78 FR 6120 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-29

..., including immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive... immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive Advantages: Binding of a new...

Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

USDA-ARS?s Scientific Manuscript database

This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

PubMed

Werle, E; Lenz, T; Strobel, G; Weicker, H

1988-07-01

The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

PubMed

Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

2003-02-01

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Competitive binding of (-)-epigallocatechin-3-gallate and 5-fluorouracil to human serum albumin: A fluorescence and circular dichroism study

NASA Astrophysics Data System (ADS)

Yuan, Lixia; Liu, Min; Liu, Guiqin; Li, Dacheng; Wang, Zhengping; Wang, Bingquan; Han, Jun; Zhang, Min

2017-02-01

Combination therapy with more than one therapeutic agent can improve therapeutic efficiency and decrease drug resistance. In this study, the interactions of human serum albumin (HSA) with individual or combined anticancer drugs, (-)-epigallocatechin-3-gallate (EGCG) and 5-fluorouracil (FU), were investigated by fluorescence and circular dichroism (CD) spectroscopy. The results demonstrated that the interaction of EGCG or FU with HSA is a process of static quenching and EGCG formed a more stable complex. The competitive experiments of site markers suggested that both anti-carcinogens mainly bound to site I (subdomain IIA). The interaction forces which play important roles in the binding process were discussed based on enthalpy and entropy changes. Moreover, the competition binding model for a ternary system was proposed so as to precisely calculate the binding parameters. The results demonstrated that one drug decreased the binding affinity of another drug with HSA, resulting in the increasing free drug concentration at the action sites. CD studies indicated that there was an alteration in HSA secondary structure due to the binding of EGCG and FU. It can be concluded that the combination of EGCG with FU may enhance anticancer efficacy. This finding may provide a theoretical basis for clinical treatments.

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

Competitive folding of RNA structures at a termination–antitermination site

PubMed Central

Ait-Bara, Soraya; Clerté, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2017-01-01

Antitermination is a regulatory process based on the competitive folding of terminator–antiterminator structures that can form in the leader region of nascent transcripts. In the case of the Bacillus subtilis licS gene involved in β-glucosides utilization, the binding of the antitermination protein LicT to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, LicT can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination–antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated licS transcript. PMID:28235843

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

PubMed

Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

2017-06-07

Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C 60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C 60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

Choline+ is a low-affinity ligand for alpha 1-adrenoceptors.

PubMed

Unelius, L; Cannon, B; Nedergaard, J

1994-10-07

The effect of choline+, a commonly used Na+ substitute, on ligand binding to alpha 1-adrenoceptors was investigated. It was found that replacement of 25% of the Na+ in a Krebs-Ringer bicarbonate buffer with choline+ led to a 3-fold decrease in the apparent affinity of [3H]prazosin for its binding site (i.e. the alpha 1-receptor) in a membrane preparation from brown adipose tissue, while no decrease in the total number of binding sites was observed. Similar effects were seen in membrane preparations from liver and brain. In competition experiments, it was found that choline+ could inhibit [3H]prazosin binding; from the inhibition curve, an affinity (Ki) of 31 mM choline+ for the [3H]prazosin-binding site could be calculated. In fully choline(+)-substituted buffers, where the level of [3H]prazosin binding was substantially reduced, both phentolamine and norepinephrine could still compete with [3H]prazosin for its binding site, with virtually unaltered affinity; thus choline+ did not substantially affect the characteristics of those receptors to which it did not bind. Choline+ did not affect the binding characteristics of the beta 1/beta 2 radioligand [3H]CGP-12177; thus, the effect on alpha 1-receptors was not due to general, unspecific effects on the membrane preparations. It is concluded that choline+ possesses characteristics similar to those of a competitive ligand for the alpha 1-adrenoceptor; it has a low affinity but the competitive type of interaction of choline may nonetheless under experimental conditions interfere with agonist interaction with the alpha 1-receptor.

Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors.

PubMed

Halámek, Jan; Makower, Alexander; Knösche, Kristina; Skládal, Petr; Scheller, Frieder W

2005-01-30

We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface. The detection of cocaine was based on a competitive assay. The change of frequency measured after 300s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35Hz (with an imprecision 3%, n = 3) while the presence of 100pmoll(-1) cocaine decreased the binding by 11%. The limit of detection was consequently below 100pmoll(-1) for cocaine. The total time of one analysis was 15min. This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay.

Mechanisms of inverse agonist action at D2 dopamine receptors

PubMed Central

Roberts, David J; Strange, Philip G

2005-01-01

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. Ki values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. Kcoupled and Kuncoupled were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTPγS binding to varying degrees in concentration–response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (−)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism. PMID:15735658

Intrinsic mineralocorticoid agonist activity of some nonsteroidal anti-inflammatory drugs. A postulated mechanism for sodium retention.

PubMed Central

Feldman, D; Couropmitree, C

1976-01-01

Because some nonsteroidal anti-inflammatory drugs (NSAID) induce salt and water retention and exhibit other steroid-like actions, studies were performed to ascertain whether these drugs possess intrinsic mineralocorticoid agonist activity. In vitro competitive binding assays utilizing tissue from adrenalectomized rats demonstrated that some NSAID can displace [3H]-aldosterone from renal cytoplasmic mineralocorticoid receptors. Displacement potency for these sites was in the sequence: aldosterone greater than spironolactone greater than phenylbutazone (PBZ) greater than aspirin (ASA) greater than indomethacin (IDM). Concentration ratios required to obtain significant displacement of [3H]aldosterone were high but clearly within the therapeutic range for PBZ and ASA but not IDM. The analogues oxyphenbutazone (OBZ) and sodium salicylate (SS) were similar in binding activity to PBZ and ASA, respectively. Lineweaver-Burk analysis revealed that the inhibition of [3H]aldosterone binding was competitive in nature. In addition, PBZ was shown to prevent the nuclear binding of [3H]aldosterone. In vivo injection of PBZ and ASA resulted in competition for [3H]aldosterone renal binding comparable to the in vitro studies. Administration of PBZ and OBZ to adrenalectomized rats resulted in significant salt retention whereas ASA and SS did not differ significantly from controls. Salt retention elicited by PBZ and OBZ was inhibited by spironolactone, a competitive mineralocorticoid antagonist. These data suggest that, despite nonsteroidal structures, PBZ and OBZ induce salt retention via a receptor-mediated mineralocorticoid pathway analogous to aldosterone action. PMID:173739

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

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

Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization ofmore » the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.« less

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

NASA Astrophysics Data System (ADS)

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-07-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment.

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

PubMed Central

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-01-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment. PMID:23881402

Peroxidative oxidation of halides catalysed by myeloperoxidase. Effect of fluoride on halide oxidation.

PubMed

Zgliczyński, J M; Stelmaszyńska, T; Olszowska, E; Krawczyk, A; Kwasnowska, E; Wróbel, J T

1983-01-01

It was found that all halides can compete with cyanide for binding with myeloperoxidase. The lower is the pH, the higher is the affinity of halides. The apparent dissociation constants (Kd) of myeloperoxidase-cyanide complex were determined in the presence of F-, Cl-, Br- and I- in the pH range of 4 to 7. In slightly acidic pH (4 - 6) fluoride and chloride exhibit a higher affinity towards the enzyme than bromide and iodide. Taking into account competition between cyanide and halides for binding with myeloperoxidase the dissociation constants of halide-myeloperoxidase complexes were calculated. All halides except fluoride can be oxidized by H2O2 in the presence of myeloperoxidase. However, since fluoride can bind with myeloperoxidase, it can competitively inhibit the oxidation of other halides. Fluoride was a competitive inhibitor with respect to other halides as well as to H2O2. Inhibition constants (Ki) for fluoride as a competitive inhibitor with respect to H2O2 increased from iodide oxidation through bromide to chloride oxidation.

May the Best Molecule Win: Competition ESI Mass Spectrometry

PubMed Central

Laughlin, Sarah; Wilson, W. David

2015-01-01

Electrospray ionization mass spectrometry has become invaluable in the characterization of macromolecular biological systems such as nucleic acids and proteins. Recent advances in the field of mass spectrometry and the soft conditions characteristic of electrospray ionization allow for the investigation of non-covalent interactions among large biomolecules and ligands. Modulation of genetic processes through the use of small molecule inhibitors with the DNA minor groove is gaining attention as a potential therapeutic approach. In this review, we discuss the development of a competition method using electrospray ionization mass spectrometry to probe the interactions of multiple DNA sequences with libraries of minor groove binding molecules. Such an approach acts as a high-throughput screening method to determine important information including the stoichiometry, binding mode, cooperativity, and relative binding affinity. In addition to small molecule-DNA complexes, we highlight other applications in which competition mass spectrometry has been used. A competitive approach to simultaneously investigate complex interactions promises to be a powerful tool in the discovery of small molecule inhibitors with high specificity and for specific, important DNA sequences. PMID:26501262

In silico investigation into the interactions between murine 5-HT3 receptor and the principle active compounds of ginger (Zingiber officinale).

PubMed

Lohning, Anna E; Marx, Wolfgang; Isenring, Liz

2016-11-01

Gingerols and shogaols are the primary non-volatile actives within ginger (Zingiber officinale). These compounds have demonstrated in vitro to exert 5-HT 3 receptor antagonism which could benefit chemotherapy-induced nausea and vomiting (CINV). The site and mechanism of action by which these compounds interact with the 5-HT 3 receptor is not fully understood although research indicates they may bind to a currently unidentified allosteric binding site. Using in silico techniques, such as molecular docking and GRID analysis, we have characterized the recently available murine 5-HT 3 receptor by identifying sites of strong interaction with particular functional groups at both the orthogonal (serotonin) site and a proposed allosteric binding site situated at the interface between the transmembrane region and the extracellular domain. These were assessed concurrently with the top-scoring poses of the docked ligands and included key active gingerols, shogaols and dehydroshogaols as well as competitive antagonists (e.g. setron class of pharmacologically active drugs), serotonin and its structural analogues, curcumin and capsaicin, non-competitive antagonists and decoys. Unexpectedly, we found that the ginger compounds and their structural analogs generally outscored other ligands at both sites. Our results correlated well with previous site-directed mutagenesis studies in identifying key binding site residues. We have identified new residues important for binding the ginger compounds. Overall, the results suggest that the ginger compounds and their structural analogues possess a high binding affinity to both sites. Notwithstanding the limitations of such theoretical analyses, these results suggest that the ginger compounds could act both competitively or non-competitively as has been shown for palonosetron and other modulators of CYS loop receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Localization of basic fibroblast growth factor binding sites in the chick embryonic neural retina.

PubMed

Cirillo, A; Arruti, C; Courtois, Y; Jeanny, J C

1990-12-01

We have investigated the localization of basic fibroblast growth factor (bFGF) binding sites during the development of the neural retina in the chick embryo. The specificity of the affinity of bFGF for its receptors was assessed by competition experiments with unlabelled growth factor or with heparin, as well as by heparitinase treatment of the samples. Two different types of binding sites were observed in the neural retina by light-microscopic autoradiography. The first type, localized mainly to basement membranes, was highly sensitive to heparitinase digestion and to competition with heparin. It was not developmentally regulated. The second type of binding site, resistant to heparin competition, appeared to be associated with retinal cells from the earliest stages studied (3-day-old embryo, stages 21-22 of Hamburger and Hamilton). Its distribution was found to vary during embryonic development, paralleling layering of the neural retina. Binding of bFGF to the latter sites was observed throughout the retinal neuroepithelium at early stages but displayed a distinct pattern at the time when the inner and outer plexiform layers were formed. During the development of the inner plexiform layer, a banded pattern of bFGF binding was observed. These bands, lying parallel to the vitreal surface, seemed to codistribute with the synaptic bands existing in the inner plexiform layer. The presence of intra-retinal bFGF binding sites whose distribution varies with embryonic development suggests a regulatory mechanism involving differential actions of bFGF on neural retinal cells.

Site Identification by Ligand Competitive Saturation (SILCS) simulations for fragment-based drug design.

PubMed

Faller, Christina E; Raman, E Prabhu; MacKerell, Alexander D; Guvench, Olgun

2015-01-01

Fragment-based drug design (FBDD) involves screening low molecular weight molecules ("fragments") that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind nonoverlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy.The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is "soaked" in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called "FragMaps" can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine "Grid Free Energies (GFEs)," which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities.

