mediates aldosterone augmentation of vascular injury. These findings suggest a non-renal mechanism for the vascular protective effects of aldosterone antagonists in humans and support targeting the vascular aldosterone/MR/PGF/Flt1 pathway as a therapeutic strategy for ischemic cardiovascular disease.

  13. O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase

    PubMed Central

    Talukdar, Arindam; Zhao, Yujie; Lv, Wei; Bacher, Adelbert; Illarionov, Boris; Fischer, Markus; Cushman, Mark

    2012-01-01

    Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction. PMID:22780198

  14. [Hypertension in the course of primary aldosteronism during pregnancy].

    PubMed

    Wyskida, Magdalena; Wyskida, Katarzyna; Olszanecka-Glinianowicz, Magdalena; Maruniak-Chudek, Iwona; Sikora, Jerzy; Chudek, Jerzy

    2015-02-15

    Hypertension is one of the most common cardiovascular diseases during pregnancy. Primary hyperaldosteronism (PHA) is the most frequent endocrinological, secondary cause of hypertension, rarely diagnosed in pregnant women. In the available literature about 50 cases of PHA in pregnant women have been described. PHA is often a cause of resistant hypertension. PHA can cause life-threatening complications both for the pregnant woman and the fetus. Diagnosis of PHA in pregnancy is difficult due to the antagonistic effect of progesterone on aldosterone, physiological increase of aldosterone release during gestation and frequent normokalaemic clinical course. Typical pharmacological treatment of PHA is limited due to the anti‑androgenic effect of spironolactone, lack of data concerning the safety of eplerenone and limited access to amiloride in Poland. Surgical treatment is a therapeutic option only in early pregnancy. This paper presents the current state of knowledge on diagnostic methods and treatment of PHA in pregnant women and a systematic review of cases described in the literature.

  15. High prevalence of thyroid ultrasonographic abnormalities in primary aldosteronism.

    PubMed

    Armanini, Decio; Nacamulli, Davide; Scaroni, Carla; Lumachi, Franco; Selice, Riccardo; Fiore, Cristina; Favia, Gennaro; Mantero, Franco

    2003-11-01

    The study was performed to evaluate the prevalence of thyroid abnormalities detected by ultrasonography and, in particular, of multinodular nontoxic goiter in primary aldosteronism. We analyzed 80 consecutive of patients with primary hyperaldosteronism (40 with unilateral adenoma and 40 with idiopathic hyperaldosteronism) and 80 normotensive healthy controls, comparable for age, sex, iodine intake, and geographical area. Blood pressure, thyroid palpation, thyroid function, and ultrasonography were evaluated. The prevalence of ultrasonographic thyroid abnormalities was 60% in primary aldosteronism and 27% in controls (p < 0.0001). There was a statistically significant difference in prevalence of these abnormalities in unilateral adenoma and idiopathic hyperaldosteronism with respect to controls (p < 0.05 and p < 0.0001, respectively). The prevalence of multinodular nontoxic goiter in idiopathic hyperaldosteronism was higher than in controls (p < 0.001) and, in particular, in female patients. From these data it seems to be worth considering the existence of primary hyperaldosteronism in patients with multinodular goiter and hypertension.

  16. Zero gravity and cardiovascular homeostasis. The relationship between endogenous hyperprolactinemia and plasma aldosterone

    NASA Technical Reports Server (NTRS)

    Haber, E.; Re, R. N.; Kourides, I. A.; Weihl, A. C.; Maloof, F.

    1978-01-01

    Prolactin, thyrotropin and aldosterone were measured by radioimmunoassay and plasma renin activity by the radioimmunoassay of angiotensin I in normal women before and after the intravenous injection of 200 micrograms of thyrotropin releasing hormone. Prolactin increased at 15 minutes following thyrotropin releasing hormone. Plasma renin activity was not different from control levels during the first hour following the administration of thyrotropin releasing hormone, nor did the plasma aldosterone concentration differ significantly from the control levels during this period. However, with upright posture, an increase in aldosterone and in plasma renin activity was noted, demonstrating a normal capacity to secrete aldosterone. Similarly, no change in aldosterone was seen in 9 patients with primary hypothyroidism given thyrotropin releasing hormone, despite the fact that the increase in prolactin was greater than normal. These data demonstrate that acutely or chronically elevated serum prolactin levels do not result in increased plasma aldosterone levels in humans.

  17. SFE/SFHTA/AFCE primary aldosteronism consensus: Introduction and handbook.

    PubMed

    Amar, Laurence; Baguet, Jean Philippe; Bardet, Stéphane; Chaffanjon, Philippe; Chamontin, Bernard; Douillard, Claire; Durieux, Pierre; Girerd, Xaxier; Gosse, Philippe; Hernigou, Anne; Herpin, Daniel; Houillier, Pascal; Jeunemaitre, Xavier; Joffre, Francis; Kraimps, Jean-Louis; Lefebvre, Hervé; Ménégaux, Fabrice; Mounier-Véhier, Claire; Nussberger, Juerg; Pagny, Jean-Yves; Pechère, Antoinette; Plouin, Pierre-François; Reznik, Yves; Steichen, Olivier; Tabarin, Antoine; Zennaro, Maria-Christina; Zinzindohoue, Franck; Chabre, Olivier

    2016-07-01

    The French Endocrinology Society (SFE) French Hypertension Society (SFHTA) and Francophone Endocrine Surgery Association (AFCE) have drawn up recommendations for the management of primary aldosteronism (PA), based on an analysis of the literature by 27 experts in 7 work-groups. PA is suspected in case of hypertension associated with one of the following characteristics: severity, resistance, associated hypokalemia, disproportionate target organ lesions, or adrenal incidentaloma with hypertension or hypokalemia. Diagnosis is founded on aldosterone/renin ratio (ARR) measured under standardized conditions. Diagnostic thresholds are expressed according to the measurement units employed. Diagnosis is established for suprathreshold ARR associated with aldosterone concentrations >550pmol/L (200pg/mL) on 2 measurements, and rejected for aldosterone concentration<240pmol/L (90pg/mL) and/or subthreshold ARR. The diagnostic threshold applied is different if certain medication cannot be interrupted. In intermediate situations, dynamic testing is performed. Genetic forms of PA are screened for in young subjects and/or in case of familial history. The patient should be informed of the results expected from medical and surgical treatment of PA before exploration for lateralization is proposed. Lateralization is explored by adrenal vein sampling (AVS), except in patients under 35 years of age with unilateral adenoma on imaging. If PA proves to be lateralized, unilateral adrenalectomy may be performed, with adaptation of medical treatment pre- and postoperatively. If PA is non-lateralized or the patient refuses surgery, spironolactone is administered as first-line treatment, replaced by amiloride, eplerenone or calcium-channel blockers if insufficiently effective or poorly tolerated.

  18. Familial or genetic primary aldosteronism and Gordon syndrome.

    PubMed

    Stowasser, Michael; Pimenta, Eduardo; Gordon, Richard D

    2011-06-01

    Salt-sensitive forms of hypertension have received considerable renewed attention in recent years. This article focuses on 2 main forms of salt-sensitive hypertension (familial or genetic primary aldosteronism [PA] and Gordon syndrome) and the current state of knowledge regarding their genetic bases. The glucocorticoid-remediable form of familial PA (familial hyperaldosteronism type I) is dealt with only briefly because it is covered in depth elsewhere.

  19. Plasma Renin Activity and Aldosterone: Correlations with Moderate Hypohydration

    DTIC Science & Technology

    1989-12-01

    circulating aldosterone, in conjunction Oith drinking systems were evaluated: the current gravity-fed sys- vasopressin ( antidiuretic hormone ), are the humoral...surprising that there is available an ex- and analyzed for fluid-electrolyte regulatory hormones . During all trials with chemical protective garments...hot ambient consumption, increased hypohydration, and elicited the great- conditions is accompanied by hormonal adaptations to est elevations in PRA and

  20. Time-dependent aldosterone metabolism in toad urinary bladder

    SciTech Connect

    Brem, A.S.; Pacholski, M.; Morris, D.J.

    1988-04-01

    Aldosterone (Aldo) metabolism was examined in the toad bladder. Bladders were incubated with (/sup 3/H)aldosterone (10(-7) M) for 5 h, 1 h, or 10 min. Tissues were analyzed for metabolites using high-pressure liquid chromatography (HPLC). In separate experiments, Na+ transport was assessed by the short-circuit current (SCC) technique. Following a 5-h tissue incubation, about 25% of the (/sup 3/H)-aldosterone was converted into metabolites including a polar monosulfate metabolite, 20 beta-dihydroaldo (20 beta-DHAldo), small quantities of 5 beta-reduced products, and a variety of 5 alpha-reduced Aldo products including 5 alpha-DHAldo, 3 alpha,5 alpha-tetrahydroaldo (3 alpha,5 alpha-THAldo), and 3 beta,5 alpha-THAldo. Tissues metabolized approximately 10% of the labeled hormone into the same compounds by 1 h. Measurable quantities of these metabolites were also synthesized by bladders exposed to Aldo for only 10 min and then incubated in buffer for an additional 50 min without Aldo. Bladders pretreated with the spironolactone, K+-canrenoate (3.5 X 10(-4) M), and stimulated with Aldo (10(-7) M) generated a peak SCC 44 +/- 6% of that observed in matched pairs stimulated with Aldo (P less than 0.001; n = 6). K+-canrenoate also markedly diminished (/sup 3/H)aldosterone metabolism at both 5 and 1 h. Thus, metabolic transformation of Aldo begins prior to hormone-induced increases in Na+ transport. Both the generation of certain metabolites (e.g., 5 alpha-reductase pathway products) and the increase in Na+ transport can be selectively inhibited by K+-canrenoate.

  1. Paradoxical glucose-induced hyperkalemia. Combined aldosterone-insulin deficiency.

    PubMed

    Goldfarb, S; Strunk, B; Singer, I; Goldberg, M

    1975-11-01

    Severe hyperkalemia associated with spontaneous hyperglycemia as well as with the intravenous infusions of glucose occurred in an insulin-requiring diabetic patient in the absence of potassium administration, the use of diuretics which inhibit urinary potassium excretion or acidemia. Metabolic balance studies revealed, in addition to diabets, the presence of isolated aldosterone deficiency of the hyporeninemic type. Intravenous glucose infusions (0.5 g/kg body weight) produced significant hyperkalemia but desoxycortisone acetate (DOCA) therapy (10 mg/day) prevented the glucose-induced hyperkalemia. In this patient, the serum potassium concentration increases after the intravenous infusions of glucose because there is insufficient aldosterone and insulin to reverse the transfer of potassium to the extracellular fluid which normally occurs after hypertonic infusions of glucose. Although DOCA replacement modifies the distribution of potassium in the extracellular fluid and blunts the hyperkalemic effect of intravenous infusions of glucose, a rise in the insulin level is required for the usual hypokalemic response to intravenously administered glucose. These studies illustrate the risk of raising blood glucose levels in patients with combined aldosterone and insulin deficiency and the tendency towards hyperkalemia in diabetic patients under certain clinical conditions.