The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

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

Humphreys, C.J.

1989-01-01

The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized,more » purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.« less

A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases

PubMed Central

Guarnieri, Michael T.; Blagg, Brian S. J.

2011-01-01

Abstract Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate competition assay beyond GHKL inhibitor screening. PMID:21050069

Putative melatonin receptors in a human biological clock

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

Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.

In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completelymore » inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.« less

Photonic crystal borax competitive binding carbohydrate sensing motif†

PubMed Central

Cui, Qingzhou; Muscatello, Michelle M. Ward; Asher, Sanford A.

2009-01-01

We developed a photonic crystal sensing method for diol containing species such as carbohydrates based on a poly(vinyl alcohol) (PVA) hydrogel containing an embedded crystalline colloidal array (CCA). The polymerized CCA (PCCA) diffracts visible light. We show that in the presence of borax the diffraction wavelength shifts as the concentration of glucose changes. The diffraction shifts result from the competitive binding of glucose to borate, which reduces the concentration of borate bound to the PVA diols. PMID:19381378

Mechanistic Insights into Xenon Inhibition of NMDA Receptors from MD Simulations

PubMed Central

Liu, Lu Tian; Xu, Yan; Tang, Pei

2010-01-01

Inhibition of N-methyl-D-aspartate (NMDA) receptors has been viewed as a primary cause of xenon anesthesia, yet the mechanism is unclear. Here, we investigated interactions between xenon and the ligand-binding domain (LBD) of a NMDA receptor and examined xenon-induced structural and dynamical changes that are relevant to functional changes of the NMDA receptor. Several comparative molecular dynamics simulations were performed on two X-ray structures representing the open- and closed-cleft LBD of the NMDA receptor. We identified plausible xenon action sites in the LBD, including those nearby agonist sites, in the hinge region, and at the interface between two subunits. The xenon binding energy varies from −5.3 to −0.7 kcal/mol. Xenon's effect on the NMDA receptor is conformation-dependent and is produced through both competitive and non-competitive mechanisms. Xenon can promote cleft opening in the absence of agonists and consequently stabilizes the closed channel. Xenon can also bind at the interface of two subunits, alter the inter-subunit interaction, and lead to a reduction of the distance between GT-links. This reduction corresponds to a rearrangement of the channel toward a direction of pore size decreasing, implying a closed or desensitized channel. In addition to these non-competitive actions, xenon was found to weaken the glutamate binding, which could lead to low agonist efficacy and appear as competitive inhibition. PMID:20560662

Thermodynamics of binding interactions between extracellular polymeric substances and heavy metals by isothermal titration microcalorimetry.

PubMed

Yan, Peng; Xia, Jia-Shuai; Chen, You-Peng; Liu, Zhi-Ping; Guo, Jin-Song; Shen, Yu; Zhang, Cheng-Cheng; Wang, Jing

2017-05-01

Extracellular polymeric substances (EPS) play a crucial role in heavy metal bio-adsorption using activated sludge, but the interaction mechanism between heavy metals and EPS remains unclear. Isothermal titration calorimetry was employed to illuminate the mechanism in this study. The results indicate that binding between heavy metals and EPS is spontaneous and driven mainly by enthalpy change. Extracellular proteins in EPS are major participants in the binding process. Environmental conditions have significant impact on the adsorption performance. Divalent and trivalent cations severely impeded the binding of heavy metal ions to EPS. Electrostatic interaction mainly attributed to competition between divalent cations and heavy metal ions; trivalent cations directly competed with heavy metal ions for EPS binding sites. Trivalent cations were more competitive than divalent cations for heavy metal ion binding because they formed complexing bonds. This study facilitates a better understanding about the interaction between heavy metals and EPS in wastewater treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adherence of oral streptococci: evidence for nonspecific adsorption to saliva-coated hydroxylapatite surfaces.

PubMed Central

Staat, R H; Peyton, J C

1984-01-01

It is proposed that binding of oral streptococci to saliva-coated hydroxylapatite (SHA) surfaces is a multifactorial process involving both specific and nonspecific receptors. In this context, specific binding is described as a high-affinity, saturable interaction between the cell and binding surface. Conversely, nonspecific binding is considered to be a nonsaturable, generalized, low-affinity reaction. Experimental differentiation of specific binding from nonspecific binding was achieved with a competition assay which utilized a large excess of nonradiolabeled bacteria to compete with the 3H-labeled cells for attachment to receptors on 1.5 mg of SHA crystals. Competition assays of Streptococcus sanguis and Streptococcus mitis adhesion clearly demonstrated that the total binding isotherm was composed of a saturable specific binding reaction and a minor nonspecific binding component. This was further substantiated by analysis of nonlinear Scatchard plots of the total binding data. The competition data for Streptococcus mutans binding indicated that ca. 50% of the S. mutans binding appeared to be specific, although saturation of the SHA surfaces with bacterial cells could not be demonstrated. Experiments measuring desorption of radiolabeled cells from SHA crystals into buffer showed that ca. 50% of the bound S. mutans cells were removed after 4 h, whereas less than 5% of the S. sanguis cells were eluted from the SHA surfaces. The kinetics of attachment were studied by using an extract of Persea americana as a noncompetitive inhibitor of adherence. The total cell binding data for these experiments suggested a very rapid binding reaction followed by a slower rate of attachment. It was concluded from these three different experimental approaches that adherence of selected oral streptococci to SHA surfaces involves specific, high-affinity and nonspecific, low-affinity binding reactions. The concept is developed that in vitro streptococcal attachment to SHA can be described as a two-reaction process in which the low-affinity interaction of the cell with the SHA surface precedes the establishment of the stronger, specific bonds needed for the maintenance of streptococci in the oral cavity. PMID:6327530

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

PubMed

Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

2004-04-01

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. Copyright 2004 Wiley-Liss, Inc.

Two-track virtual screening approach to identify both competitive and allosteric inhibitors of human small C-terminal domain phosphatase 1

NASA Astrophysics Data System (ADS)

Park, Hwangseo; Lee, Hye Seon; Ku, Bonsu; Lee, Sang-Rae; Kim, Seung Jun

2017-08-01

Despite a wealth of persuasive evidence for the involvement of human small C-terminal domain phosphatase 1 (Scp1) in the impairment of neuronal differentiation and in Huntington's disease, small-molecule inhibitors of Scp1 have been rarely reported so far. This study aims to the discovery of both competitive and allosteric Scp1 inhibitors through the two-track virtual screening procedure. By virtue of the improvement of the scoring function by implementing a new molecular solvation energy term and by reoptimizing the atomic charges for the active-site Mg2+ ion cluster, we have been able to identify three allosteric and five competitive Scp1 inhibitors with low-micromolar inhibitory activity. Consistent with the results of kinetic studies on the inhibitory mechanisms, the allosteric inhibitors appear to be accommodated in the peripheral binding pocket through the hydrophobic interactions with the nonpolar residues whereas the competitive ones bind tightly in the active site with a direct coordination to the central Mg2+ ion. Some structural modifications to improve the biochemical potency of the newly identified inhibitors are proposed based on the binding modes estimated with docking simulations.

Site Identification by Ligand Competitive Saturation (SILCS) Simulations for Fragment-Based Drug Design

PubMed Central

Faller, Christina E.; Raman, E. Prabhu; MacKerell, Alexander D.; Guvench, Olgun

2015-01-01

Fragment-based drug design (FBDD) involves screening low molecular weight molecules (“fragments”) that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind non-overlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy. The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is “soaked” in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called “FragMaps” can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine “Grid Free Energies (GFEs),” which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities. PMID:25709034

[Studying specific effects of nootropic drugs on glutamate receptors in the rat brain].

PubMed

Firstova, Iu Iu; Vasil'eva, E V; Kovalev, G I

2011-01-01

The influence of nootropic drugs of different groups (piracetam, phenotropil, nooglutil, noopept, semax, meclofenoxate, pantocalcine, and dimebon) on the binding of the corresponding ligands to AMPA, NMDA, and mGlu receptors of rat brain has been studied by the method of radio-ligand binding in vitro. It is established that nooglutil exhibits pharmacologically significant competition with a selective agonist of AMPA receptors ([G-3H]Ro 48-8587) for the receptor binding sites (with IC50 = 6.4 +/- 0.2 microM), while the competition of noopept for these receptor binding sites was lower by an order of magnitude (IC50 = 80 +/- 5.6 microM). The heptapeptide drug semax was moderately competitive with [G-3H]LY 354740 for mGlu receptor sites (IC50 = 33 +/- 2.4 microM). Dimebon moderately influenced the specific binding of the ligand of NMDA receptor channel ([G-3H]MK-801) at IC50 = 59 +/- 3.6 microM. Nootropic drugs of the pyrrolidone group (piracetam, phenotropil) as well as meclofenoxate, pantocalcine (pantogam) in a broad rage of concentrations (10(-4)-10(-10) M) did not affect the binding of the corresponding ligands to glutamate receptors (IC50 100 pM). Thus, the direct neurochemical investigation was used for the first time to qualitatively characterize the specific binding sites for nooglutil and (to a lower extent) noopept on AMPA receptors, for semax on metabotropic glutamate receptors, and for dimebon on the channel region of NMDA receptors. The results are indicative of a selective action of some nootropes on the glutamate family.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Interaction of fenoterol stereoisomers with β2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

PubMed

Reinartz, Michael T; Kälble, Solveig; Wainer, Irving W; Seifert, Roland

2015-05-01

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the β2-adrenoceptor (β2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using β2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay.

PubMed

Le Saux, Thomas; Hisamoto, Hideaki; Terabe, Shigeru

2006-02-03

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.

Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

EPA Science Inventory

The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

High glucose disrupts oligosaccharide recognition function via competitive inhibition: a potential mechanism for immune dysregulation in diabetes mellitus.

PubMed

Ilyas, Rebecca; Wallis, Russell; Soilleux, Elizabeth J; Townsend, Paul; Zehnder, Daniel; Tan, Bee K; Sim, Robert B; Lehnert, Hendrik; Randeva, Harpal S; Mitchell, Daniel A

2011-01-01

Diabetic complications include infection and cardiovascular disease. Within the immune system, host-pathogen and regulatory host-host interactions operate through binding of oligosaccharides by C-type lectin. A number of C-type lectins recognise oligosaccharides rich in mannose and fucose - sugars with similar structures to glucose. This raises the possibility that high glucose conditions in diabetes affect protein-oligosaccharide interactions via competitive inhibition. Mannose-binding lectin, soluble DC-SIGN and DC-SIGNR, and surfactant protein D, were tested for carbohydrate binding in the presence of glucose concentrations typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were performed in high glucose. DC-SIGN and DC-SIGNR expression in adipose tissues was examined via immunohistochemistry. High glucose inhibited C-type lectin binding to high-mannose glycoprotein and binding of DC-SIGN to fucosylated ligand (blood group B) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high trehalose - a nonreducing sugar with glucoside stereochemistry. DC-SIGN staining was seen on cells with DC morphology within omental and subcutaneous adipose tissues. We conclude that high glucose disrupts C-type lectin function, potentially illuminating new perspectives on susceptibility to infectious and inflammatory disease in diabetes. Mechanisms involve competitive inhibition of carbohydrate binding within sets of defined proteins, in contrast to broadly indiscriminate, irreversible glycation of proteins. Copyright © 2010 Elsevier GmbH. All rights reserved.

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

PubMed

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

2017-05-04

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

Apo-states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain

PubMed Central

Findeisen, Felix; Rumpf, Christine; Minor, Daniel L.

2013-01-01

In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation (CDI) and limits calcium entry, whereas CaBP1 blocks CDI and allows sustained calcium influx. Here, we combine isothermal titration calorimetry (ITC) with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca2+/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium binding properties. The observation that the apo-forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. PMID:23811053

Identification and quantification of human kidney atrial natriuretic peptide receptors.

PubMed

Kahana, L; Yechiely, H; Mecz, Y; Lurie, A

1995-04-01

The present study determined 125I-label atrial natriuretic peptide (ANP) binding sites in human kidney glomerular and papillary membranes. The membranes were prepared from non-malignant renal tissue obtained at nephrectomy of patients with renal carcinoma. To evaluate the proportion of ANP receptor classes ANP-R1 (ANPR-A, -B) versus ANP-R2 (ANPR-C), competitive binding studies were performed using [125I]-ANP in the presence of increasing concentrations of ANP or an internally ring-deleted analog, des(Gln116, Ser117, Gly118, Leu119, Gly120)ANP(102-121), called C-ANP, which binds selectively to ANPR-C receptors. Analysis of the competitive binding curve with ANP in glomerular membranes suggested the presence of one group of high-affinity receptors with dissociation constant Kd = 26 +/- 12 pmol/l and density Bmax = 101 +/- 47 nmol/kg protein. A decrease of 10-30% in Bmax with no change in Kd was obtained in the presence of excess (10(-6) mol/l) C-ANP, suggesting the existence of a small amount of a second class of receptors, the ANPR-C class. The densities of ANPR-A, -B versus ANPR-C receptors in human glomeruli, calculated from competitive inhibition experiments, were 75 +/- 42 and 22 +/- 16 nmol/kg protein (N = 8). Autoradiography of the sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions showed two bands: a highly labeled 130kD band and a weakly labeled 66 kD band, both displaced by ANP. Only the 66-kD band was displaced by the C-ANP analog. Human papilla membrane, as shown by competition binding studies and SDS gel electrophoresis, presented only one class of receptors with Kd = 40 +/- 23 pmol/l (mean +/- SD, N = 3) and Bmax = 17 +/- 6.3 nmol/kg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of Protein G Microcolumns in Chromatographic Immunoassays: A Comparison of Competitive Binding Formats

PubMed Central

Pfaunmiller, Erika L.; Anguizola, Jeanethe A.; Milanuk, Mitchell L.; Carter, NaTasha; Hage, David S.

2016-01-01

Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limits of detection, and analysis time. All these methods gave detection limits in the range of 8–19 ng/mL and precisions ranging from ± 5% to ± 10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest. PMID:26777776

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

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

Skafar, D.F.

1991-11-12

The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with ({sup 3}H)progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020. This indicated that a difference in the binding mechanism of RU486more » and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable. These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.« less

Differential transcriptional regulation by alternatively designed mechanisms: A mathematical modeling approach.

PubMed

Yildirim, Necmettin; Aktas, Mehmet Emin; Ozcan, Seyma Nur; Akbas, Esra; Ay, Ahmet

2017-01-01

Cells maintain cellular homeostasis employing different regulatory mechanisms to respond external stimuli. We study two groups of signal-dependent transcriptional regulatory mechanisms. In the first group, we assume that repressor and activator proteins compete for binding to the same regulatory site on DNA (competitive mechanisms). In the second group, they can bind to different regulatory regions in a noncompetitive fashion (noncompetitive mechanisms). For both competitive and noncompetitive mechanisms, we studied the gene expression dynamics by increasing the repressor or decreasing the activator abundance (inhibition mechanisms), or by decreasing the repressor or increasing the activator abundance (activation mechanisms). We employed delay differential equation models. Our simulation results show that the competitive and noncompetitive inhibition mechanisms exhibit comparable repression effectiveness. However, response time is fastest in the noncompetitive inhibition mechanism due to increased repressor abundance, and slowest in the competitive inhibition mechanism by increased repressor level. The competitive and noncompetitive inhibition mechanisms through decreased activator abundance show comparable and moderate response times, while the competitive and noncompetitive activation mechanisms by increased activator protein level display more effective and faster response. Our study exemplifies the importance of mathematical modeling and computer simulation in the analysis of gene expression dynamics.

Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe.

PubMed

Ren, Xiao M; Guo, Liang-Hong

2012-04-17

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein-thyroxine (F-T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F-T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F-T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F-T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F-T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F-T4 bound to the proteins at the T4 binding site. By using F-T4 as the fluorescence probe in competitive binding assays, 11 OH-PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH-BDE-47 and 3'-OH-BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.

Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

PubMed

Kyriakis, Efthimios; Stravodimos, George A; Kantsadi, Anastassia L; Chatzileontiadou, Demetra S M; Skamnaki, Vassiliki T; Leonidas, Demetres D

2015-07-08

We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 μM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

‘Partial’ competition of heterobivalent ligand binding may be mistaken for allosteric interactions: a comparison of different target interaction models

PubMed Central

Vauquelin, Georges; Hall, David; Charlton, Steven J

2015-01-01

Background and Purpose Non-competitive drugs that confer allosteric modulation of orthosteric ligand binding are of increasing interest as therapeutic agents. Sought-after advantages include a ceiling level to drug effect and greater receptor-subtype selectivity. It is thus important to determine the mode of interaction of newly identified receptor ligands early in the drug discovery process and binding studies with labelled orthosteric ligands constitute a traditional approach for this. According to the general allosteric ternary complex model, allosteric ligands that exhibit negative cooperativity may generate distinctive ‘competition’ curves: they will not reach baseline levels and their nadir will increase in par with the orthosteric ligand concentration. This behaviour is often considered a key hallmark of allosteric interactions. Experimental Approach The present study is based on differential equation-based simulations. Key Results The differential equation-based simulations revealed that the same ‘competition binding’ pattern was also obtained when a monovalent ligand binds to one of the target sites of a heterobivalent ligand, even if this process is exempt of allosteric interactions. This pattern was not strictly reciprocal when the binding of each of the ligands was recorded. The prominence of this phenomenon may vary from one heterobivalent ligand to another and we suggest that this phenomenon may take place with ligands that have been proposed to bind according to ‘two-domain’ and ‘charnière’ models. Conclusions and Implications The present findings indicate a familiar experimental situation where bivalency may give rise to observations that could inadvertently be interpreted as allosteric binding. Yet, both mechanisms could be differentiated based on alternative experiments and structural considerations. PMID:25537684

Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

PubMed Central

Bosson, Andrew D.; Zamudio, Jesse R.; Sharp, Phillip A.

2016-01-01

SUMMARY Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ~3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo. PMID:25449132

Selective binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid to peroxisome proliferator-activated receptor gamma allows ligand identification and characterization.

PubMed

Zorrilla, Silvia; Garzón, Beatriz; Pérez-Sala, Dolores

2010-04-01

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily involved in insulin sensitization, atherosclerosis, inflammation, and carcinogenesis. PPARgamma transcriptional activity is modulated by specific ligands that promote conformational changes allowing interaction with coactivators. Here we show that the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) binds to PPARgamma-LBD (ligand binding domain), displaying negligible interaction with other nuclear receptors such as PPARalpha and retinoid X receptor alpha (RXRalpha). ANS binding is competed by PPARgamma agonists such as rosiglitazone, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), and 9,10-dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) (CAY10410). Moreover, the affinity of PPARgamma for these ligands, determined through ANS competition titrations, is within the range of that reported previously, thereby suggesting that ANS competition could be useful in the screening and characterization of novel PPARgamma agonists. In contrast, gel-based competition assays showed limited performance with noncovalently bound ligands. We applied the ANS binding assay to characterize a biotinylated analog of 15d-PGJ(2) that does not activate PPAR in cells. We found that although this compound bound to PPARgamma with low affinity, it failed to promote PPARgamma interaction with a fluorescent SRC-1 peptide, indicating a lack of receptor activation. Therefore, combined approaches using ANS and fluorescent coactivator peptides to monitor PPARgamma binding and interactions may provide valuable strategies to fully understand the role of PPARgamma ligands. Copyright 2009 Elsevier Inc. All rights reserved.

Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function.

PubMed

Xie, Y; Cohen, J B

2001-01-26

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.

In vitro DNA binding studies of Aspartame, an artificial sweetener.

PubMed

Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

2013-03-05

A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of bradykinin receptors in human lung fibroblasts using the binding of 3[H][Des-Arg10,Leu9]kallidin and [3H]NPC17731.

PubMed

Zhang, S P; Codd, E E

1998-01-01

Bradykinin (BK) receptors are involved in pain and inflammation. Two BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. Both B1 and B2 receptors are G-protein coupled membrane receptors. B1 receptors are present in smooth muscle tissue, whereas B2 receptors are found in both smooth muscle tissue and neurons. [Des-Arg10,Leu9]kallidin (DALKD) is a selective B1 receptor antagonist, and NPC17731 is a selective B2 receptor antagonist. To develop binding assays for the two known BK receptor subtypes, [3H]DALKD and [3H]NPC17731 were used as selective ligands for B1 and B2 receptors respectively. Both ligands bound to the CCD-16 human lung fibroblast membranes reaching equilibrium at 25 degrees C within 30 min. Binding was stable for at least 60 min. The Kd of [3H]DALKD was 0.33 nM and Bmax was 52 fmol/mg membrane protein. The Kd of [3H]NPC17731 was 0.39 nM and Bmax was 700 fmol/mg membrane protein. Competition for [3H]DALKD binding with BK receptor agonists was in the order: [des-Arg10]KD (DAKD) > KD > [des-Arg9]BK (DABK) > BK, and competition for [3H]DALKD binding with BK receptor antagonists was in the order: DALKD > [des-Arg10]Hoe 140 (DAHoe 140) > [des-Arg9,Leu8]BK (DALBK) > NPC17731 > Hoe 140 > DNMFBK, suggesting that [3H]DALKD bound selectively to B1 receptors. By contrast, competition for [3H]NPC17731 binding by BK agonists was in the order: BK > KD > DAKD > DABK, and competition for [3H]NPC17731 binding by BK antagonists was in the order: NPC17731 = Hoe 140 > DNMFBK > DAHoe 140 > DALBK > DALKD, indicating that [3H]NPC17731 labeled B2 receptors selectively. These results demonstrate that [3H]DALKD and [3H]NPC17731 can be used with CCD-16 human lung fibroblast membranes to provide a pair of binding assays for the simultaneous evaluation of B1 and B2 BK receptor subtypes.

Apo states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain.

PubMed

Findeisen, Felix; Rumpf, Christine H; Minor, Daniel L

2013-09-09

In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation and limits calcium entry, whereas CaBP1 blocks calcium-dependent inactivation (CDI) and allows sustained calcium influx. Here, we combine isothermal titration calorimetry with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca(2+)/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium-binding properties. The observation that the apo forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Simulation of a model nanopore sensor: Ion competition underlies device behavior.

PubMed

Mádai, Eszter; Valiskó, Mónika; Dallos, András; Boda, Dezső

2017-12-28

We study a model nanopore sensor with which a very low concentration of analyte molecules can be detected on the basis of the selective binding of the analyte molecules to the binding sites on the pore wall. The bound analyte ions partially replace the current-carrier cations in a thermodynamic competition. This competition depends both on the properties of the nanopore and the concentrations of the competing ions (through their chemical potentials). The output signal given by the device is the current reduction caused by the presence of the analyte ions. The concentration of the analyte ions can be determined through calibration curves. We model the binding site with the square-well potential and the electrolyte as charged hard spheres in an implicit background solvent. We study the system with a hybrid method in which we compute the ion flux with the Nernst-Planck (NP) equation coupled with the Local Equilibrium Monte Carlo (LEMC) simulation technique. The resulting NP+LEMC method is able to handle both strong ionic correlations inside the pore (including finite size of ions) and bulk concentrations as low as micromolar. We analyze the effect of bulk ion concentrations, pore parameters, binding site parameters, electrolyte properties, and voltage on the behavior of the device.

Simulation of a model nanopore sensor: Ion competition underlies device behavior

NASA Astrophysics Data System (ADS)

Mádai, Eszter; Valiskó, Mónika; Dallos, András; Boda, Dezső

2017-12-01

We study a model nanopore sensor with which a very low concentration of analyte molecules can be detected on the basis of the selective binding of the analyte molecules to the binding sites on the pore wall. The bound analyte ions partially replace the current-carrier cations in a thermodynamic competition. This competition depends both on the properties of the nanopore and the concentrations of the competing ions (through their chemical potentials). The output signal given by the device is the current reduction caused by the presence of the analyte ions. The concentration of the analyte ions can be determined through calibration curves. We model the binding site with the square-well potential and the electrolyte as charged hard spheres in an implicit background solvent. We study the system with a hybrid method in which we compute the ion flux with the Nernst-Planck (NP) equation coupled with the Local Equilibrium Monte Carlo (LEMC) simulation technique. The resulting NP+LEMC method is able to handle both strong ionic correlations inside the pore (including finite size of ions) and bulk concentrations as low as micromolar. We analyze the effect of bulk ion concentrations, pore parameters, binding site parameters, electrolyte properties, and voltage on the behavior of the device.