  2. Reverse remodeling and recovery from cachexia in rats with aldosteronism.

    PubMed

    Cheema, Yaser; Zhao, Wenyuan; Zhao, Tieqiang; Khan, M Usman; Green, Kelly D; Ahokas, Robert A; Gerling, Ivan C; Bhattacharya, Syamal K; Weber, Karl T

    2012-08-15

    The congestive heart failure (CHF) syndrome with soft tissue wasting, or cachexia, has its pathophysiologic origins rooted in neurohormonal activation. Mechanical cardiocirculatory assistance reveals the potential for reverse remodeling and recovery from CHF, which has been attributed to device-based hemodynamic unloading whereas the influence of hormonal withdrawal remains uncertain. This study addresses the signaling pathways induced by chronic aldosteronism in normal heart and skeletal muscle at organ, cellular/subcellular, and molecular levels, together with their potential for recovery (Recov) after its withdrawal. Eight-week-old male Sprague-Dawley rats were examined at 4 wk of aldosterone/salt treatment (ALDOST) and following 4-wk Recov. Compared with untreated, age-/sex-/strain-matched controls, ALDOST was accompanied by 1) a failure to gain weight, reduced muscle mass with atrophy, and a heterogeneity in cardiomyocyte size across the ventricles, including hypertrophy and atrophy at sites of microscopic scarring; 2) increased cardiomyocyte and mitochondrial free Ca(2+), coupled to oxidative stress with increased H(2)O(2) production and 8-isoprostane content, and increased opening potential of the mitochondrial permeability transition pore; 3) differentially expressed genes reflecting proinflammatory myocardial and catabolic muscle phenotypes; and 4) reversal to or toward recovery of these responses with 4-wk Recov. Aldosteronism in rats is accompanied by cachexia and leads to an adverse remodeling of the heart and skeletal muscle at organ, cellular/subcellular, and molecular levels. However, evidence presented herein implicates that these tissues retain their inherent potential for recovery after complete hormone withdrawal.

  3. Regulation of aldosterone secretion by Cav1.3

    PubMed Central

    Xie, Catherine B.; Haris Shaikh, Lalarukh; Garg, Sumedha; Tanriver, Gizem; Teo, Ada E. D.; Zhou, Junhua; Maniero, Carmela; Zhao, Wanfeng; Kang, Soosung; Silverman, Richard B.; Azizan, Elena A. B.; Brown, Morris J.

    2016-01-01

    Aldosterone-producing adenomas (APAs) vary in phenotype and genotype. Zona glomerulosa (ZG)-like APAs frequently have mutations of an L-type calcium channel (LTCC) CaV1.3. Using a novel antagonist of CaV1.3, compound 8, we investigated the role of CaV1.3 on steroidogenesis in the human adrenocortical cell line, H295R, and in primary human adrenal cells. This investigational drug was compared with the common antihypertensive drug nifedipine, which has 4.5-fold selectivity for the vascular LTCC, CaV1.2, over CaV1.3. In H295R cells transfected with wild-type or mutant CaV1.3 channels, the latter produced more aldosterone than wild-type, which was ameliorated by 100 μM of compound 8. In primary adrenal and non-transfected H295R cells, compound 8 decreased aldosterone production similar to high concentration of nifedipine (100 μM). Selective CaV1.3 blockade may offer a novel way of treating primary hyperaldosteronism, which avoids the vascular side effects of CaV1.2-blockade, and provides targeted treatment for ZG-like APAs with mutations of CaV1.3. PMID:27098837

  4. Plasma aldosterone and sweat sodium concentrations after exercise and heat acclimation

    NASA Technical Reports Server (NTRS)

    Kirby, C. R.; Convertino, V. A.

    1986-01-01

    The relationship between plasma aldosterone levels and sweat sodium excretion after chronic exercise and heat acclimation was investigated, using subjects exercised, at 40 C and 45 percent humidity, for 2 h/day on ten consecutive days at 45 percent of their maximal oxygen uptake. The data indicate that, following heat acclimation, plasma aldosterone concentrations decrease, and that the eccrine gland responsiveness to aldosterone, as represented by sweat sodium reabsorption, may be augmented through exercise and heat acclimation.

  5. Development and Binding Mode Assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic acid (BGC 945), a Novel Thymidylate Synthase Inhibitor that Targets Tumor Cells

    PubMed Central

    Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv; O’Connell, Joseph D.; Griner, Sarah; Finer-Moore, Janet S.; Stroud, Robert M.

    2013-01-01

    N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in Phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with α-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through α-folate receptor (α-FR) transport. The α-FR, a cell-surface receptor glycoprotein, which is over expressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2’-deoxyuridine-5’-monophosphate, and a model for a similar complex with human TS. PMID:23710599

  6. The Influence of a Heparin-Like Compound on Hypertension, Electrolytes and Aldosterone in Man

    PubMed Central

    Abbott, E. C.; Gornall, A. G.; Sutherland, D. J. A.; Stiefel, M.; Laidlaw, J. C.

    1966-01-01

    The mechanisms of the aldosterone-inhibiting and antihypertensive actions of heparin and certain other sulfated mucopolysaccharides were investigated in 13 hypertensive patients. N-formyl chitosan polysulfuric acid (RO 1-8307) was used rather than heparin because of its low anticoagulant activity. RO 1-8307 lowered aldosterone secretion in one patient with proved and one with probable primary aldosteronism; this indicates that the mucopolysaccharide does not inhibit aldosterone secretion via the renin-angiotensin mechanism. In six hypertensive patients RO 1-8307 caused a fall in the secretion of aldosterone and its probable immediate precursor, 18-hydroxycorticosterone; therefore it does not act at the last step in the synthesis of aldosterone. RO 1-8307 produced a prolonged clinical and biochemical remission in the two patients considered to have primary aldosteronism but did not influence blood pressure in five of seven patients in whom excessive aldosterone was probably not a major factor in the hypertension. It is concluded that the antihypertensive action of RO 1-8307, and perhaps of heparin as well, is largely due to suppression of aldosterone secretion. ImagesFig. 6 PMID:4222823

  7. Assessment of the Quantitative Value Usefulness of the Aldosterone-Renin Ratio (ARR) for Primary Aldosteronism (AQUARR) Study.

    PubMed

    Maiolino, Giuseppe; Mareso, Sara; Bisogni, Valeria; Rossitto, Giacomo; Azzolini, Matteo; Cesari, Maurizio; Seccia, Teresa Maria; Calò, Lorenzo; Rossi, Gian Paolo

    2016-03-01

    Current guidelines recommend use of the aldosterone-renin ratio (ARR) for the case detection of primary aldosteronism (PA), the most common cause of secondary hypertension, in selected hypertensive patients. "Confirmatory" tests are then recommended in patients who tested positive at the ARR to exclude from further diagnostic work-up false positive results. Based on our experience we hypothesized that the ARR carries quantitative information, which can avoid the need of confirmatory tests. We herein describe a study protocol to identify the ARR cut-off value with a high specificity for the exclusion of aldosterone-producing adenoma (APA) based on analysis of two large prospectively collected datasets of patients in which a conclusive diagnosis of APA was made by the four corners criteria. This will also serve to investigate the diagnostic gain provided over this ARR cut-off value by one confirmatory test, the captopril challenge test. Hence, with this protocol we expect to identify an ARR cut-off value that might prevent further testing in patients with either a low or a high probability of APA. This could translate in a higher diagnostic accuracy and, by preventing unnecessary invasive testing, into a substantial saving of money, time, and resources.

  8. Genomic and rapid effects of aldosterone: what we know and do not know thus far.

    PubMed

    Hermidorff, Milla Marques; de Assis, Leonardo Vinícius Monteiro; Isoldi, Mauro César

    2017-01-01

    Aldosterone is the most known mineralocorticoid hormone synthesized by the adrenal cortex. The genomic pathway displayed by aldosterone is attributed to the mineralocorticoid receptor (MR) signaling. Even though the rapid effects displayed by aldosterone are long known, our knowledge regarding the receptor responsible for such event is still poor. It is intense that the debate whether the MR or another receptor-the "unknown receptor"-is the receptor responsible for the rapid effects of aldosterone. Recently, G protein-coupled estrogen receptor-1 (GPER-1) was elegantly shown to mediate some aldosterone-induced rapid effects in several tissues, a fact that strongly places GPER-1 as the unknown receptor. It has also been suggested that angiotensin receptor type 1 (AT1) also participates in the aldosterone-induced rapid effects. Despite this open question, the relevance of the beneficial effects of aldosterone is clear in the kidneys, colon, and CNS as aldosterone controls the important water reabsorption process; on the other hand, detrimental effects displayed by aldosterone have been reported in the cardiovascular system and in the kidneys. In this line, the MR antagonists are well-known drugs that display beneficial effects in patients with heart failure and hypertension; it has been proposed that MR antagonists could also play an important role in vascular disease, obesity, obesity-related hypertension, and metabolic syndrome. Taken altogether, our goal here was to (1) bring a historical perspective of both genomic and rapid effects of aldosterone in several tissues, and the receptors and signaling pathways involved in such processes; and (2) critically address the controversial points within the literature as regarding which receptor participates in the rapid pathway display by aldosterone.

  9. AMP-activated protein kinase inhibits TGF-β-, angiotensin II-, aldosterone-, high glucose-, and albumin-induced epithelial-mesenchymal transition.

    PubMed

    Lee, Jang Han; Kim, Ji Hyun; Kim, Ja Seon; Chang, Jai Won; Kim, Soon Bae; Park, Jung Sik; Lee, Sang Koo

    2013-03-15

    The epithelial-mesenchymal transition (EMT) is a novel mechanism that promotes renal fibrosis. Transforming growth factor-β (TGF-β), angiotensin II, aldosterone, high glucose, and urinary albumin are well-known causes of EMT and renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed EMT induced by the above agents in tubular epithelial cells. All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. Exposure of tubular cells to TGF-β (10 ng/ml), angiotensin II (1 μM), aldosterone (100 nM), high glucose (30 mM), and albumin (5 mg/ml) for 5 days induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ROS and NADPH oxidase 4 (Nox4) expression were increased, and antioxidants such as tiron and N-acetylcysteine inhibited EMT induction. Metformin (the best known clinical activator of AMPK) suppressed EMT induction through inhibition of ROS via induction of heme oxygenase-1 and endogenous antioxidant thioredoxin. An AMPK inhibitor (compound C) and AMPK small interfering RNA blocked the effect of metformin, and another AMPK activator [5-aminoimidazole-4-carboxamide-1β riboside (AICAR)] exerted the same effects as metformin. In conclusion, AMPK activation might be beneficial in attenuating the tubulointerstitial fibrosis induced by TGF-β, angiotensin II, aldosterone, high glucose, and urinary albumin.

  10. SFE/SFHTA/AFCE Consensus on Primary Aldosteronism, part 2: First diagnostic steps.

    PubMed

    Douillard, Claire; Houillier, Pascal; Nussberger, Juerg; Girerd, Xavier

    2016-07-01

    In patients with suspected primary aldosteronism (PA), the first diagnostic step, screening, must have high sensitivity and negative predictive value. The aldosterone-to-renin ratio (ARR) is used because it has higher sensitivity and lower variability than other measures (serum potassium, plasma aldosterone, urinary aldosterone). ARR is calculated from the plasma aldosterone (PA) and plasma renin activity (PRA) or direct plasma renin (DR) values. These measurements must be taken under standard conditions: in the morning, more than 2hours after awakening, in sitting position after 5 to 15minutes, with normal dietary salt intake, normal serum potassium level and without antihypertensive drugs significantly interfering with the renin-angiotensin-aldosterone system. To rule out ARR elevation due to very low renin values, ARR screening is applied only if aldosterone is>240pmol/l (90pg/ml); DR values<5mIU/l are assimilated to 5mIU/l and PRA values<0.2ng/ml/h to 0.2ng/ml/h. We propose threshold ARR values depending on the units used and a conversion factor (pg to mIU) for DR. If ARR exceeds threshold, PA should be suspected and exploration continued. If ARR is below threshold or if plasma aldosterone is<240pmol/l (90pg/ml) on two measurements, diagnosis of PA is excluded.

  11. Effect of exposure to hypoxia from birth on aldosterone in rabbits: role of unesterified fatty acids.

    PubMed

    Raff, H; Jankowski, B M; Goodfriend, T L; Baker, J E; Papanek, P E

    1997-04-01

    Hypoxia and fluid and electrolyte disturbances are serious risks to normal postnatal development. Because a decrease in inspired O2 (hypoxic hypoxia) inhibits aldosterone synthesis in the adult and aldosterone controls water and electrolyte balance, we studied adrenocortical function in rabbits exposed to normobaric normoxia or hypoxic hypoxia (fraction of inspired O2 0.09) from birth. At 21 days of age, rabbits were anesthetized, the adrenals were rapidly removed, and the adrenal capsules containing mostly zona glomerulosa cells were separated. Cells were dispersed with collagenase and studied in vitro. Hypoxia in vivo resulted in a 73% decrease in basal aldosterone release and a 86% decrease in adenosine 3',5'-cyclic monophosphate-stimulated aldosterone release in vitro. We hypothesized that increased unesterified fatty acids could be partly responsible for inhibition of aldosterone synthesis. Total serum unesterified fatty acids in hypoxic kits were significantly increased (298 +/- 14 micromol/l) compared with normoxic kits (184 +/- 31 micromol/l). When cells from hypoxic rabbits were washed with fatty acid-free albumin and studied under conditions devoid of fatty acids, aldosterone production was partially restored. Corticosterone production was not affected by washing. Washing had no effect on aldosterone synthesis by cells from normoxic rats. Finally, exposing washed zona glomerulosa cells to oleic acid (10-50 microM) inhibited aldosteronogenesis. We conclude that exposure to hypoxia from birth attenuates aldosterone production in part due to an increase in levels of unesterified fatty acid levels.

  12. Role of aldosterone in the remnant kidney model in the rat.

    PubMed Central

    Greene, E L; Kren, S; Hostetter, T H

    1996-01-01

    The renin-angiotensin-aldosterone system (RAAS) participates in the injury sustained by the remnant kidney. Our studies assessed the importance of aldosterone in that model and the response of aldosterone to drugs interfering with the RAAS. Initially, four groups of rats were studied: SHAM-operated rats, untreated remnant rats (REM), REM rats treated with losartan and enalapril (REM AIIA), and REM AIIA rats infused with exogenous aldosterone (REM AIIA + ALDO). The last group was maintained with aldosterone levels comparable to those in untreated REM rats by constant infusion of exogenous aldosterone. REM rats had larger adrenal glands and a > 10-fold elevation in plasma aldosterone compared to SHAM. REM AIIA rats demonstrated significant suppression of the hyperaldosteronism as well as marked attenuation of proteinuria, hypertension, and glomerulosclerosis compared to REM. REM AIIA + ALDO rats manifested greater proteinuria, hypertension, and glomerulosclerosis than REM AIIA rats. Indeed, by 4 wk of observation all of these features of the experimental disease were similar in magnitude in REM AIIA + ALDO and untreated REM. In separate REM rats spironolactone administration did not reduce glomerular sclerosis but did transiently reduce proteinuria, lowered arterial pressure, and lessened cardiac hypertrophy. In summary, aldosterone contributes to hypertension and renal injury in the remnant kidney model. PMID:8770880

  13. TASK channels are not required to mount an aldosterone secretory response to metabolic acidosis in mice

    PubMed Central

    Guagliardo, Nick A.; Yao, Junlan; Bayliss, Douglas A.; Barrett, Paula Q.

    2010-01-01

    The stimulation of aldosterone production by acidosis enhances proton excretion and serves to limit disturbances in systemic acid-base equilibrium. Yet, the mechanisms by which protons stimulate aldosterone production from cells of the adrenal cortex remain largely unknown. TWIK-related acid sensitive K channels (TASK) are inhibited by extracellular protons within the physiological range and have emerged as important regulators of aldosterone production in the adrenal cortex. Here we show that congenic C57BL/6J mice with genetic deletion of TASK-1 (K2P3.1) and TASK-3 (K2P9.1) channel subunits overproduce aldosterone and display an enhanced sensitivity to steroidogenic stimuli, including a more pronounced steroidogenic response to chronic NH4Cl loading. Thus, we conclude that TASK channels are not required for the stimulation of aldosterone production by protons but their inhibition by physiological acidosis may contribute to full expression of the steroidogenic response. PMID:21111026

  14. Clinical Practice Guideline for Management of Primary Aldosteronism: What is New in the 2016 Update?

    PubMed Central

    Romero, Damian G; Yanes Cardozo, Licy L

    2016-01-01

    Primary Aldosteronism is the single most common cause of secondary hypertension and is associated with increased target organ injury. The Endocrine Society has recently released the updated Clinical Practice Guideline for Primary Aldosteronism entitled “The Management of Primary Aldosteronism: Case Detection, Diagnosis, and Treatment: An Endocrine Society Clinical Practice Guideline”. We review the updated Clinical Practice Guideline, highlighting the new recommendations and the implications that they may have in clinical practice. The recognition by the Endocrine Society’s Task Force that Primary Aldosteronism is a public health issue and that the population at risk for screening should be significantly expanded will surely have an impact in the clinical practice which hopefully will translate in better detection, diagnosis and treatment of patients with Primary Aldosteronism. PMID:28018978

  15. mPGES-1 deletion impairs aldosterone escape and enhances sodium appetite.

    PubMed

    Jia, Zhanjun; Aoyagi, Toshinori; Kohan, Donald E; Yang, Tianxin

    2010-07-01

    Aldosterone (Aldo) is a major sodium-retaining hormone that reduces renal sodium excretion and also stimulates sodium appetite. In the face of excess Aldo, the sodium-retaining action of this steroid is overridden by an adaptive regulatory mechanism, a phenomenon termed Aldo escape. The underlying mechanism of this phenomenon is not well defined but appeared to involve a number of natriuretic factors such prostaglandins (PGs). Here, we investigated the role of microsomal prostaglandin E synthase-1 (mPGES-1) in the response to excess Aldo. A 14-day Aldo infusion at 0.35 mg x kg(-1) x day(-1) via an osmotic minipump in conjunction with normal salt intake did not produce obvious disturbances in fluid metabolism in WT mice as suggested by normal sodium and water balance, plasma sodium concentration, hematocrit, and body weight, despite the evidence of a transient sodium accumulation on days 1 or 2. In a sharp contrast, the 14-day Aldo treatment in mPGES-1 knockoute (KO) mice led to increased sodium and water balance, persistent reduction of hematocrit, hypernatremia, and body weight gain, all evidence of fluid retention. The escaped wild-type (WT) mice displayed a remarkable increase in urinary PGE(2) excretion in parallel with coinduction of mPGES-1 in the proximal tubules, accompanied by a remarkable, widespread downregulation of renal sodium and water transporters. The increase in urinary PGE(2) excretion together with the downregulation of renal sodium and water transporters were all significantly blocked in the KO mice. Interestingly, compared with WT controls, the KO mice exhibited consistent increases in sodium and water intake during Aldo infusion. Together, these results suggest an important role of mPGES-1 in antagonizing the sodium-retaining action of Aldo at the levels of both the central nervous system and the kidney.

  16. Pathogenesis of hypertension: interactions among sodium, potassium, and aldosterone.

    PubMed

    Büssemaker, Eckhart; Hillebrand, Uta; Hausberg, Martin; Pavenstädt, Hermann; Oberleithner, Hans

    2010-06-01

    Arterial hypertension is a major cause of disease-related morbidity and mortality worldwide. It is nearly absent in populations that consume natural foods low in sodium. However, in industrial countries, where the individual intake of sodium is at least 10 times higher, the prevalence of hypertension is approximately 40%. Major population-based studies link a high-sodium and low-potassium diet to an increase in blood pressure. A hallmark of arterial hypertension is endothelial dysfunction characterized by decreased synthesis of nitric oxide (NO). Plasma sodium and potassium are major determinants for the mechanical stiffness of endothelial cells. High plasma sodium levels stiffen endothelial cells and block NO synthesis. Aldosterone is a prerequisite for this action. However, high plasma potassium levels soften endothelial cells and activate NO release. There is increasing evidence that sodium can be stored transiently in considerable amounts and osmotically inactive in the interstitium. Taken together, it is recommended to maintain plasma sodium levels in the low physiologic range and potassium levels in the high physiologic range while suppressing plasma aldosterone as much as possible. A restriction in sodium intake that is accompanied by increased intake of potassium can profoundly improve the prevalence of hypertension and cardiovascular disease.