Effects of competitive red blood cell binding and reduced hematocrit on the blood and plasma levels of (/sup 14/C)Indapamide in the rat

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

Lettieri, J.T.; Portelli, S.T.

1983-02-01

The effects of chlorthalidone and acetazolamide on the red blood cell binding of indapamide were investigated. Both drugs caused a substantial decrease in the amount of indapamide bound to the erythrocytes in vitro. This effect was demonstrated by a change in the indapamide blood/plasma ratio from approximately 6 in control samples, to a value of 1 when either of the displacing agents was added. Coadministration of acetazolamide with /sup 14/C-labeled indapamide to rats, resulted in a 5-fold drop in the blood levels of total radioactivity, relative to rats dosed with (/sup 14/C)indapamide alone. Concomitantly, there was a 2-fold increase inmore » the plasma levels of total radioactivity after acetazolamide coadministration. In rats whose hematocrits had been reduced by extensive bleeding, there were only minor alterations in the blood/plasma partitioning of (/sup 14/C)indapamide. Thus, chlorthalidone and acetazolamide were able to displace indapamide from erythrocytes in vitro and in vivo, possibly by competition at a carbonic anhydrase binding site. The pharmacokinetics of drugs which are extensively bound to erythrocytes may be significantly altered by the presence of other agents capable of competitive binding.« less

Binding specificity of the juvenile hormone carrier protein from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

PubMed

Peterson, R C; Reich, M F; Dunn, P E; Law, J H; Katzenellnbogen, J A

1977-05-17

A series of analogues of insect juvenile hormone (four geometric isomers of methyl epoxyfarnesenate, several para-substituted epoxygeranyl phenyl ethers, and epoxyfarnesol and its acetate and haloacetate derivatives) was prepared to investigate the binding specificity of the hemolymph juvenile hormone binding protein from the tobacco hornworm Manduct sexta. The relative binding affinities were determined by a competition assay against radiolabeled methyl (E,E)-3,11-dimethyl-7-ethyl-cis-10,11-epoxytrideca-2,6-dienoate (JH I). The ratio of dissociation constants was estimated by plotting competitor data according to a linear transformation of the dissociation equations describing competition of two ligands for a binding protein. The importance of the geometry of the sesquiterpene hydrocarbon chain is indicated by the fact that the binding affinity is decreased as Z (cis) double bonds are substituted for E (trans) double bonds in the methyl epoxyfarnesenate series; the unepoxidized analogues do not bind. A carboxylic ester function is important although its orientation can be reversed, as indicated by the good binding of epoxyfarnesyl acetate. In the monoterpene series, methyl epoxygeranoate shows no affinity for the binding protein, but substitution of a phenyl or p-carbomethoxyphenyl ether for the ester function imparts a low, but significant affinity. These data taken together with earlier results indicate that the binding site for juvenile hormone in the hemolymph binding protein is characterized by a sterically defined hydrophobic region with polar sites that recognize the epoxide and the ester functions.

Competitive fluorescent pseudo-immunoassay exploiting molecularly imprinted polymers for the detection of biogenic amines in fish matrix.

PubMed

Mattsson, Leena; Xu, Jingjing; Preininger, Claudia; Tse Sum Bui, Bernadette; Haupt, Karsten

2018-05-01

We developed a competitive fluorescent molecularly imprinted polymer (MIP) assay to detect biogenic amines in fish samples. MIPs synthesized by precipitation polymerization using histamine as template were used in a batch binding assay analogous to competitive fluoroimmunoassays. Introducing a complex sample matrix, such as fish extract, into the assay changes the environment and the binding conditions, therefore the importance of the sample preparation is extensively discussed. Several extraction and purification methods for fish were comprehensively studied, and an optimal clean-up procedure for fish samples using liquid-liquid extraction was developed. The feasibility of the competitive MIP assay was shown in the purified fish extract over a broad histamine range (1 - 430µM). The MIP had the highest affinity towards histamine, but recognized also the structurally similar biogenic amines tyramine and tryptamine, as well as spermine and spermidine, providing simultaneous analysis and assessment of the total amount of biogenic amines. Copyright © 2018 Elsevier B.V. All rights reserved.

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

PubMed Central

Presley, Tennille; Vedam, Kaushik; Liu, Xiaoping; Zweier, Jay L.

2009-01-01

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42°C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV. PMID:19412660

From non-covalent binding to irreversible DNA lesions: nile blue and nile red as photosensitizing agents

PubMed Central

Gattuso, Hugo; Besancenot, Vanessa; Grandemange, Stéphanie; Marazzi, Marco; Monari, Antonio

2016-01-01

We report a molecular modeling study, coupled with spectroscopy experiments, on the behavior of two well known organic dyes, nile blue and nile red, when interacting with B-DNA. In particular, we evidence the presence of two competitive binding modes, for both drugs. However their subsequent photophysical behavior is different and only nile blue is able to induce DNA photosensitization via an electron transfer mechanism. Most notably, even in the case of nile blue, its sensitization capabilities strongly depend on the environment resulting in a single active binding mode: the minor groove. Fluorescence spectroscopy confirms the presence of competitive interaction modes for both sensitizers, while the sensitization via electron transfer, is possible only in the case of nile blue. PMID:27329409

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

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

Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with boundmore » ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.« less

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

PubMed Central

Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea

2017-01-01

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity. PMID:28760974

Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.

PubMed

Yang, Rui-Nan; Li, Dong-Zhen; Yu, Guangqiang; Yi, Shan-Cheng; Zhang, Yinan; Kong, De-Xin; Wang, Man-Qun

2017-12-01

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand.

PubMed

De Souza, Melissa; Matthews, Hayden; Lee, Jodi A; Ranson, Marie; Kelso, Michael J

2011-04-15

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Competition effects in cation binding to humic acid: Conditional affinity spectra for fixed total metal concentration conditions

NASA Astrophysics Data System (ADS)

David, Calin; Mongin, Sandrine; Rey-Castro, Carlos; Galceran, Josep; Companys, Encarnació; Garcés, José Luis; Salvador, José; Puy, Jaume; Cecilia, Joan; Lodeiro, Pablo; Mas, Francesc

2010-09-01

Information on the Pb and Cd binding to a purified Aldrich humic acid (HA) is obtained from the influence of different fixed total metal concentrations on the acid-base titrations of this ligand. NICA (Non-Ideal Competitive Adsorption) isotherm has been used for a global quantitative description of the binding, which has then been interpreted by plotting the Conditional Affinity Spectra of the H + binding at fixed total metal concentrations (CAScTM). This new physicochemical tool, here introduced, allows the interpretation of binding results in terms of distributions of proton binding energies. A large increase in the acidity of the phenolic sites as the total metal concentration increases, especially in presence of Pb, is revealed from the shift of the CAScTM towards lower affinities. The variance of the CAScTM distribution, which can be used as a direct measure of the heterogeneity, also shows a significant dependence on the total metal concentration. A discussion of the factors that influence the heterogeneity of the HA under the conditions of each experiment is provided, so that the smoothed pattern exhibited by the titration curves can be justified.

Selected wheat seed defense proteins exhibit competitive binding to model microbial lipid interfaces.

PubMed

Sanders, Michael R; Clifton, Luke A; Neylon, Cameron; Frazier, Richard A; Green, Rebecca J

2013-07-17

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. This study examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b, and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and β-purothionin (β-Pth) model anionic lipid layers using a combination of surface pressure measurements, external reflection FTIR spectroscopy, and neutron reflectometry. Results highlighted differences in the protein binding mechanisms and in the competitive binding and penetration of lipid layers between respective Pins and β-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of β-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with β-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of β-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity.

Multi-Laboratory Study of Five Methods for the Determination of Brevetoxins in Shellfish Tissue Extracts.

PubMed

Dickey, Robert W; Plakas, Steven M; Jester, Edward L E; El Said, Kathleen R; Johannessen, Jan N; Flewelling, Leanne J; Scott, Paula; Hammond, Dan G; Van Dolah, Frances M; Leighfield, Tod A; Bottein Dachraoui, Marie-Yasmine; Ramsdell, John S; Pierce, Richard H; Henry, Mike S; Poli, Mark A; Walker, Calvin; Kurtz, Jan; Naar, Jerome; Baden, Daniel G; Musser, Steve M; White, Kevin D; Truman, Penelope; Miller, Aaron; Hawryluk, Timothy P; Wekell, Marleen M; Stirling, David; Quilliam, Michael A; Lee, Jung K

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

PubMed Central

Pfister, Klaus; Radosevich, Steven R.; Arntzen, Charles J.

1979-01-01

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H2O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (14C-radiolabel assays) with binding constants (K) of 1.4 × 10−8 molar and 4 × 10−8 molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10−8 molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts. We conclude that triazine resistance of both intact plants and isolated chloroplasts of Senecio vulgaris L. is based upon a minor modification of the protein in the photosystem II complex which is responsible for herbicide binding. This change results in a specific loss of atrazine (triazine)-binding capacity. PMID:16661120

International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

EPA Science Inventory

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

PubMed Central

Sauvé, K; Nachman, M; Spence, C; Bailon, P; Campbell, E; Tsien, W H; Kondas, J A; Hakimi, J; Ju, G

1991-01-01

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. PMID:2052547

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

PubMed Central

Ramsey, Simeon J; Attkins, Neil J; Fish, Rebecca; van der Graaf, Piet H

2011-01-01

BACKGROUND AND PURPOSE A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF1) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. EXPERIMENTAL APPROACH A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF1 antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF1 receptor binding of novel compounds can be predicted on the basis of in vitro assays. KEY RESULTS The non-competitive antagonist behaviour appeared to be correlated to the CRF1 receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. CONCLUSIONS AND IMPLICATIONS This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF1 receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF1 receptor antagonists in an efficient manner in a drug discovery setting. PMID:21449919

Periplasmic binding protein-based detection of maltose using liposomes: a new class of biorecognition elements in competitive assays.

PubMed

Edwards, Katie A; Baeumner, Antje J

2013-03-05

A periplasmic binding protein (PBP) was investigated as a novel binding species in a similar manner to an antibody in a competitive enzyme linked immunosorbent assay (ELISA), resulting in a highly sensitive and specific assay utilizing liposome-based signal amplification. PBPs are located at high concentrations (10(-4) M) between the inner and outer membranes of gram negative bacteria and are involved in the uptake of solutes and chemotaxis of bacteria toward nutrient sources. Previous sensors relying on PBPs took advantage of the change in local environment or proximity of site-specific fluorophore labels resulting from the significant conformational shift of these proteins' two globular domains upon target binding. Here, rather than monitoring conformational shifts, we have instead utilized the maltose binding protein (MBP) in lieu of an antibody in an ELISA. To our knowledge, this is the first PBP-based sensor without the requirement for engineering site-specific modifications within the protein. MBP conjugated fluorescent dye-encapsulating liposomes served to provide recognition and signal amplification in a competitive assay for maltose using amylose magnetic beads in a microtiter plate-based format. The development of appropriate binding buffers and competitive surfaces are described, with general observations expected to extend to PBPs for other analytes. The resulting assay was specific for d-(+)-maltose versus other sugar analogs including d-(+)-raffinose, sucrose, d-trehalose, d-(+)-xylose, d-fructose, 1-thio-β-d-glucose sodium salt, d-(+)-galactose, sorbitol, glycerol, and dextrose. Cross-reactivity with d-lactose and d-(+)-glucose occurred only at concentrations >10(4)-fold greater than d-(+)-maltose. The limit of detection was 78 nM with a dynamic range covering over 3 orders of magnitude. Accurate detection of maltose as an active ingredient in a pharmaceutical preparation was demonstrated. This method offers a significant improvement over existing enzymatic detection approaches that cannot discriminate between maltose and glucose and over existing fluorescence resonance energy transfer (FRET)-based detection methods that are sensitivity limited. In addition, it opens up a new strategy for the development of biosensors to difficult analytes refractory to immunological detection.