  17. Long term outcome of Aldosteronism after target treatments

    PubMed Central

    Wu, Vin-Cent; Wang, Shuo-Meng; Chang, Chia-Hui; Hu, Ya-Hui; Lin, Lian-Yu; Lin, Yen-Hung; Chueh, Shih-Chieh Jeff; Chen, Likwang; Wu, Kwan-Dun

    2016-01-01

    There exists a great knowledge gap in terms of long-term effects of various surgical and pharmacological treatments on outcomes among primary aldosteronism (PA) patients. Using a validated algorithm, we extracted longitudinal data for all PA patients diagnosed in 1997–2010 and treated in the Taiwan National Health Insurance. We identified 3362 PA patients for whom the mean length of follow-up was 5.75 years. PA has higher major cardiovascular events (MACE) than essential hypertension (23.3% vs 19.3%, p = 0.015). Results from the Cox model suggest a strong effect of adrenalectomy on lowering mortality (HR = 0.23 with residual hypertension and 0.21 with resolved hypertension). While need for receptor antagonist (MRA) MRA after diagnosis suggests that a defined daily dose (DDD) of MRA between 12.5 and 50 mg may alleviate risk of death in a U-shape pattern. A specificity test identified patients who has aldosterone producing adenoma (HR = 0.50, p = 0.005) also confirmed adrenalectomy attenuated all-cause mortality. Adrenalectomy decreases long-term all-cause mortality independently from PA cure from hypertension. Prescription corresponding to a DDD between 12.5 and 50 mg may decrease mortality for patients needing MRA. It calls for more attention on early diagnosis, early treatment and prescription of appropriate dosage of MRA for PA patients. PMID:27586402

  18. Primary aldosteronism and impaired natriuresis in mice underexpressing TGFβ1

    PubMed Central

    Kakoki, Masao; Pochynyuk, Oleh M.; Hathaway, Catherine M.; Tomita, Hirofumi; Hagaman, John R.; Kim, Hyung-Suk; Zaika, Oleg L.; Mamenko, Mykola; Kayashima, Yukako; Matsuki, Kota; Hiller, Sylvia; Li, Feng; Xu, Longquan; Grant, Ruriko; Bertorello, Alejandro M.; Smithies, Oliver

    2013-01-01

    To uncover the potential cardiovascular effects of human polymorphisms influencing transforming growth factor β1 (TGFβ1) expression, we generated mice with Tgfb1 mRNA expression graded in five steps from 10% to 300% normal. Adrenal expression of the genes for mineralocorticoid-producing enzymes ranged from 50% normal in the hypermorphs at age 12 wk to 400% normal in the hypomorphs accompanied with proportionate changes in plasma aldosterone levels, whereas plasma volumes ranged from 50% to 150% normal accompanied by marked compensatory changes in plasma angiotensin II and renin levels. The aldosterone/renin ratio ranged from 0.3 times normal in the 300% hypermorphs to six times in the 10% hypomorphs, which have elevated blood pressure. Urinary output of water and electrolytes are markedly decreased in the 10% hypomorphs without significant change in the glomerular filtration rate. Renal activities for the Na+, K+-ATPase, and epithelial sodium channel are markedly increased in the 10% hypomorphs. The hypertension in the 10% hypomorphs is corrected by spironolactone or amiloride at doses that do not change blood pressure in wild-type mice. Thus, changes in Tgfb1 expression cause marked progressive changes in multiple systems that regulate blood pressure and fluid homeostasis, with the major effects being mediated by changes in adrenocortical function. PMID:23503843

  19. ATP synthase: a molecular therapeutic drug target for antimicrobial and antitumor peptides.

    PubMed

    Ahmad, Zulfiqar; Okafor, Florence; Azim, Sofiya; Laughlin, Thomas F

    2013-01-01

    In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimicrobial/ antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F(1) sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented.

  20. Kinetic characteristics of nitric oxide synthase from rat brain.

    PubMed Central

    Knowles, R G; Palacios, M; Palmer, R M; Moncada, S

    1990-01-01

    The relationship between the rate of synthesis of nitric oxide (NO) and guanylate cyclase stimulation was used to characterize the kinetics of the NO synthase from rat forebrain and of some inhibitors of this enzyme. The NO synthase had an absolute requirement for L-arginine and NADPH and did not require any other cofactors. The enzyme had a Vmax. of 42 pmol of NO formed.min-1.mg of protein-1 and a Km for L-arginine of 8.4 microM. Three analogues of L-arginine, namely NG-monomethyl-L-arginine, NG-nitro-L-arginine and NG-iminoethyl-L-ornithine inhibited the brain NO synthase. All three compounds were competitive inhibitors of the enzyme with Ki values of 0.7, 0.4 and 1.2 microM respectively. PMID:1695842

  1. Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats

    PubMed Central

    Martín-Fernández, Beatriz; Rubio-Navarro, Alfonso; Cortegano, Isabel; Ballesteros, Sandra; Alía, Mario; Cannata-Ortiz, Pablo; Olivares-Álvaro, Elena; Egido, Jesús; de Andrés, Belén; Gaspar, María Luisa; de las Heras, Natalia; Lahera, Vicente; Moreno, Juan Antonio

    2016-01-01

    We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt–treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt–treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation. PMID:26730742

  2. Ménage à trois: aldosterone, sodium and nitric oxide in vascular endothelium.

    PubMed

    Fels, Johannes; Oberleithner, Hans; Kusche-Vihrog, Kristina

    2010-12-01

    Aldosterone, a mineralocorticoid hormone mainly synthesized in the adrenal cortex, has been recognized to be a regulator of cell mechanics. Recent data from a number of laboratories implicate that, besides kidney, the cardiovascular system is an important target for aldosterone. In the endothelium, it promotes the expression of epithelial sodium channels (ENaC) and modifies the morphology of cells in terms of mechanical stiffness, surface area and volume. Additionally, it renders the cells highly sensitive to small changes in extracellular sodium and potassium. In this context, the time course of aldosterone action is pivotal. In the fast (seconds to minutes), non-genomic signalling pathway vascular endothelial cells respond to aldosterone with transient swelling, softening and insertion of ENaC in the apical plasma membrane. In parallel, nitric oxide (NO) is released from the cells. In the long-term (hours), aldosterone has opposite effects: The mechanical stiffness increases, the cells shrink and NO production decreases. This leads to the conclusion that both the physiology and pathophysiology of aldosterone action in the vascular endothelium are closely related. Aldosterone, at concentrations in the physiological range and over limited time periods can stabilize blood pressure and regulate tissue perfusion while chronically high concentrations of this hormone over extended time periods impair sodium homeostasis promoting endothelial dysfunction and the development of tissue fibrosis.

  3. Interleukin-33/ST2 system attenuates aldosterone-induced adipogenesis and inflammation.

    PubMed

    Martínez-Martínez, Ernesto; Cachofeiro, Victoria; Rousseau, Elodie; Álvarez, Virginia; Calvier, Laurent; Fernández-Celis, Amaya; Leroy, Céline; Miana, María; Jurado-López, Raquel; Briones, Ana M; Jaisser, Frederic; Zannad, Faiez; Rossignol, Patrick; López-Andrés, Natalia

    2015-08-15

    Interleukin-33 (IL-33) but not soluble ST2 (sST2) exerts anti-inflammatory and protective effects in several tissues. Aldosterone, a proinflammatory mediator which promotes adipogenesis, is elevated in obese patients. The aim of this study was to investigate the interactions between IL-33/ST2 system and Aldosterone in adipose tissue. Rats fed a high fat diet presented increased sST2 expression, diminished IL-33/sST2 ratio and enhanced levels of differentiation and inflammation in adipose tissue as compared to controls. A similar pattern was observed in adipose tissue from C57BL/6 Aldosterone-treated mice. In both animal models, Aldosterone was correlated with sST2. Treatment of 3T3-L1 adipocytes with IL-33 delayed adipocyte differentiation diminished lipid accumulation and decreased inflammation. Aldosterone decreased IL-33 and increased sST2 expressions in differentiated adipocytes. Aldosterone-induced adipocyte differentiation and inflammation were blocked by IL-33 treatment, but sST2 did not exert any effects. The crosstalk between IL-33/ST2 and Aldosterone could be relevant in the metabolic consequences of obesity.

  4. Angiotensin II-Activated Protein Kinase D Mediates Acute Aldosterone Secretion

    PubMed Central

    Shapiro, Brian A.; Olala, Lawrence; Arun, Senthil Nathan; Parker, Peter M.; George, Mariya V.; Bollag, Wendy B.

    2009-01-01

    Summary Dysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24 hours) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time- and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutively-active PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion. PMID:19961896

  5. Interleukin-18 deficiency protects against renal interstitial fibrosis in aldosterone/salt-treated mice.

    PubMed

    Tanino, Akiko; Okura, Takafumi; Nagao, Tomoaki; Kukida, Masayoshi; Pei, Zuowei; Enomoto, Daijiro; Miyoshi, Ken-Ichi; Okamura, Haruki; Higaki, Jitsuo

    2016-10-01

    Interleukin (IL)-18 is a member of the IL-1 family of cytokines and was described originally as an interferon γ-inducing factor. Aldosterone plays a central role in the regulation of sodium and potassium homoeostasis by binding to the mineralocorticoid receptor and contributes to kidney and cardiovascular damage. Aldosterone has been reported to induce IL-18, resulting in cardiac fibrosis with induced IL-18-mediated osteopontin (OPN). We therefore hypothesized that aldosterone-induced renal fibrosis via OPN may be mediated by IL-18. To verify this hypothesis, we compared mice deficient in IL-18 and wild-type (WT) mice in a model of aldosterone/salt-induced hypertension. IL-18(-/-) and C57BL/6 WT mice were used for the uninephrectomized aldosterone/salt hypertensive model, whereas NRK-52E cells (rat kidney epithelial cells) were used in an in vitro model. In the present in vivo study, IL-18 protein expression was localized in medullary tubules in the WT mice, whereas in aldosterone-infused WT mice this expression was up-regulated markedly in the proximal tubules, especially in injured and dilated tubules. This renal damage caused by aldosterone was attenuated significantly by IL-18 knockout with down-regulation of OPN expression. In the present in vitro study, aldosterone directly induced IL-18 gene expression in renal tubular epithelial cells in a concentration- and time-dependent manner. These effects were inhibited completely by spironolactone. IL-18 may be a key mediator of aldosterone-induced renal fibrosis by inducing OPN, thereby exacerbating renal interstitial fibrosis. Inhibition of IL-18 may therefore provide a potential target for therapeutic intervention aimed at preventing the progression of renal injury.

  6. Aldosterone Activates Transcription Factor Nrf2 in Kidney Cells Both In Vitro and In Vivo

    PubMed Central

    Oteiza, Patricia I.; Link, Samuel; Hey, Valentin; Stopper, Helga; Schupp, Nicole

    2014-01-01

    Abstract Aims: An increased kidney cancer risk was found in hypertensive patients, who frequently exhibit hyperaldosteronism, known to contribute to kidney injury, with oxidative stress playing an important role. The capacity of kidney cells to up-regulate transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2), a key regulator of the cellular antioxidative defense, as a prevention of aldosterone-induced oxidative damage was investigated both in vitro and in vivo. Results: Aldosterone activated Nrf2 and increased the expression of enzymes involved in glutathione (GSH) synthesis and detoxification. This activation depended on the mineralocorticoid receptor (MR) and oxidative stress. In vitro, Nrf2 activation, GSH amounts, and target gene levels decreased after 24 h, while oxidant levels remained high. Nrf2 activation could not protect cells against oxidative DNA damage, as aldosterone-induced double-strand breaks and 7,8-dihydro-8-oxo-guanine (8-oxodG) lesions steadily rose. The Nrf2 activator sulforaphane enhanced the Nrf2 response both in vitro and in vivo, thereby preventing aldosterone-induced DNA damage. In vivo, Nrf2 activation further had beneficial effects on the aldosterone-caused blood pressure increase and loss of kidney function. Innovation: This is the first study showing the activation of Nrf2 by aldosterone. Moreover, the results identify sulforaphane as a substance that is capable of preventing aldosterone-induced damage both in vivo and in vitro. Conclusion: Aldosterone-induced Nrf2 adaptive response cannot neutralize oxidative actions of chronically increased aldosterone, which, therefore could be causally involved in the increased cancer incidence of hypertensive individuals. Enhancing the cellular antioxidative defense with sulforaphane might exhibit beneficial effects. Antioxid. Redox Signal. 21, 2126–2142. PMID:24512358

  7. Investigation of potential inhibitors of chorismate-utilizing enzymes.

    PubMed

    Švarcová, Markéta; Krátký, Martin; Vinšova, Jarmila

    2015-01-01

    Chorismate-utilizing enzymes (CUE) such as chorismate mutase, anthranilate synthase, chorismate pyruvate-lyase, 4-amino-4-deoxychorismate synthase, isochorismate synthase and salicylate synthase are responsible for converting chorismate into various products necessary for the survival of bacteria. The absence of these enzymes in humans and their importance in the virulence and survival of bacteria make them suitable targets for potential antimicrobial compounds. Furthermore, the CUE have significant structural homology and similar catalytic mechanisms, enabling the strategy of affecting multiple enzymes with one single inhibitor. This review follows up the investigation of mechanisms of CUE-catalysed reactions and the concurrent development of CUE inhibitors. Many active compounds were found amongst the structures mimicking the transition state of chorismate during the reaction. Most recently, high nanomolar and low micromolar inhibitors against isochorismate-pyruvate lyase were identified, which were also effective against chorismate mutase and salicylate synthase and belong to the most active inhibitors reported up to date.

  8. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  9. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  10. Evaluation of insulin sensitivity and secretion in primary aldosteronism.

    PubMed

    Watanabe, Daisuke; Yatabe, Midori; Ichihara, Atsuhiro

    In primary aldosteronism (PA), insulin response to glucose is not fully understood. Insulin action was elucidated using indices in 32 PA and 21 essential hypertension (EH) patients. These patients were evaluated using homeostasis model assessment (HOMA) indices, quantitative insulin sensitivity check index (QUICKI), and insulinogenic index (IGI), which were expressed for insulin sensitivity/secretion and the early phase of insulin secretion. Insulin sensitivity and early phase of insulin secretion were decreased in PA, and there was a negative correlation between serum potassium concentration and insulin sensitivity indices. These findings suggest that glucose intolerance in PA may be caused by hypokalemia-induced insulin resistance and hypokalemia-independent impairment of early-phase insulin secretion.

  11. Hypokalemic myopathy in primary aldosteronism: A case report

    PubMed Central

    Wu, Chuifen; Xin, Jun; Xin, Minghua; Zou, Hai; Jing, Lie; Zhu, Caoyong; Lei, Wenhui

    2016-01-01

    Primary aldosteronism (PA) is a rare disorder. The majority of patients with PA present with typical features and are easily diagnosed. This disorder is usually diagnosed with hypokalemia, hypertension or an adrenal mass. However, patients with atypical symptoms may present a challenge for diagnosis and treatment. In the present study, a case of PA is described that presented with hypokalemic myopathy simulating polymyositis. The patient was a 44-year-old woman who presented with weakness and difficulty walking. The patient was initially suspected to have PM and was treated with methylprednisolone. The patient was found to have hypokalemia which persisted despite high-dose supplementation of potassium. Adrenal computed tomography revealed a right adrenal mass. Surgical adrenalectomy was conducted. The final pathological diagnosis was benign adrenocortical adenoma. The serum tests remained normal and the patient's symptoms were resolved during the 8-month follow-up. PMID:28101185

  12. SP 01-3 ALDOSTERONE ANTAGONISTS IN HEART FAILURE.

    PubMed

    Johnston, Colin

    2016-09-01

    Aldosterone's deleterious pathophysiological effects on the cardiovascular system if blocked by mineralcorticord antagonists (MRAs) logically should lead to improvement in heart function and outcomes in heart failure (HF). The first trial to test this hypothesis was tthe RALES trial in 1999 which treated patients with class III-IV HF with spironolactone. It showed significant reduction in mortality and cardiovascular hospitalzation rates. This was confirmed & extended in EMHASIS-HF RCT with classs II-III being treated with ACEIs & BB who received placebo or elperinone (a MRA) with again a statistically significant fall in mortality & hospitalization.The possible cardioprotective effects of MRA post acute myocardial infarct (MI) is less clear. The EPHESUS RCT in 2003 demostrated that elperinone given 3-14 days AMI in patients with early signs of HF reduced mortality & morbidity. However in the ALBTROSS trial using spironolactone 2 days after AMI showed no benfit in patients without HF but in a subgroup with ST elevation there was a 80% reduction in mortality after 6 months. However a recent meta-analysis from 25 RCT with data invovling 19,333 patients with either HF or post MI assigned aldosterone antagonists (AA)or placebo showed a 18% reduction in mortality including a 20% fall in CV mortality and a 19% reduction in SCD.The role of AA in HFPEF is even even more contraversial. The TOPCAT RCT of 3445 patients with symptomatc HFPEF randomised to spironolactone failed to meet the primary composite end point of death, aborted cardiac arrest or hospitalization although there was a reduction in hospitalization for HF (HR 0.83 P = 0.04).The differences between selective or non-selective MRAs, their ADRs & off target effects will also be discussed.

  13. A case of primary aldosteronism combined with acquired nephrogenic diabetes insipidus.

    PubMed

    Kim, Kitae; Lee, Jae Hyoung; Kim, Sun Chul; Cha, Dae Ryong; Kang, Young Sun

    2014-12-01

    Aldosterone-producing adrenal adenoma can induce various clinical manifestations as a result of chronic exposure to aldosterone. We report a rare case of a 37-year-old man who complained of general weakness and polyuria. He was diagnosed with aldosterone-producing adrenal adenoma and nephrogenic diabetes insipidus. Aldosterone enhances the secretion of potassium in the collecting duct, which can lead to hypokalemia. By contrast, nephrogenic diabetes insipidus, which manifests as polyuria and polydipsia, can occur in several clinical conditions such as acquired tubular disease and those attributed to toxins and congenital causes. Among them, hypokalemia can also damage tubular structures in response to vasopressin. The patient's urine output was >3 L/d and was diluted. Owing to the ineffectiveness of vasopressin, we eventually made a diagnosis of nephrogenic diabetes insipidus. Laparoscopic adrenalectomy and intraoperative kidney biopsy were subsequently performed. The pathologic finding of kidney biopsy revealed a decrease in aquaporin-2 on immunohistochemical stain.

  14. Is the vascular endothelium under the control of aldosterone? Facts and hypothesis.

    PubMed

    Oberleithner, Hans

    2007-05-01

    Fluid and electrolyte balance in the human organism is controlled by aldosterone, a mineralocorticoid hormone of the suprarenal glands. The major target cells are localized in the kidney where the hormone controls transepithelial salt transport. Over the past few years, evidence has been accumulated that cells of the cardiovascular system are also targeted by the hormone. As an example, endothelial cells resemble similar mechanisms triggered by aldosterone as shown for the kidney. Although the pathological alterations induced by aldosterone excess are obvious, the physiological changes are largely unknown. On the basis of recent experiments, using atomic force microscopy as an imaging tool and a mechanical sensor, I present a hypothesis on the physiological role of aldosterone in endothelial function and its potential implications in the control of blood pressure.

  15. Effects of aldosterone and potassium-sparing diuretics on electrical potential differences across the distal nephron.

    PubMed Central

    Gross, J B; Kokko, J P

    1977-01-01

    We have previously shown that the transtubular potential of the rabbit cortical collecting tubule varies in concert with changes in plasma mineralocorticoid levels, while the potential of the distal convoluted tubule is invariant with such changes. In the present studies we have examined the effects of in vitro addition of d-aldosterone to isolated tubules, as well as the effects of triamterene and spirolactone. d-Aldosterone (0.2 mum added to the perfusate or 1 muM added to the bathing medium) resulted in a marked stimulation of the transtubular potential difference (lumen-negative) after a short latent period. d-Aldosterone had no effect on the potential difference of distal convoluted tubules of intact or adrenalectomized rabbits. Both the magnitude of the response and the length of the latent period in the cortical collecting tubule after aldosterone were markedly temperature-dependent. Triamterene caused a gradual but reversible inhibition of the potential difference in the cortical collecting tubule but had no effect in the distal tubule. Spirolactone, when added before aldosterone, blocked the electrical response to the hormone in the cortical collecting tubule, and produced a gradual inhibition of the potential difference in mineralocorticoid-stimulated tubules. Spirolactone had no effect on the potential difference of the distal tubule. We conclude that (a) the influence of aldosterone on the potential across the distal nephron is restricted to the distal convoluted tubule, (b) the electrical response to aldosterone and the latent period are temperature-dependent, (c) the response to aldosterone is blocked by spirolactone, and (d) triamterene inhibits the potential difference only in the cortical collecting tubule. PMID:830667

  16. An inhibitor of the δPKC interaction with the d subunit of F1Fo ATP synthase reduces cardiac troponin I release from ischemic rat hearts: utility of a novel ammonium sulfate precipitation technique.

    PubMed

    Ogbi, Mourad; Obi, Ijeoma; Johnson, John A

    2013-01-01

    We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F1Fo ATPase (dF1Fo) interaction inhibitor [NH2-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-COOH] in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH4)2SO4) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH4)2SO4 saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n=5) percentage of maximal cTnI release was 30 ± 7 and 60 ± 17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF1Fo interaction inhibitor, prior to 20 min of ischemia and 60-150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH4)2SO4 and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF1Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts.