Automated data processing and radioassays.

PubMed

Samols, E; Barrows, G H

1978-04-01

Radioassays include (1) radioimmunoassays, (2) competitive protein-binding assays based on competition for limited antibody or specific binding protein, (3) immunoradiometric assay, based on competition for excess labeled antibody, and (4) radioreceptor assays. Most mathematical models describing the relationship between labeled ligand binding and unlabeled ligand concentration have been based on the law of mass action or the isotope dilution principle. These models provide useful data reduction programs, but are theoretically unfactory because competitive radioassay usually is not based on classical dilution principles, labeled and unlabeled ligand do not have to be identical, antibodies (or receptors) are frequently heterogenous, equilibrium usually is not reached, and there is probably steric and cooperative influence on binding. An alternative, more flexible mathematical model based on the probability or binding collisions being restricted by the surface area of reactive divalent sites on antibody and on univalent antigen has been derived. Application of these models to automated data reduction allows standard curves to be fitted by a mathematical expression, and unknown values are calculated from binding data. The vitrues and pitfalls are presented of point-to-point data reduction, linear transformations, and curvilinear fitting approaches. A third-order polynomial using the square root of concentration closely approximates the mathematical model based on probability, and in our experience this method provides the most acceptable results with all varieties of radioassays. With this curvilinear system, linear point connection should be used between the zero standard and the beginning of significant dose response, and also towards saturation. The importance is stressed of limiting the range of reported automated assay results to that portion of the standard curve that delivers optimal sensitivity. Published methods for automated data reduction of Scatchard plots for radioreceptor assay are limited by calculation of a single mean K value. The quality of the input data is generally the limiting factor in achieving good precision with automated as it is with manual data reduction. The major advantages of computerized curve fitting include: (1) handling large amounts of data rapidly and without computational error; (2) providing useful quality-control data; (3) indicating within-batch variance of the test results; (4) providing ongoing quality-control charts and between assay variance.

p-( sup 125 I)iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

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

Gerhardt, M.A.; Wade, S.M.; Neubig, R.R.

1990-08-01

The binding properties of p-(125I)iodoclonidine (( 125I)PIC) to human platelet membranes and the functional characteristics of PIC are reported. (125I)PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific (125I)PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. (125I)PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist (3H) bromoxidine (UK14,304), which representedmore » approximately 40% of the sites bound by the antagonist (3H)yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of (125I)PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for (125I)PIC binding was stereoselective. Competition for (3H)bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for (3H)yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. (125I)PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM.« less

Development of a Competitive Binding Assay System with Recombinant Estrogen Receptors from Multiple Species

EPA Science Inventory

ABSTRACT In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human (hERα), quai...

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

PubMed Central

Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

2017-01-01

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. DOI: http://dx.doi.org/10.7554/eLife.23326.001 PMID:28425915

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos.

PubMed

Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

2017-04-20

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin TransporterⓈ

PubMed Central

Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H.; Ecker, Gerhard F.; Freissmuth, Michael; Sucic, Sonja

2015-01-01

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

The effects of pargyline and 2-phenylethylamine on D1-like dopamine receptor binding.

PubMed

Berry, Mark D

2011-07-01

2-Phenylethylamine (PE) potentiates neuronal responses to dopamine by an unknown post-synaptic mechanism. Here, whether PE modifies D1-like receptor binding was examined. An unexpected effect of the monoamine oxidase inhibitor pargyline was observed, which did not involve competition for ligand binding. PE did not affect ligand binding in the presence or absence of pargyline. It is concluded that the effect of pargyline does not involve elevation of endogenous PE, and PE effects on dopaminergic neurotransmission are not due to altered D1-like receptor binding.

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

Inhibition of /sup 3/H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

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

Aharony, D.; Falcone, R.C.; Krell, R.D.

1987-12-01

The specific binding of (/sup 3/H)5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ((/sup 3/H)LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. (/sup 3/H)LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound (/sup 3/H)LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2more » less than 5 min) and full dissociation. Equilibrium saturation analysis of (/sup 3/H)LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of (/sup 3/H)LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.« less

Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads.

PubMed

Frégeau, Chantal J; De Moors, Anick

2012-09-01

The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain.

PubMed Central

Stevenson, S C; Rollence, M; White, B; Weaver, L; McClelland, A

1995-01-01

The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber head domain with novel ligands may permit adenovirus vectors with new receptor specificities which could be useful for targeted gene delivery in vivo to be engineered. PMID:7707507

Competitive binding experiments can reduce the false positive results of affinity-based ultrafiltration-HPLC: A case study for identification of potent xanthine oxidase inhibitors from Perilla frutescens extract.

PubMed

Wang, Zhiqiang; Kwon, Shin Hwa; Hwang, Seung Hwan; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

2017-03-24

The purpose of this study was to assess the possibility of using competitive binding experiments with ultrafiltration-HPLC analysis to identify potent xanthine oxidase (XO) inhibitors from the Perilla frutescens extract as an attempt to reduce the number of false positive results. To isolate the enzyme-ligand complex from unbound compounds, the P. frutescens extract was either incubated in the absence of XO, in the presence of XO, or with the active site blocked XO before the ultrafiltration was performed. Allopurinaol was used as the XO active site blocker. The unbound compounds were subjected to HPLC analysis. The degree of total binding (TBD) and degree of specific binding (SBD) of each compound were calculated using the peak areas. TBD represents the binding affinities of compounds from the P. frutescens extract for the XO binding site. SBD represents the XO competitive binding between allopurinol and ligands from the extract samples. Two criteria were applied to select putative targets that could help avoid false positives. These include TBD>30% and SBD>10%. Using that approach, kaempferol-3-O-rutinoside, rosmarinic acid, methyl-rosmarinic acid, apigenin, and 4',5,7-trimethoxyflavone were identified, from total 11 compounds, as potent XO inhibitors. Finally, apigenin, 4',5,7-trimethoxyflavone, and luteolin were XO inhibitors verified through an XO inhibition assay and structural simulation of the complex. These results showed that the newly developed strategy has the advantage that the number of targets identified via ultrafiltration-HPLC can be narrowed from many false positives. However, not all false positives can be eliminated with this approach. Some potent inhibitors might also be excluded with the use of this method. The limitations of this method are also discussed herein. Copyright © 2017 Elsevier B.V. All rights reserved.

Harmaline competitively inhibits [3H]MK-801 binding to the NMDA receptor in rabbit brain.

PubMed

Du, W; Aloyo, V J; Harvey, J A

1997-10-03

Harmaline, a beta-carboline derivative, is known to produce tremor through a direct activation of cells in the inferior olive. However, the receptor(s) through which harmaline acts remains unknown. It was recently reported that the tremorogenic actions of harmaline could be blocked by the noncompetitive NMDA channel blocker, MK-801. This study examined whether the blockade of harmaline's action, in the rabbit, by MK-801 was due to a pharmacological antagonism at the MK-801 binding site. This was accomplished by measurement of [3H]MK-801 binding in membrane fractions derived from tissue containing the inferior olivary nucleus and from cerebral cortex. Harmaline completely displaced saturable [3H]MK-801 binding in both the inferior olive and cortex with apparent IC50 values of 60 and 170 microM, respectively. These IC50 values are consistent with the high doses of harmaline required to produce tremor, e.g., 10-30 mg/kg. Non-linear curve fitting analysis of [3H]MK-801 saturation experiments indicated that [3H]MK-801 bound to a single site and that harmaline's displacement of [3H]MK-801 binding to the NMDA receptor was competitive as indicated by a shift in Kd but not in Bmax. In addition, a Schild plot gave a slope that was not significantly different from 1 indicating that harmaline was producing a displacement of [3H]MK-801 from its binding site within the NMDA cation channel and not through an action at the glutamate or other allosteric sites on the NMDA receptor. These findings provide in vitro evidence that the competitive blockade of harmaline-induced tremor by MK-801 occurs within the calcium channel coupled to the NMDA receptor. Our hypothesis is that harmaline produces tremor by acting as an inverse agonist at the MK-801 binding site and thus opening the cation channel.

Effect of buffer at nanoscale molecular recognition interfaces - electrostatic binding of biological polyanions.

PubMed

Rodrigo, Ana C; Laurini, Erik; Vieira, Vânia M P; Pricl, Sabrina; Smith, David K

2017-10-19

We investigate the impact of an over-looked component on molecular recognition in water-buffer. The binding of a cationic dye to biological polyanion heparin is shown by isothermal calorimetry to depend on buffer (Tris-HCl > HEPES > PBS). The heparin binding of self-assembled multivalent (SAMul) cationic micelles is even more buffer dependent. Multivalent electrostatic molecular recognition is buffer dependent as a result of competitive interactions between the cationic binding interface and anions present in the buffer.

Sodium and potassium competition in potassium-selective and non-selective channels

NASA Astrophysics Data System (ADS)

Sauer, David B.; Zeng, Weizhong; Canty, John; Lam, Yeeling; Jiang, Youxing

2013-11-01

Potassium channels selectively conduct K+, primarily to the exclusion of Na+, despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na+ and K+ binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K+ occupancy within the filter upon Na+ competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K+-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K+ sites. A double-barrier mechanism is proposed to explain K+ channel selectivity at low K+ concentrations.

Verifying the competition between haloperidol and biperiden in serum albumin through a model based on spectrofluorimetry

NASA Astrophysics Data System (ADS)

Muniz da Silva Fragoso, Viviane; Patrícia de Morais e Coura, Carla; Paulino, Erica Tex; Valdez, Ethel Celene Narvaez; Silva, Dilson; Cortez, Celia Martins

2017-11-01

The aim of this work was to apply mathematical-computational modeling to study the interactions of haloperidol (HLP) and biperiden (BPD) with human (HSA) and bovine (BSA) serum albumin in order to verify the competition of these drugs for binding sites in HSA, using intrinsic tryptophan fluorescence quenching data. The association constants estimated for HPD-HSA was 2.17(±0.05) × 107 M-1, BPD-HSA was 2.01(±0.03) × 108 M-1 at 37 °C. Results have shown that drugs do not compete for the same binding sites in albumin.

A competitive binding model predicts the response of mammalian olfactory receptors to mixtures

NASA Astrophysics Data System (ADS)

Singh, Vijay; Murphy, Nicolle; Mainland, Joel; Balasubramanian, Vijay

Most natural odors are complex mixtures of many odorants, but due to the large number of possible mixtures only a small fraction can be studied experimentally. To get a realistic understanding of the olfactory system we need methods to predict responses to complex mixtures from single odorant responses. Focusing on mammalian olfactory receptors (ORs in mouse and human), we propose a simple biophysical model for odor-receptor interactions where only one odor molecule can bind to a receptor at a time. The resulting competition for occupancy of the receptor accounts for the experimentally observed nonlinear mixture responses. We first fit a dose-response relationship to individual odor responses and then use those parameters in a competitive binding model to predict mixture responses. With no additional parameters, the model predicts responses of 15 (of 18 tested) receptors to within 10 - 30 % of the observed values, for mixtures with 2, 3 and 12 odorants chosen from a panel of 30. Extensions of our basic model with odorant interactions lead to additional nonlinearities observed in mixture response like suppression, cooperativity, and overshadowing. Our model provides a systematic framework for characterizing and parameterizing such mixing nonlinearities from mixture response data.

Hapten syntheses and antibody generation for the development of a polybrominated flame retardant ELISA.

PubMed

Shelver, Weilin L; Keum, Young-Soo; Kim, Hee-Joo; Rutherford, Drew; Hakk, Heldur H; Bergman, Ake; Li, Qing X

2005-05-18

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are increasingly an environmental concern. Several antibodies were developed for the polybrominated diphenyl ether flame retardant BDE-47 (1), often found in the highest concentration in human milk, plasma, and adipose tissue. Four haptens with different bromine and linker substitution patterns were synthesized and utilized to generate five polyclonal antibodies from goats and two polyclonal antibodies from rabbits. Competition was assessed using four different coating antigens for all seven antibodies. The coating antigen showed marked effects on competition. When the same hapten was used for antibody and the coating antigen less competition was observed. The effect of BDE structure on competition was evaluated by using BDE-47 (1), BDE-99 (2), BDE-100 (3), BDE-153 (4), and BDE-183 (5). None of the compounds showed high competition with antibody I-KLH, presumably because steric hindrance prevented formation of an efficient binding site. As predicted from structural considerations, BDE-47 (1) competed well with the remaining antibodies, whereas BDE-100 (3) competed well with only II-KLH. The remaining congeners (BDE-99 (2), BDE-153 (4), and BDE-183 (5)) contain bromines that cannot be positioned in binding sites and thus cross-react poorly. The competition study demonstrated that a bromine substitution on the congener could occupy a position analogous to the linker's position.