  17. An Inhibitor of the δPKC Interaction with the d Subunit of F1Fo ATP Synthase Reduces Cardiac Troponin I Release from Ischemic Rat Hearts: Utility of a Novel Ammonium Sulfate Precipitation Technique

    PubMed Central

    Ogbi, Mourad; Obi, Ijeoma; Johnson, John A.

    2013-01-01

    We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F1Fo ATPase (dF1Fo) interaction inhibitor [NH2-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-COOH] in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH4)2SO4) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH4)2SO4 saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n = 5) percentage of maximal cTnI release was 30±7 and 60±17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF1Fo interaction inhibitor, prior to 20 min of ischemia and 60–150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH4)2SO4 and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF1Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts. PMID:23936451

  18. Aldosterone induces rapid sodium intake by a nongenomic mechanism in the nucleus tractus solitarius

    PubMed Central

    Qiao, Hu; Hu, Bo; Zhou, Hong; Yan, Jianqun; Jia, Ru; Lu, Bo; Sun, Bo; Luo, Xiao; Fan, Yuanyuan; Wang, Nan

    2016-01-01

    The purpose of this study was to determine whether aldosterone has a rapid action in the nucleus tractus solitarius (NTS) that increases sodium intake, and to examine whether this effect of aldosterone, if present, is mediated by G protein-coupled estrogen receptor (GPER). Adult male Sprague-Dawley rats with a stainless-steel cannula in the NTS were used. Aldosterone was injected into the NTS at the doses of 1, 5, 10 and 20 ng 0.1 μl−1. A rapid dose-related increase of 0.3 M NaCl intake was induced within 30 min and this increase was not suppressed by the mineralocorticoid receptor (MR) antagonist spironolactone (10 ng 0.1 μl−1). Water intake was not affected by aldosterone. The GPER agonist G-1 produced a parallel and significant increase in sodium intake, while pre-treatment with GPER antagonist G15 (10 ng 0.1 μl−1) blocked the G-1 or aldosterone-induced rapid sodium intake. In addition, sodium intake induced by sodium depletion or low-sodium diet fell within 30 min after injection into the NTS of the MR antagonist spironolactone, while G15 had no effect. Our results confirm previous reports, and support the hypothesis that aldosterone evokes rapid sodium intake through a non-genomic mechanism involving GPER in NTS. PMID:27934887

  19. Plasma aldosterone and glomerular filtration in hypertensive patients with preserved renal function.

    PubMed

    Roldán, Julián; Morillas, Pedro; Castillo, Jesús; Andrade, Helder; Guillén, Silvia; Núñez, Daniel; Quiles, Juan; Bertomeu, Vicente

    2010-01-01

    There is increasing interest in the role of aldosterone in the pathophysiology of hypertension, cardiovascular disease and deteriorating renal function. The aim of this study was to investigate the relationship between aldosterone and the glomerular filtration rate (GFR) in hypertensive patients with preserved renal function. The study involved 186 consecutive hypertensive patients with a GFR >60 mL/min. The GFR was determined using the Modification of Diet in Renal Disease (MDRD) equation and the patients' plasma aldosterone levels were measured. Patients with a GFR between 60-89 mL/min had a significantly higher plasma aldosterone level than those with a GFR >90 mL/min (20.02 ng/dL vs. 15.3 ng/dL; P< .05). Multivariate analysis showed that the plasma aldosterone level was independently associated with the GFR (B=-7.36; P< .001). In hypertensive patients with preserved kidney function, the plasma aldosterone level was observed to increase as the GFR decreased.

  20. Effect of a multistage ultraendurance triathlon on aldosterone, vasopressin, extracellular water and urine electrolytes.

    PubMed

    Knechtle, B; Morales, N P Hernández; González, E Ruvalcaba; Gutierrez, A A Aguirre; Sevilla, J Noriega; Gómez, R Amézquita; Robledo, A R Estrada; Rodríguez, A L Marroquín; Fraire, O Salas; Andonie, J L; Lopez, L C; Kohler, G; Rosemann, T

    2012-02-01

    Prolonged endurance exercise over several days induces increase in extracellular water (ECW). We aimed to investigate an association between the increase in ECW and the change in aldosterone and vasopressin in a multistage ultraendurance triathlon, the 'World Challenge Deca Iron Triathlon' with 10 Ironman triathlons within 10 days. Before and after each Ironman, body mass, ECW, urinary [Na(+)], urinary [K(+)], urinary specific gravity, urinary osmolality and aldosterone and vasopressin in plasma were measured. The 11 finishers completed the total distance of 38 km swimming, 1800 km cycling and 422 km running within 145.5 (18.8) hours and 25 (22) minutes. ECW increased by 0.9 (1.1) L from 14.6 (1.5) L prerace to 15.5 (1.9) L postrace (P < 0.0001). Aldosterone increased from 70.8 (104.5) pg/mL to 102.6 (104.6) pg/mL (P = 0.033); vasopressin remained unchanged. The increase in ECW was related neither to postrace aldosterone nor to postrace vasopressin. In conclusion, ECW and aldosterone increased after this multistage ultraendurance triathlon, but vasopressin did not. The increase in ECW and the increase in aldosterone were not associated.

  1. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

    PubMed Central

    Leopold, Jane A.; Dam, Aamir; Maron, Bradley A.; Scribner, Anne W.; Liao, Ronglih; Handy, Diane E.; Stanton, Robert C.; Pitt, Bertram; Loscalzo, Joseph

    2013-01-01

    Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanism by which aldosterone promotes endothelial dysfunction remains unknown. Glucose-6-phosphate dehydrogenase (G6pd), the principal source of Nadph, modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). In these studies, we show that aldosterone (10−9-10−7 mol/l) decreases endothelial G6pd expression and activity in vitro resulting in increased oxidant stress and decreased cGMP levels similar to what is observed in G6pd-deficient cells. Aldosterone decreases G6pd expression by protein kinase A activation to increase expression of Crem, which interferes with Creb binding to the G6pd promoter. In vivo, infusion of aldosterone decreases vascular G6pd expression and impairs vascular reactivity. These effects are abrogated by spironolactone or vascular gene transfer of G6pd. These studies demonstrate that aldosterone induces a G6pd-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6pd activity. PMID:17273168

  2. Hybrid polyketide synthases

    SciTech Connect

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  3. Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A waterhemp population (MCR) previously characterized as resistant to 4-hyroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors was found to have two different resistance responses to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistan...

  4. Renin angiotensin aldosterone system altered in resistant hypertension in Sub-Saharan African diabetes patients without evidence of primary hyperaldosteronism

    PubMed Central

    Edinga-Melenge, Bertille Elodie; Ama Moor, Vicky J; Nansseu, Jobert Richie N; Nguetse Djoumessi, Romance; Mengnjo, Michel K; Katte, Jean-Claude; Noubiap, Jean Jacques N

    2017-01-01

    Background The renin-angiotensin-aldosterone system may be altered in patients with resistant hypertension. This study aimed to evaluate the relation between renin-angiotensin-aldosterone system activity and resistant hypertension in Cameroonian diabetes patients with resistant hypertension. Methods We carried out a case-control study including 19 diabetes patients with resistant hypertension and 19 diabetes patients with controlled hypertension matched to cases according to age, sex and duration of hypertension since diagnosis. After collection of data, fasting blood was collected for measurement of sodium, potassium, chloride, active renin and plasma aldosterone of which the aldosterone-renin ratio was derived to assess the activity of renin-angiotensin-aldosterone system. Then, each participant received 2000 ml infusion of saline solution after which plasma aldosterone was re-assayed. Results Potassium levels were lower among cases compared to controls (mean: (4.10 ± 0.63 mmol/l vs. 4.47 ± 0.58 mmol/l), though nonsignificant (p = 0.065). Active renin, plasma aldosterone both before and after the dynamic test and aldosterone-renin ratio were comparable between cases and controls (all p values > 0.05). Plasma aldosterone significantly decreased after the dynamic test in both groups (p < 0.001), but no participant exhibited a post-test value>280 pmol/l. We found a significant negative correlation between potassium ion and plasma aldosterone (ρ = −0.324; p = 0.047), the other correlations being weak and unsignificant. Conclusion Although this study failed to show an association between RH and primary hyperaldosteronism in our context, there was a hyperactivity of renin-angiotensin-aldosterone system. Moreover, this study confirms the importance of potassium dosage when screening the renin-angiotensin-aldosterone system. PMID:28321294

  5. Single Agents with Designed Combination Chemotherapy Potential: Synthesis and Evaluation of Substituted Pyrimido[4,5-b]indoles as Receptor Tyrosine Kinase and Thymidylate Synthase Inhibitors and as Antitumor Agents

    PubMed Central

    Gangjee, Aleem; Zaware, Nilesh; Raghavan, Sudhir; Ihnat, Michael; Shenoy, Satyendra; Kisliuk, Roy L.

    2010-01-01

    Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise and several such clinical trials are currently underway. We have designed, synthesized and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet derived growth factor receptor-beta (PDGFR-β) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-β and hTS is better than or close to standards. In a COLO-205 xenograft mouse model one of the analogs significantly decreased tumor growth (TGI = 76% at 35 mg/kg), liver metastases and tumor blood vessels compared to a standard drug and to control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents. PMID:20092323

  6. Renin–angiotensin–aldosterone system related gene polymorphisms and urinary total arsenic is related to chronic kidney disease

    SciTech Connect

    Chen, Wei-Jen; Huang, Ya-Li; Shiue, Horng-Sheng; Chen, Tzen-Wen; Lin, Yuh-Feng; Huang, Chao-Yuan; Lin, Ying-Chin; Han, Bor-Cheng; Hsueh, Yu-Mei

    2014-09-01

    A recent study demonstrated that an increased risk of chronic kidney disease (CKD) was associated with high urinary total arsenic levels. However, whether genomic instability is related to CKD remains unclear. An association between CKD and genetic polymorphisms of regulation enzymes of the renin–angiotensin–aldosterone system (RAAS), such as angiotensin-converting enzyme (ACE), angiotensinogen (AGT), angiotensin II type I receptor (AT1R), and aldosterone synthase (CYP11B2) has not been shown. The aim of the present study was to investigate the relationship between arsenic, genetic polymorphisms of RAAS enzymes and CKD. A total of 233 patients and 449 age- and gender-matched controls were recruited from the Taipei Medical University Hospital, Taipei Municipal Wan Fang Hospital and the Shin Kong Wu Ho-Su Memorial Hospital. Concentrations of urinary arsenic were determined by a high-performance liquid chromatography-linked hydride generator, and atomic absorption spectrometry. Polymorphisms of ACE(I/D), AGT(A[− 20]C), (T174M), (M235T), AT1R(A1166C) and CYP11B2(C[− 344]T) were examined by polymerase chain reaction and restriction fragment length polymorphism. Subjects carrying the CYP11B2 TT genotype had a higher odds ratio (OR), 1.39 (0.96–2.01), of CKD; while those with the AGT(A[− 20]C) CC genotype had an inverse OR of CKD (0.20 (0.05–0.81)), and a high-risk genotype was defined as A/A + A/C for AGT(A[− 20C]) and T/T for CYP11B2(C[− 344]T). The trend test showed a higher OR for CKD in patients who had either high urinary total arsenic levels or carried the high-risk genotype, or both, compared to patients with low urinary total arsenic levels, who carried the low-risk genotype, and could also be affected by the hypertension or diabetes status. - Highlights: • AGT(− 20 C) and CYP11B2(− 344 T) genotypes were significantly associated with CKD. • Combined effect of high-risk genotypes and high urinary total arsenic on OR of CKD. • Combined