Relative Chemical Binding Affinities for Trout and Human Estrogen Receptor Using Different Competitive Binding Assays

EPA Science Inventory

Rainbow trout-based assays for estrogenicity are currently being used for development of predictive models based upon quantitative structure activity relationships. A predictive model based on a single species raises the question of whether this information is valid for other spe...

CONSTANTS FOR MERCURY BINDING BY DISSOLVED ORGANIC MATTER ISOLATES FROM THE FLORIDA EVERGLADES. (R827653)

EPA Science Inventory

Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand appro...

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors.

PubMed

Nongonierma, Alice B; Mooney, Catherine; Shields, Denis C; FitzGerald, Richard J

2014-07-01

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure-activity relationship modeling, rather than on docking, in computationally selecting peptides for screening. Copyright © 2014 Elsevier Inc. All rights reserved.

The investigation of the binding of 6-mercaptopurine to site I on human serum albumin.

PubMed

Sochacka, Jolanta; Baran, Wojciech

2012-12-01

6-Mercaptopurine (6-MP) is one of a large series of purine analogues which has been found active against human leukemias. The equilibrium dialysis, circular dichroism (CD) and molecular docking were employed to study the binding of 6-MP to human serum albumin (HSA). The binding of 6-MP to HSA in the equilibrium dialysis experiment was detected by measuring the displacement of 6-MP by specific markers for site I on HSA, warfarin (RWF), phenylbutazone (PhB) and n-butyl p-aminobenzoate (ABE). It was shown, according to CD data, that binding of 6-MP to HSA leads to alteration of HSA secondary structure. Based on the findings from displacement experiment and molecular docking simulation it was found that 6-MP was located within binding cavity of subdomain IIA and the space occupied by site markers overlapped with that of 6-MP. Displacement of 6-MP by the RWF or PhB was not up the level expected for a competitive mechanism, therefore displacement of 6-MP was rather by non-cooperative than that the direct competition. Instead, in case of the interaction between ABE and 6-MP, when the little enhancement of the binding of ABE by 6-MP was found, the interaction could be via a positively cooperative mechanism.

A Sphingosine 1-phosphate receptor 2 selective allosteric agonist

PubMed Central

Satsu, Hideo; Schaeffer, Marie-Therese; Guerrero, Miguel; Saldana, Adrian; Eberhart, Christina; Hodder, Peter; Cayanan, Charmagne; Schürer, Stephan; Bhhatarai, Barun; Roberts, Ed; Rosen, Hugh; Brown, Steven J.

2013-01-01

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. PMID:23849205

In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125I Radiolabeling

PubMed Central

Chakroun, Maissa

2014-01-01

Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the 125I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops. PMID:25002420

Interaction of phenylbutazone and colchicine in binding to serum albumin in rheumatoid therapy: 1H NMR study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

2009-09-01

The monitoring of drug concentration in blood serum is necessary in multi-drug therapy. Mechanism of drug binding with serum albumin (SA) is one of the most important factors which determine drug concentration and its transport to the destination tissues. In rheumatoid diseases drugs which can induce various adverse effects are commonly used in combination therapy. Such proceeding may result in the enhancement of those side effects due to drug interaction. Interaction of phenylbutazone and colchicine in binding to serum albumin and competition between them in gout has been studied by proton nuclear magnetic resonance ( 1H NMR) technique. The aim of the study was to determine the low affinity binding sites, the strength and kind of interaction between serum albumin and drugs used in combination therapy. The study of competition between phenylbutazone and colchicine in binding to serum albumin points to the change of their affinity to serum albumin in the ternary systems. This should be taken into account in multi-drug therapy. This work is a subsequent part of the spectroscopic study on Phe-COL-SA interactions [A. Sułkowska, et al., J. Mol. Struct. 881 (2008) 97-106].

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

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

Middleton, R.E.; Cohen, J.B.

1991-07-16

The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with highmore » affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.« less

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

PubMed

Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

2007-08-15

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

The action of stress hormones on the structure and function of erythrocyte membrane.

PubMed

Mokrushnikov, Pavel V; Panin, Lev E; Zaitsev, Boris N

2015-07-01

The action of a mixture of hormones (cortisol and adrenaline) on erythrocyte membrane during their binding was investigated. Changes in the membrane structure were elucidated by atomic force microscopy; microviscosity of the lipid bilayer and changes in the activity of Na(+),K(+)-ATPase at different concentrations of the hormones in erythrocyte suspension were estimated by the fluorescence method. Cortisol and adrenaline were shown to compete for the binding sites. A hormone that managed to bind nonspecifically to the membrane hindered the binding of another hormone. In a mixture of these hormones, cortisol won a competition for the binding sites; therewith, microviscosity of the membranes increased by 25%, which corresponds to a change in microviscosity produced by the action of cortisol alone. The competitive relationships affected also the Na(+),K(+)-ATPase activity, which was indicated by appearance of the second maximum of enzyme activity. It is assumed that an increase in microviscosity of erythrocyte membrane first raises the Na(+),K(+)-ATPase activity due to a growth of the maximum energy of membrane phonons, and then decreases the activity due to hindering of conformational transitions in the enzyme molecule.

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

DOE PAGES

Ng, Simon; Lin, Edith; Kitov, Pavel I.; ...

2015-04-10

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

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

Ng, Simon; Lin, Edith; Kitov, Pavel I.

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Copper speciation and binding by organic matter in copper-contaminated streamwater

USGS Publications Warehouse

Breault, R.F.; Colman, J.A.; Aiken, G.R.; McKnight, D.

1996-01-01

Fulvic acid binding sites (1.3-70 ??M) and EDTA (0.0017-0.18 ??M) accounted for organically bound Cu in seven stream samples measured by potentiometric titration. Cu was 84-99% organically bound in filtrates with 200 nM total Cu. Binding of Cu by EDTA was limited by competition from other trace metals. Water hardness was inversely related to properties of dissolved organic carbon (DOC) that enhance fulvic acid binding: DOC concentration, percentage of DOC that is fulvic acid, and binding sites per fulvic acid carbon. Dissolved trace metals, stabilized by organic binding, occurred at increased concentration in soft water as compared to hard water.

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

Fedynyshyn, J.P.

The opioid binding characteristics of the rat (PAG) and the signal transduction mechanisms of the opioid receptors were examined with in vitro radioligand binding, GTPase, adenylyl cyclase, and inositol phosphate assays. The nonselective ligand {sup 3}H-ethylketocyclazocine (EKC), the {mu} and {delta} selective ligand {sup 3}H-(D-Ala{sup 2}, D-Leu{sup 5}) enkephalin (DADLE), the {mu} selective ligand {sup 3}H-(D-Ala{sup 2}, N-methyl Phe{sup 4}, Glyol{sup 5}) enkephalin (DAGO), and the {delta} selective ligand {sup 3}H-(D-Pen{sup 2}, D-Pen{sup 5}) enkephalin (DPDPE) were separately used as tracer ligands to label opioid binding sites in rat PAG enriched P{sub 2} membrane in competition with unlabeled DADLE, DAGO,more » DPDPE, or the {kappa} selective ligand trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide, methane sulfonate, hydrate (U50, 488H). Only {mu} selective high affinity opioid binding was observed. No high affinity {delta} or {kappa} selective binding was detected. {sup 3}H-DAGO was used as a tracer ligand to label {mu} selective high affinity opioid binding sites in PAG enriched P{sub 2} membrane in competition with unlabeled {beta}-endorphin, dynorphin A (1-17), BAM-18, methionine enkephalin, dynorphin A (1-8), and leucine enkephalin. Of these endogenous opioid peptides only those with previously reported high affinity {mu} type opioid binding activity competed with {sup 3}H-DAGO for binding sites in rat PAG enriched P{sub 2} membrane with affinities similar to that of unlabeled DAGO.« less

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase

NASA Astrophysics Data System (ADS)

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-01

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2‧,3‧-O-(2,4,6-trinitrophenyl)adenosine 5‧-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

PubMed

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-05

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase. Copyright © 2016 Elsevier B.V. All rights reserved.

Gallium as a Therapeutic Agent: A Thermodynamic Evaluation of the Competition between Ga(3+) and Fe(3+) Ions in Metalloproteins.

PubMed

Nikolova, Valia; Angelova, Silvia; Markova, Nikoleta; Dudev, Todor

2016-03-10

Gallium has been employed (in the form of soluble salts) to fight various forms of cancer, infectious, and inflammatory diseases. The rationale behind this lies in the ability of Ga(3+) cation to mimic closely in appearance the native ferric ion, Fe(3+), thus interfering with the biological processes requiring ferric cofactors. However, Ga(3+) ion cannot participate in redox reactions and, when substituting for the "native" Fe(3+) ion in the enzyme active site, renders it inactive. Although a significant body of information on the Ga(3+)-Fe(3+) competition in biological systems has been accumulated, the intimate mechanism of the process is still not well understood and several questions remain: What are the basic physical principles governing the competition between the two trivalent cations in proteins? What type of metal centers are the most likely targets for gallium therapy? To what extent are the Fe(3+)-binding sites in the key enzyme ribonucleotide reductase vulnerable to Ga(3+) substitution? Here, we address these questions by studying the competition between Ga(3+) and Fe(3+) ions in model metal binding sites of various compositions and charge states. The results obtained are in line with available experimental data and shed light on the intimate mechanism of the Ga(3+)/Fe(3+) selectivity in various model metal binding sites and biological systems such as serum transferrin and ribonucleotide reductase.

Prostate cell membrane chromatography-liquid chromatography-mass spectrometry for screening of active constituents from Uncaria rhynchophylla.

PubMed

He, Jianyu; Han, Shengli; Yang, Fangfang; Zhou, Nan; Wang, Sicen

2013-01-01

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

Effects of Competition and Mode of Fire on Physiological Responses, Psychological Stress Reactions, and Shooting Performance

DTIC Science & Technology

1991-07-01

Alterations in serum cortisol and its binding characteristics in anorexia nervosa . J.urnal o•f Q n1raI £ndcrinaloag and M -bo.liam, Ui, 406-411. Clement...91 43. Comparison of Mean Pre-stress MAACL-R Depression Scores for SS Competition and SS Control Groups on Record-Fire Day With Those for Subjects in...independent non-stress contzol condition) .................. 92 5 44. Comparison of Mean Post-stress MAACL-R Depression Scores for SS Competition and SS

Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

PubMed

Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

2007-11-01

We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment

PubMed Central

Lee-Sayer, Sally S. M.; Dougan, Meghan N.; Cooper, Jesse; Sanderson, Leslie; Dosanjh, Manisha; Maxwell, Christopher A.