  7. Association of aldosterone excess and apnea-hypopnea index in patients with resistant hypertension

    PubMed Central

    Ke, Xiao; Guo, Wenyu; Peng, Hu; Hu, Chengheng; Zhang, Henghong; Peng, Changnong; Wang, Xiaoqing

    2017-01-01

    The present study was to investigate the association of aldosterone excess and apnea-hypopnea index (AHI) in patients with resistant hypertension. Patients with resistant hypertension were enrolled and baseline characteristics including plasma aldosterone concentration (PAC) and 24 h-urine aldosterone levels were collected and compared between groups with different degrees of AHI as assessed by polysomnography. Association of key variables and AHI was then evaluated by univariate and multiple linear regression analysis. A total of 534 patients with resistant hypertension were enrolled and mean age was 57 ± 11 years. Overall, mean number of AHI was 21.7 ± 9.6 and nearly 92.3% of resistant hypertensive patients had obstructive sleep apnea (OSA). Mean PAC and 24 h-urine aldosterone level was 12.4 ± 6.3 ng/dL and 13.1 ± 6.8 ug, respectively. Compared with other groups, participants in the severe OSA group (AHI ≥ 30) had significantly higher PAC and 24 h-urine aldosterone level. Multiple linear regression analysis showed that PAC and 24 h-urine aldosterone levels were positively associated with AHI, while spironolactone was negatively associated with AHI, independent of age, gender, body mass index, smoking, plasma renin activity and diuretics. OSA is highly prevalent in patients with resistant hypertension and both PAC and 24 h-urine aldosterone level are significantly associated with AHI. PMID:28327653

  8. Aldosterone-cortisol imbalance immediately after fontan operation with implications for abnormal fluid homeostasis.

    PubMed

    Saiki, Hirofumi; Kuwata, Seiko; Kurishima, Clara; Iwamoto, Yoichi; Ishido, Hirotaka; Masutani, Satoshi; Senzaki, Hideaki

    2014-11-15

    Abnormal water metabolism is frequently observed after Fontan surgery. We hypothesized that patients' adrenal hormones show unique responses immediately after Fontan operation and that such a hormonal profile is related to postoperative hemodynamics and water imbalance. Twenty-eight patients who underwent a Fontan operation (n = 16) or a non-Fontan type operation (n = 12; controls) under cardiopulmonary bypass were studied. Postoperative urine cortisol and aldosterone levels were measured daily to minimize the influence of circadian rhythms and temporal hemodynamic variations. Cortisol excretion was markedly elevated on postoperative day (POD) 0 in controls, consistent with a stress-induced cortisol response. Cortisol excretion was not high on POD 0 in Fontan patients and was markedly lower than that in the controls (444 ± 150 vs 34 ± 6 μg/m(2)/day, p <0.05), indicating an adrenal insufficiency status. Conversely, aldosterone levels were significantly higher in Fontan patients than in controls immediately after surgery and remained so thereafter. The cortisol-to-aldosterone ratio was significantly lower in Fontan patients on POD 0 (p <0.05 vs controls); low cortisol-to-aldosterone ratios were associated with a longer pleural drainage duration and intensive care unit stay. Daily cortisol and aldosterone levels were significantly associated with postoperative hemodynamics; low cortisol levels correlated with low cardiac and urine outputs, whereas high aldosterone levels correlated with low cardiac output and increased blood pressure and central venous pressure. Thus, aldosterone-to-cortisol imbalance occurred specifically after the Fontan operation. This unique hormonal profile significantly affected patients' postoperative water balance and hemodynamics. Modulation of the adrenal hormone could be useful for reducing postoperative complications after the Fontan operation.

  9. Evaluation of the effects of occupational noise exposure on serum aldosterone and potassium among industrial workers

    PubMed Central

    Zare, Sajad; Nassiri, Parvin; Monazzam, Mohammad Reza; Pourbakht, Akram; Azam, Kamal; Golmohammadi, Taghi

    2016-01-01

    The existing literature indicates that occupational exposure to noise may have adverse effects on workers’ health. The aim of this study was to evaluate the possible effects of exposure to different sound pressure levels (SPLs) on serum aldosterone and potassium concentration among Iranian blue collar workers in Golgohar Mining and Industrial Company in Sirjan, Kerman Province, Iran. This case-control study was performed on 45 workers of Golgohar Mining and Industrial Company. The subjects consisted of 30 workers from manufacturing departments and 15 office employees of the mining company. The controls, mainly with administrative jobs were exposed to 72 dBA SPL. Cases, in two separate groups, were exposed to noise levels of 88 dBA and 103 dBA, respectively. Noise intensity was measured at the desired locations. Noise measurements were performed according to the International Organization for Standardization (ISO) 9612. To measure the serum aldosterone and potassium concentrations, a 5 mL blood sample was taken from each worker at the specified time intervals and aldosterone concentration was determined using enzyme-linked immunosorbent assay (ELISA) test in the laboratory. Repeated measurement and Spearman's correlation coefficient analysis were used with α = 0.05. Exposure to the different levels of sound pressure resulted in different aldosterone concentrations and meanwhile an increase in the SPL did not affect the concentration of potassium. From 10:00 AM to 10:30 AM, as SPL increased, aldosterone concentrations did not increase significantly but from 13:30 PM to 14:00 PM, raised SPL led to a significant increase in aldosterone concentration. However, there was no correlation between the concentration of potassium and different factors. This study indicated that increases in SPLs affect aldosterone concentration but at the same time do not have significant effects on serum potassium level. PMID:26780955

  10. Prostaglandins, catecholamines, renin and aldosterone during hypertensive and normotensive pregnancy.

    PubMed

    Pedersen, E B; Christensen, N J; Christensen, P; Johannesen, P; Kornerup, H J; Kristensen, S; Lauritsen, J G; Leyssac, P P; Rasmussen, A B; Wohlert, M

    1982-01-01

    Urinary excretion of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha), plasma concentrations of renin (PRC), aldosterone (PAC), noradrenaline (PNA) and adrenaline (PA) were determined in the third trimester of pregnancy, 5 days and 3 months after delivery in preeclampsia and normotensive pregnant and non-pregnant control subjects. PGE2 was higher in pregnant control subjects than in non-pregnant subjects, but reduced to non-pregnant level in preeclampsia. PGF2 alpha was the same in preeclampsia and normotensive pregnancy but higher than in the non-pregnant group. PRC and PAC were increased during pregnancy, but considerably lesser in preeclampsia than during normotensive pregnancy. PNA and PA were the same in all three groups. All parameters were normal 3 months after delivery. There were no correlations between any of the hormones and blood pressure in any of the groups. PGE2 was positively correlated to PRC. The lack of renal PGE2 in preeclampsia might be responsible for the decrease in renal blood flow and sodium excretion, and the changes in PRC and PAC are supposed to be secondary to changes in PGE2. It is hypothesised that preeclampsia is a state of prostaglandin deficiency.

  11. Salt, aldosterone, and insulin resistance: impact on the cardiovascular system.

    PubMed

    Lastra, Guido; Dhuper, Sonal; Johnson, Megan S; Sowers, James R

    2010-10-01

    Hypertension and type 2 diabetes mellitus (T2DM) are powerful risk factors for cardiovascular disease (CVD) and chronic kidney disease (CKD), both of which are leading causes of morbidity and mortality worldwide. Research into the pathophysiology of CVD and CKD risk factors has identified salt sensitivity and insulin resistance as key elements underlying the relationship between hypertension and T2DM. Excess dietary salt and caloric intake, as commonly found in westernized diets, is linked not only to increased blood pressure, but also to defective insulin sensitivity and impaired glucose homeostasis. In this setting, activation of the sympathetic nervous system and the renin-angiotensin-aldosterone system (RAAS), as well as increased signaling through the mineralocorticoid receptor (MR), result in increased production of reactive oxygen species and oxidative stress, which in turn contribute to insulin resistance and impaired vascular function. In addition, insulin resistance is not limited to classic insulin-sensitive tissues such as skeletal muscle, but it also affects the cardiovascular system, where it participates in the development of CVD and CKD. Current clinical knowledge points towards an impact of salt restriction, RAAS blockade, and MR antagonism on cardiovascular and renal protection, but also on improved insulin sensitivity and glucose homeostasis.

  12. Emergence and evolution of the renin-angiotensin-aldosterone system.

    PubMed

    Fournier, David; Luft, Friedrich C; Bader, Michael; Ganten, Detlev; Andrade-Navarro, Miguel A

    2012-05-01

    The renin-angiotensin-aldosterone system (RAAS) is not the sole, but perhaps the most important volume regulator in vertebrates. To gain insights into the function and evolution of its components, we conducted a phylogenetic analysis of its main related genes. We found that important parts of the system began to appear with primitive chordates and tunicates and that all major components were present at the divergence of bony fish, with the exception of the Mas receptor. The Mas receptor first appears after the bony-fish/tetrapod divergence. This phase of evolutionary innovation happened about 400 million years ago. We found solid evidence that angiotensinogen made its appearance in cartilage fish. The presence of several RAAS genes in organisms that lack all the components shows that these genes have had other ancestral functions outside of their current role. Our analysis underscores the utility of sequence comparisons in the study of evolution. Such analyses may provide new hypotheses as to how and why in today's population an increased activity of the RAAS frequently leads to faulty salt and volume regulation, hypertension, and cardiovascular diseases, opening up new and clinically important research areas for evolutionary medicine.