2018-01-01

CD44 is a widely expressed cell adhesion molecule that binds to the extracellular matrix component, hyaluronan. However, this interaction is not constitutive in most immune cells at steady state, as the ability of CD44 to engage hyaluronan is highly regulated. While activated T cells and macrophages gain the ability to bind hyaluronan by CD44, the status in other immune cells is less studied. Here we found a percentage of murine eosinophils, natural killer and natural killer T cells were capable of interacting with hyaluronan at steady state. To further investigate the consequences of hyaluronan binding by CD44 in the hematopoietic system, point mutations of CD44 that either cannot bind hyaluronan (LOF-CD44) or have an increased affinity for hyaluronan (GOF-CD44) were expressed in CD44-deficient bone marrow. Competitive bone marrow reconstitution of irradiated mice revealed an early preference for GOF-CD44 over WT-CD44 expressing cells, and for WT-CD44 over LOF-CD44 expressing cells, in the hematopoietic progenitor cell compartment. The advantage of the hyaluronan-binding cells was observed in the hematopoietic stem and progenitor populations, and was maintained throughout the immune system. Hematopoietic stem cells bound minimal hyaluronan at steady state, and this was increased when the cells were induced to proliferate whereas multipotent progenitors had an increased ability to bind hyaluronan at steady state. In vitro, the addition of hyaluronan promoted their proliferation. Thus, proliferating hematopoietic progenitors bind hyaluronan, and hyaluronan binding cells have a striking competitive advantage in bone marrow engraftment. PMID:29684048

Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters

PubMed Central

Hirayama, Bruce A; Díez-Sampedro, Ana; Wright, Ernest M

2001-01-01

Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7×12Å. Important hydrogen bond interactions occur at five positions bordering this surface. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site ∼8Å from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors. PMID:11588102

An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates*

PubMed Central

Fredslund, Folmer; Vujičić Žagar, Andreja; Andersen, Thomas Lars; Svensson, Birte; Slotboom, Dirk Jan

2016-01-01

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria. PMID:27502277

A Computational Model for Docking of Noncompetitive Neuraminidase Inhibitors and Probing their Binding Interactions with Neuraminidase of Influenza Virus H5N1.

PubMed

Chintakrindi, Anand S; Martis, Elvis A F; Gohil, Devanshi J; Kothari, Sweta T; Chowdhary, Abhay S; Coutinho, Evans C; Kanyalkar, Meena A

2016-01-01

With cases of emergence of drug resistance to the current competitive inhibitors of neuraminidase (NA) such as oseltamivir and zanamavir, there is a present need for an alternative approach in the treatment of avian influenza. With this in view, some flavones and chalcones were designed based on quercetin, the most active naturally occurring noncompetitive inhibitor. We attempt to understand the binding of quercetin to H5N1-NA, and synthetic analogs of quercetin namely flavones and its precursors the chalcones using computational tools. Molecular docking was done using Libdock. Molecular dynamics (MD) simulations were performed using Amber14. We synthesized the two compounds; their structures were confirmed by infrared spectroscopy, 1H-NMR, and mass spectrometry. These molecules were then tested for H5N1-NA inhibition and kinetics of inhibition. Molecular docking studies yielded two compounds i.e., 4'-methoxyflavone and 2'-hydroxy-4-methoxychalcone, as promising leads which identified them as binders of the 150-cavity of NA. Furthermore, MD simulation studies revealed that quercetin and the two compounds bind and hold the 150 loop in its open conformation, which ultimately perturbs the binding of sialic acid in the catalytic site. Estimation of the free energy of binding by MM-PBSA portrays quercetin as more potent than chalcone and flavone. These molecules were then determined as non-competitive inhibitors from the Lineweaver-Burk plots rendered from the enzyme kinetic studies. We conclude that non-competitive type of inhibition, as shown in this study, can serve as an effective method to block NA and evade the currently seen drug resistance.

Dialysis, Albumin Binding, and Competitive Binding: A Laboratory Lesson Relating Three Chemical Concepts to Healthcare

ERIC Educational Resources Information Center

Domingo, Jennifer P.; Abualia, Mohammed; Barragan, Diana; Schroeder, Lianne; Wink, Donald J.; King, Maripat; Clark, Ginevra A.

2017-01-01

Introductory Chemistry laboratories must go beyond "cookbook" methods to illustrate how chemistry concepts apply to complex, real-world problems. In our case, we are preparing students to use their chemistry knowledge in the healthcare profession. The experiment described here explicitly models three important chemical concepts: dialysis…

Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening

EPA Science Inventory

The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across differ...

Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays, oral

EPA Science Inventory

The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

PubMed

Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

2017-01-27

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Competition between Anion Binding and Dimerization Modulates Staphylococcus aureus Phosphatidylinositol-specific Phospholipase C Enzymatic Activity*

PubMed Central

Cheng, Jiongjia; Goldstein, Rebecca; Stec, Boguslaw; Gershenson, Anne; Roberts, Mary F.

2012-01-01

Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) is a secreted virulence factor for this pathogenic bacterium. A novel crystal structure shows that this PI-PLC can form a dimer via helix B, a structural feature present in all secreted, bacterial PI-PLCs that is important for membrane binding. Despite the small size of this interface, it is critical for optimal enzyme activity. Kinetic evidence, increased enzyme specific activity with increasing enzyme concentration, supports a mechanism where the PI-PLC dimerization is enhanced in membranes containing phosphatidylcholine (PC). Mutagenesis of key residues confirm that the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the S. aureus enzyme that stabilizes monomeric protein. Despite its structural and sequence similarity to PI-PLCs from other Gram-positive pathogenic bacteria, S. aureus PI-PLC appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane. PMID:23038258

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

PubMed

Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

2018-02-01

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

PubMed Central

Jakubík, J; Janíčková, H; El-Fakahany, EE; Doležal, V

2011-01-01

BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [35S]GTPγS and [3H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [3H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPγS binding. PMID:20958290

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

PubMed

Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

2011-03-01

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-09-19

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-07-11

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

Antagonistic Rgg regulators mediate quorum sensing via competitive DNA binding in Streptococcus pyogenes.

PubMed

Lasarre, Breah; Aggarwal, Chaitanya; Federle, Michael J

2013-01-02

Recent studies have established the fact that multiple members of the Rgg family of transcriptional regulators serve as key components of quorum sensing (QS) pathways that utilize peptides as intercellular signaling molecules. We previously described a novel QS system in Streptococcus pyogenes which utilizes two Rgg-family regulators (Rgg2 and Rgg3) that respond to neighboring signaling peptides (SHP2 and SHP3) to control gene expression and biofilm formation. We have shown that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes, and that SHPs function to activate the QS system. The mechanisms by which Rgg proteins regulate both QS-dependent and QS-independent processes remain poorly defined; thus, we sought to further elucidate how Rgg2 and Rgg3 mediate gene regulation. Here we provide evidence that S. pyogenes employs a unique mechanism of direct competition between the antagonistic, peptide-responsive proteins Rgg2 and Rgg3 for binding at target promoters. The highly conserved, shared binding sites for Rgg2 and Rgg3 are located proximal to the -35 nucleotide in the target promoters, and the direct competition between the two regulators results in concentration-dependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP. These results suggest that exclusionary binding of target promoters by Rgg3 may prevent Rgg2 binding under SHP-limiting conditions, thereby preventing premature induction of the quorum sensing circuit. Rgg-family transcriptional regulators are widespread among low-G+C Gram-positive bacteria and in many cases contribute to bacterial physiology and virulence. Only recently was it discovered that several Rgg proteins function in cell-to-cell communication (quorum sensing [QS]) via direct interaction with signaling peptides. The mechanism(s) by which Rgg proteins mediate regulation is poorly understood, and further insight into Rgg function is anticipated to be of great importance for the understanding of both regulatory-network architecture and intercellular communication in Rgg-containing species. The results of this study on the Rgg2/3 QS circuit of S. pyogenes demonstrate that DNA binding of target promoters by the activator Rgg2 is directly inhibited by competitive binding by the repressor Rgg3, thereby preventing transcriptional activation of the target genes and premature induction of the QS circuit. This is a unique regulatory mechanism among Rgg proteins and other peptide-responsive QS regulators.

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

PubMed

Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-10-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor

PubMed Central

Bertini, R; Barcelos, LS; Beccari, AR; Cavalieri, B; Moriconi, A; Bizzarri, C; Di Benedetto, P; Di Giacinto, C; Gloaguen, I; Galliera, E; Corsi, MM; Russo, RC; Andrade, SP; Cesta, MC; Nano, G; Aramini, A; Cutrin, JC; Locati, M; Allegretti, M; Teixeira, MM

2012-01-01

BACKGROUND AND PURPOSE DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [35S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys99 on CXCR1 and the non-conserved residue Asp293 on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases. PMID:21718305

Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host

PubMed Central

Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-01-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants.

PubMed Central

Small, J M; Mitchell, T G

1986-01-01

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with 125I, and used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal. PMID:3536747

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants

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

Small, J.M.; Mitchell, T.G.

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with /sup 125/I, andmore » used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal.« less

Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

PubMed Central

Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael

2016-01-01

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. PMID:26976641

Characterization of 12 GnRH peptide agonists - a kinetic perspective.

PubMed

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak-Reppel, Katrin; Fernández-Montalván, Amaury E; IJzerman, Adriaan P; Heitman, Laura H

2016-01-01

Drug-target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin-releasing hormone (GnRH) receptor for the treatment of hormone-dependent diseases. Surprisingly, the kinetic receptor-binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor-binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. A novel radioligand-binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [(125) I]-triptorelin. In addition to radioligand-binding studies, a homogeneous time-resolved FRET Tag-lite™ method was developed as an alternative assay for the same purpose. Two novel competition association assays were successfully developed and applied to determine the kinetic receptor-binding characteristics of 12 high-affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. © 2015 The British Pharmacological Society.

Characterization of 12 GnRH peptide agonists – a kinetic perspective

PubMed Central

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak‐Reppel, Katrin; Fernández‐Montalván, Amaury E.; IJzerman, Adriaan P.

2015-01-01

Background and Purpose Drug‐target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin‐releasing hormone (GnRH) receptor for the treatment of hormone‐dependent diseases. Surprisingly, the kinetic receptor‐binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor‐binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. Experimental Approach A novel radioligand‐binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [125I]‐triptorelin. In addition to radioligand‐binding studies, a homogeneous time‐resolved FRET Tag‐lite™ method was developed as an alternative assay for the same purpose. Key Results Two novel competition association assays were successfully developed and applied to determine the kinetic receptor‐binding characteristics of 12 high‐affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Conclusions and Implications Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. PMID:26398856

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues.

PubMed

Skyrianou, Kalliopi C; Psycharis, Vassilis; Raptopoulou, Catherine P; Kessissoglou, Dimitris P; Psomas, George

2011-01-01

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2'-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid. Copyright © 2010 Elsevier Inc. All rights reserved.

International Validation of Two Human Recombinant Estrogen ...

EPA Pesticide Factsheets

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay using a ligand-binding domain of the human ER. Twenty three compounds were tested in 6 laboratories for the FW assay and 5 for the CERJ assay, which included three controls (used with every run), 9 uncoded, and 14 coded chemicals across 3 subtasks. The overall goal of this validation study was to demonstrate the ability of each of the two assays to reliably classify the test chemicals as binders or non-binders. Laboratories had little trouble with the ER binders that produced a full binding curve when using either the CERI or FW assays. As is typical with all ER competitive binding assays, the weak binders proved to be more challenging. However, overall results from both the FW and CERI assays were consistent and in agreement with expected classifications regardless of the form of the hrER (i.e., full length ER versus an ER ligand binding domain) or the subtle differences in the protocols for conducting each assay. The reproducibility and accuracy for classification of chemicals as potential ER binders and non- binders using the FW and CERI hrER binding assays were comparable to that of the U.S.EPA’s existing ER binding test guideline OPPTS 890.1250, while providing an improved, highe

Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques.

PubMed

Yang, Hongqin; Liu, Jiuyang; Huang, Yanmei; Gao, Rui; Tang, Bin; Li, Shanshan; He, Jiawei; Li, Hui

2017-03-30

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 10 5  M -1 ) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA.

Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques

PubMed Central

Yang, Hongqin; Liu, Jiuyang; Huang, Yanmei; Gao, Rui; Tang, Bin; Li, Shanshan; He, Jiawei; Li, Hui

2017-01-01

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 105 M−1) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA. PMID:28358124

Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker ( sup 3 H)GBR-12935

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

Kalow, W.; Tyndale, R.F.; Niznik, H.B.