  13. Autophagy is involved in aldosterone-induced mesangial cell proliferation

    PubMed Central

    Yang, Min; Wang, Bin; Miao, Liying; Xu, Xianlin; He, Xiaozhou

    2016-01-01

    The aim of the present study was to investigate whether autophagy is involved in aldosterone (Aldo)-induced mesangial cell (MC) proliferation. MCs were incubated with 10−7 M Aldo for 24 h. Proliferation of MCs, and the underlying mechanisms, were subsequently analyzed using [3H]thymidine assay, cell counting assay, western blotting and RNA interference (RNAi). Aldo was revealed to induce autophagy, as indicated by the increased conversion from microtubule-associated protein 1A/1B-light chain 3 (LC3)-I to LC3-II, the increased expression levels of autophagy-related gene 7 (Atg7) and the increased degradation of p62, which was accompanied by MC proliferation. Notably, pharmacological inhibition of autophagy or RNAi-mediated knockdown of Atg7 attenuated Aldo-induced MC proliferation, suggesting that autophagy was at least partially responsible for this effect. The results of the present study provided evidence that autophagy is critical for regulating Aldo-induced MC proliferation. PMID:27748808

  14. SbnG, a Citrate Synthase in Staphylococcus aureus

    PubMed Central

    Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.; Heinrichs, David E.; Murphy, Michael E. P.

    2014-01-01

    In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. PMID:25336653

  15. Mammalian ceramide synthases.

    PubMed

    Levy, Michal; Futerman, Anthony H

    2010-05-01

    In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their structure and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases.

  16. Effects of a novel ACE inhibitor, 3-(3-thienyl)-l-alanyl-ornithyl-proline, on endothelial vasodilation and hepatotoxicity in l-NAME-induced hypertensive rats.

    PubMed

    Seth, Mahesh Kumar; Hussain, M Ejaz; Pasha, Santosh; Fahim, Mohammad

    2016-01-01

    Nitric oxide (NO) is a widespread biological mediator involved in many physiological and pathological processes, eg, in the regulation of vascular tone and hypertension. Chronic inhibition of NO synthase by N(G)-nitro-l-arginine methyl ester (l-NAME) hydrochloride results in the development of hypertension accompanied by an increase in vascular responsiveness to adrenergic stimuli. Recently, we developed a novel sulfur-containing angiotensin-converting enzyme inhibitor: 3-(3-thienyl)-l-alanyl-ornithyl-proline (TOP). Our previous studies indicated a superior nature of the molecule as an antihypertensive agent in spontaneously hypertensive rats (showing the involvement of renin-angiotensin-aldosterone system) in comparison to captopril. The aim of the present study was to investigate the effect of TOP on NO pathway in l-NAME-induced hypertensive rats, and captopril was included as the standard treatment group. Treatment with both TOP (20 mg/kg) and captopril (40 mg/kg) prevented the development of hypertension in l-NAME model, but TOP showed better restoration of NO and normal levels of angiotensin-converting enzyme. In addition, in vitro vasorelaxation assay showed an improvement in endothelium-dependent vasodilation in both the cases. Further, the biochemical (malondialdehyde, alanine aminotransferase, and aspartate aminotransferase) and the histopathological effects of TOP on rat liver tissues revealed a protective nature of TOP in comparison to captopril in the l-NAME model. In conclusion, TOP at 50% lesser dose than captopril was found to be better in the l-NAME model.

  17. Monoterpene synthases from common sage (Salvia officinalis)

    DOEpatents

    Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  18. Aldosterone and myocardial extracellular matrix expansion in type 2 diabetes mellitus.

    PubMed

    Rao, Ajay D; Shah, Ravi V; Garg, Rajesh; Abbasi, Siddique A; Neilan, Tomas G; Perlstein, Todd S; Di Carli, Marcelo F; Jerosch-Herold, Michael; Kwong, Raymond Y; Adler, Gail K

    2013-07-01

    Myocardial extracellular matrix expansion and reduced coronary flow reserve (CFR) occur in patients with type 2 diabetes mellitus without heart failure or coronary artery disease. Because aldosterone is implicated in the pathophysiology of cardiac fibrosis and vascular injury, the aim of this study was to test the hypothesis that aldosterone is associated with extracellular matrix expansion and reduced CFR in type 2 diabetes mellitus. Patients with type 2 diabetes mellitus without evidence of coronary artery disease were recruited. Blood pressure, lipid management, and glycemic control were optimized over 3 months. Cardiac magnetic resonance imaging with T1 mapping was used to measure myocardial extracellular volume (ECV). Cardiac positron emission tomography was used to assess CFR. On a liberal, 250 mEq/day sodium diet, 24-hour urinary aldosterone and change in serum aldosterone with angiotensin II stimulation were measured. Fifty-three participants with type 2 diabetes (68% men, mean age 53 ± 7 years, mean body mass index 32.2 ± 4.3 kg/m², mean glycosylated hemoglobin 6.8 ± 0.7%, mean systolic blood pressure 126 ± 14 mm Hg) without infarction or ischemia by cardiac magnetic resonance and positron emission tomography were studied. Subjects had impaired CFR (2.51 ± 0.83) and elevated ECV (0.36 ± 0.05), despite normal echocardiographic diastolic function and normal left ventricular function. Myocardial ECV, but not CFR, was positively associated with 24-hour urinary aldosterone excretion (r = 0.37, p = 0.01) and angiotensin II-stimulated aldosterone increase (r = 0.35, p = 0.02). In a best-overall multivariate model (including age, gender, body mass index, glycosylated hemoglobin, and blood pressure), 24-hour urinary aldosterone was the strongest predictor of myocardial ECV (p = 0.004). In conclusion, in patients with type 2 diabetes mellitus without coronary artery disease, aldosterone is associated with myocardial extracellular matrix expansion. These

  19. Aldosterone induces fibrosis, oxidative stress and DNA damage in livers of male rats independent of blood pressure changes

    SciTech Connect

    Queisser, Nina; Happ, Kathrin; Link, Samuel; Jahn, Daniel; Zimnol, Anna; Geier, Andreas; Schupp, Nicole

    2014-11-01

    Mineralocorticoid receptor blockers show antifibrotic potential in hepatic fibrosis. The mechanism of this protective effect is not known yet, although reactive oxygen species seem to play an important role. Here, we investigated the effects of elevated levels of aldosterone (Ald), the primary ligand of the mineralocorticoid receptor, on livers of rats in a hyperaldosteronism model: aldosterone-induced hypertension. Male Sprague–Dawley rats were treated for 4 weeks with aldosterone. To distinguish if damage caused in the liver depended on increased blood pressure or on increased Ald levels, the mineralocorticoid receptor antagonist spironolactone was given in a subtherapeutic dose, not normalizing blood pressure. To investigate the impact of oxidative stress, the antioxidant tempol was administered. Aldosterone induced fibrosis, detected histopathologically, and by expression analysis of the fibrosis marker, α-smooth muscle actin. Further, the mRNA amount of the profibrotic cytokine TGF-β was increased significantly. Fibrosis could be reduced by scavenging reactive oxygen species, and also by blocking the mineralocorticoid receptor. Furthermore, aldosterone treatment caused oxidative stress and DNA double strand breaks in livers, as well as the elevation of DNA repair activity. An increase of the transcription factor Nrf2, the main regulator of the antioxidative response could be observed, and of its target genes heme oxygenase-1 and γ-glutamylcysteine synthetase. All these effects of aldosterone were prevented by spironolactone and tempol. Already after 4 weeks of treatment, aldosteroneinfusion induced fibrosis in the liver. This effect was independent of elevated blood pressure. DNA damage caused by aldosterone might contribute to fibrosis progression when aldosterone is chronically increased. - Highlights: • Aldosterone has direct profibrotic effects on the liver independent of blood pressure. • Fibrosis is mediated by the mineralocorticoid receptor and

  20. A decreased metabolic clearance rate of aldosterone in benign essential hypertension

    PubMed Central

    Nowaczynski, W.; Kuchel, O.; Genest, J.

    1971-01-01

    Aldosterone secretion rate, metabolic clearance rate, and/or plasma concentration were determined in 16 patients with benign, uncomplicated essential hypertension and compared with those of control subjects. The mean metabolic clearance rate of aldosterone in 10 patients was significantly (P < 0.001) lower (mean 867 liters of plasma/day per m2 ±270 SD) than in a group of 7 healthy subjects (mean 1480 liters/day per m2 ±265 SD). Secretion rates in 13 patients (including the 10 already mentioned) tended to be low (83 ±43 vs. 109 ±54 μg/day) and plasma concentrations tended to be high (13.6 ±4.6 vs. 7.5 ±4.8 ng/100 ml), but neither of these differences was statistically significant. The lower metabolic clearance rate could account for elevated plasma concentrations of aldosterone even when the secretion rate is normal or low. Measurement of secretion rate or urinary excretion only is therefore insufficient to establish the presence and/or mode of evolution of hyperaldosteronism. Failure of the aldosterone secretion to adapt fully to a decreased aldosterone metabolic clearance rate (MCR) could explain the state of relative hyperaldosteronism in patients with benign essential hypertension, even when the secretion rate and the urinary excretion rate are in the normal range. PMID:5116208

  1. Renal denervation attenuates aldosterone expression and associated cardiovascular pathophysiology in angiotensin II-induced hypertension

    PubMed Central

    Chen, Dong-Rui; Ruan, Cheng-Chao; Xu, Jian-Zhong; Chen, Jing; Wu, Yong-Jie; Ma, Yu; Zhu, Ding-Liang; Gao, Ping-Jin

    2016-01-01

    The sympathetic nervous system interacts with the renin-angiotensin-aldosterone system (RAAS) contributing to cardiovascular diseases. In this study, we sought to determine if renal denervation (RDN) inhibits aldosterone expression and associated cardiovascular pathophysiological changes in angiotensin II (Ang II)-induced hypertension. Bilateral RDN or SHAM operation was performed before chronic 14-day Ang II subcutaneous infusion (200ng/kg/min) in male Sprague-Dawley rats. Bilateral RDN blunted Ang II-induced hypertension and ameliorated the mesenteric vascular dysfunction. Cardiovascular hypertrophy in response to Ang II was significantly attenuated by RDN as shown by histopathology and transthoracic echocardiography. Moreover, Ang II-induced vascular and myocardial inflammation and fibrosis were suppressed by RDN with concurrent decrease in fibronectin and collagen deposition, macrophage infiltration, and MCP-1 expression. Interestingly, RDN also inhibited Ang II-induced aldosterone expression in the plasma, kidney and heart. This was associated with the reduction of calcitonin gene-related peptide (CGRP) in the adrenal gland. Ang II promoted aldosterone secretion which was partly attenuated by CGRP in the adrenocortical cell line, suggesting a protective role of CGRP in this model. Activation of transforming growth factor-β (TGF-β)/Smad and mitogen-activated protein kinases (MAPKs) signaling pathway was both inhibited by RDN especially in the heart. These results suggest that the regulation of the renal sympathetic nerve in Ang II-induced hypertension and associated cardiovascular pathophysiological changes is likely mediated by aldosterone, with CGRP involvement. PMID:27661131