1990-02-26

P450IID6 (human sparteine monooxygenase) metabolizes many drugs including neuroleptics, antidepressants, and beta-blockers. The P450IID6 exists in human, bovine, rat and canine brains, but in very low quantities causing methodological difficulties in its assessment. Work with ({sup 3}H)GBR-12935; 1-(2-(diphenylmethoxy) ethyl)-4-(3-phenyl propyl) piperazine has shown that it binds a neuronal/hepatic protein with high affinity ({approximately}7nM) and a rank order of inhibitory potency suggesting that the binding protein is cytochrome P450IID6. The binding was used to predict that d-amphetamine and methamphetamine would interact with P450IID6. Inhibition studies indicated that these compounds were competitive inhibitors of P450IID6. Haloperidol (HAL) and it's metabolite hydroxy-haloperidol (RHAL)more » are both competitive inhibitors of P450IID6 activity and were found to inhibit ({sup 3}H)GBR-12935 binding. K{sub i} values of twelve compounds (known to interact with the DA transporter or P450IID6) for ({sup 3}H)GRB-12935 binding and P450IID6 activity. The techniques are now available for measurements of cytochrome P450IID6 in healthy and diseased brain/liver tissue using radio-receptor binding assay techniques with ({sup 3}H)GBR-12935.« less

Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques

NASA Astrophysics Data System (ADS)

Yang, Hongqin; Liu, Jiuyang; Huang, Yanmei; Gao, Rui; Tang, Bin; Li, Shanshan; He, Jiawei; Li, Hui

2017-03-01

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 105 M-1) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA.

Integrated Summary Report: Validation of Two Binding Assays ...

EPA Pesticide Factsheets

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (hrERα), to identify chemicals that may impact estrogen signaling through binding to the ER. The purpose of the ISR is to support the peer review of the findings obtained during the validation process.The two assays evaluated during this validation process are: The Freyberger-Wilson Assay (FW) using a full length human ER, and The Chemical Evaluation and Research Institute (CERI) Assay using a ligand-binding domain of the human ER.The two assays are mechanistically and functionally similar in that each measures the ability of a test chemical to competitively inhibit binding of [3H]17β-estradiol to the human recombinant ER. The essential elements of the FW and the CERI assays were developed at the laboratories of Bayer Pharma AG, Wuppertal, Germany (Freyberger et al., 2010) and CERI, Tokyo, Japan (Akahori et al., 2008), respectively.The ER competitive binding assay has long been in use, and is a well characterized approach, but historically uses rodent or other animal tissues as a source of the ER. Validation of the FW and CERI assays using human recombinant estrogen receptors ( subtype) will provide an updated alternative for the Agency’s current test guideline (OPPTS 89

Enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin.

PubMed

Gao, Shunxiang; Zheng, Xin; Hu, Bo; Sun, Mingjuan; Wu, Jihong; Jiao, Binghua; Wang, Lianghua

2017-03-15

In this study, we coupled biolayer interferometry (BLI) with competitive binding assay through an enzyme-linked aptamer and developed a real-time, ultra-sensitive, rapid quantitative method for detection of the marine biotoxin palytoxin. Horseradish peroxidase-labeled aptamers were used as biorecognition receptors to competitively bind with palytoxin, which was immobilized on the biosensor surface. The palytoxin: horseradish peroxidase-aptamer complex was then submerged in a 3,3'-diaminobenzidine solution, which resulted in formation of a precipitated polymeric product directly on the biosensor surface and a large change in the optical thickness of the biosensor layer. This change could obviously shift the interference pattern and generate a response profile on the BLI biosensor. The biosensor showed a broad linear range for palytoxin (200-700pg/mL) with a low detection limit (0.04pg/mL). Moreover, the biosensor was applied to the detection of palytoxin in spiked extracts and showed a high degree of selectivity for palytoxin, good reproducibility, and stability. This enzyme-linked, aptamer-based, competitive BLI biosensor offers a promising method for rapid and sensitive detection of palytoxin and other analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

Binding Mode Analyses and Pharmacophore Model Development for Stilbene Derivatives as a Novel and Competitive Class of α-Glucosidase Inhibitors

PubMed Central

Kim, Jun Young; Arooj, Mahreen; Kim, Siu; Hwang, Swan; Kim, Byeong-Woo; Park, Ki Hun; Lee, Keun Woo

2014-01-01

Stilbene urea derivatives as a novel and competitive class of non-glycosidic α-glucosidase inhibitors are effective for the treatment of type II diabetes and obesity. The main purposes of our molecular modeling study are to explore the most suitable binding poses of stilbene derivatives with analyzing the binding affinity differences and finally to develop a pharmacophore model which would represents critical features responsible for α-glucosidase inhibitory activity. Three-dimensional structure of S. cerevisiae α-glucosidase was built by homology modeling method and the structure was used for the molecular docking study to find out the initial binding mode of compound 12, which is the most highly active one. The initial structure was subjected to molecular dynamics (MD) simulations for protein structure adjustment at compound 12-bound state. Based on the adjusted conformation, the more reasonable binding modes of the stilbene urea derivatives were obtained from molecular docking and MD simulations. The binding mode of the derivatives was validated by correlation analysis between experimental Ki value and interaction energy. Our results revealed that the binding modes of the potent inhibitors were engaged with important hydrogen bond, hydrophobic, and π-interactions. With the validated compound 12-bound structure obtained from combining approach of docking and MD simulation, a proper four featured pharmacophore model was generated. It was also validated by comparison of fit values with the Ki values. Thus, these results will be helpful for understanding the relationship between binding mode and bioactivity and for designing better inhibitors from stilbene derivatives. PMID:24465730

SP transcription factor paralogs and DNA-binding sites coevolve and adaptively converge in mammals and birds.

PubMed

Yokoyama, Ken Daigoro; Pollock, David D

2012-01-01

Functional modification of regulatory proteins can affect hundreds of genes throughout the genome, and is therefore thought to be almost universally deleterious. This belief, however, has recently been challenged. A potential example comes from transcription factor SP1, for which statistical evidence indicates that motif preferences were altered in eutherian mammals. Here, we set out to discover possible structural and theoretical explanations, evaluate the role of selection in SP1 evolution, and discover effects on coregulatory proteins. We show that SP1 motif preferences were convergently altered in birds as well as mammals, inducing coevolutionary changes in over 800 regulatory regions. Structural and phylogenic evidence implicates a single causative amino acid replacement at the same SP1 position along both lineages. Furthermore, paralogs SP3 and SP4, which coregulate SP1 target genes through competitive binding to the same sites, have accumulated convergent replacements at the homologous position multiple times during eutherian and bird evolution, presumably to preserve competitive binding. To determine plausibility, we developed and implemented a simple model of transcription factor and binding site coevolution. This model predicts that, in contrast to prevailing beliefs, even small selective benefits per locus can drive concurrent fixation of transcription factor and binding site mutants under a broad range of conditions. Novel binding sites tend to arise de novo, rather than by mutation from ancestral sites, a prediction substantiated by SP1-binding site alignments. Thus, multiple lines of evidence indicate that selection has driven convergent evolution of transcription factors along with their binding sites and coregulatory proteins.

SP Transcription Factor Paralogs and DNA-Binding Sites Coevolve and Adaptively Converge in Mammals and Birds

PubMed Central

Yokoyama, Ken Daigoro; Pollock, David D.

2012-01-01

Functional modification of regulatory proteins can affect hundreds of genes throughout the genome, and is therefore thought to be almost universally deleterious. This belief, however, has recently been challenged. A potential example comes from transcription factor SP1, for which statistical evidence indicates that motif preferences were altered in eutherian mammals. Here, we set out to discover possible structural and theoretical explanations, evaluate the role of selection in SP1 evolution, and discover effects on coregulatory proteins. We show that SP1 motif preferences were convergently altered in birds as well as mammals, inducing coevolutionary changes in over 800 regulatory regions. Structural and phylogenic evidence implicates a single causative amino acid replacement at the same SP1 position along both lineages. Furthermore, paralogs SP3 and SP4, which coregulate SP1 target genes through competitive binding to the same sites, have accumulated convergent replacements at the homologous position multiple times during eutherian and bird evolution, presumably to preserve competitive binding. To determine plausibility, we developed and implemented a simple model of transcription factor and binding site coevolution. This model predicts that, in contrast to prevailing beliefs, even small selective benefits per locus can drive concurrent fixation of transcription factor and binding site mutants under a broad range of conditions. Novel binding sites tend to arise de novo, rather than by mutation from ancestral sites, a prediction substantiated by SP1-binding site alignments. Thus, multiple lines of evidence indicate that selection has driven convergent evolution of transcription factors along with their binding sites and coregulatory proteins. PMID:23019068

The antimalarial drugs quinine, chloroquine and mefloquine are antagonists at 5-HT3 receptors

PubMed Central

Thompson, A J; Lochner, M; Lummis, S C R

2007-01-01

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT3 receptor antagonists. They may therefore act at 5-HT3 receptors. Experimental Approach: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT3A receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site. Key Results: 5-HT3 responses were blocked with IC 50 values of 13.4 μM, 11.8 μM and 9.36 μM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA 2 values of 4.92 (K B=12.0 μM) and 4.97 (K B=16.4 μM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [3H]granisetron with K i values of 15.0, 24.2 and 35.7 μ M. Docking of quinine into a homology model of the 5-HT3 receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226. Conclusions and Implications: This study shows that quinine and chloroquine competitively inhibit 5-HT3 receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT3 receptor antagonists. PMID:17502851

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

Competition between Hydrogen Bonding and Proton Transfer during Specific Anion Recognition by Dihomooxacalix[4]arene Bidentate Ureas.

PubMed

Martínez-González, Eduardo; González, Felipe J; Ascenso, José R; Marcos, Paula M; Frontana, Carlos

2016-08-05

Competition between hydrogen bonding and proton transfer reactions was studied for systems composed of electrogenerated dianionic species from dinitrobenzene isomers and substituted dihomooxacalix[4]arene bidentate urea derivatives. To analyze this competition, a second-order ErCrCi mechanism was considered where the binding process is succeeded by proton transfer and the voltammetric responses depend on two dimensionless parameters: the first related to hydrogen bonding reactions, and the second one to proton transfer processes. Experimental results indicated that, upon an increase in the concentration of phenyl-substituted dihomooxacalix[4]arene bidentate urea, voltammetric responses evolve from diffusion-controlled waves (where the binding process is at chemical equilibrium) into irreversible kinetic responses associated with proton transfer. In particular, the 1,3-dinitrobenzene isomer showed a higher proton transfer rate constant (∼25 M(-1) s(-1)) compared to that of the 1,2-dinitrobenzene (∼5 M(-1) s(-1)), whereas the 1,4-dinitrobenzene did not show any proton transfer effect in the experimental conditions employed.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET.

PubMed

Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2013-02-01

The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.

Uncertainty analysis of the nonideal competitive adsorption-donnan model: effects of dissolved organic matter variability on predicted metal speciation in soil solution.

PubMed

Groenenberg, Jan E; Koopmans, Gerwin F; Comans, Rob N J

2010-02-15

Ion binding models such as the nonideal competitive adsorption-Donnan model (NICA-Donnan) and model VI successfully describe laboratory data of proton and metal binding to purified humic substances (HS). In this study model performance was tested in more complex natural systems. The speciation predicted with the NICA-Donnan model and the associated uncertainty were compared with independent measurements in soil solution extracts, including the free metal ion activity and fulvic (FA) and humic acid (HA) fractions of dissolved organic matter (DOM). Potentially important sources of uncertainty are the DOM composition and the variation in binding properties of HS. HS fractions of DOM in soil solution extracts varied between 14 and 63% and consisted mainly of FA. Moreover, binding parameters optimized for individual FA samples show substantial variation. Monte Carlo simulations show that uncertainties in predicted metal speciation, for metals with a high affinity for FA (Cu, Pb), are largely due to the natural variation in binding properties (i.e., the affinity) of FA. Predictions for metals with a lower affinity (Cd) are more prone to uncertainties in the fraction FA in DOM and the maximum site density (i.e., the capacity) of the FA. Based on these findings, suggestions are provided to reduce uncertainties in model predictions.

Convection, diffusion and reaction in a surface-based biosensor: modeling of cooperativity and binding site competition on the surface and in the hydrogel.

PubMed

Lebedev, Konstantin; Mafé, Salvador; Stroeve, Pieter

2006-04-15

We study theoretically the transport and kinetic processes underlying the operation of a biosensor (particularly the surface plasmon sensor "Biacore") used to study the surface binding kinetics of biomolecules in solution to immobilized receptors. Unlike previous studies, we concentrate mainly on the modeling of system-specific phenomena rather than on the influence of mass transport limitations on the intrinsic kinetic rate constants determined from binding data. In the first problem, the case of two-site binding where each receptor unit on the surface can accommodate two analyte molecules on two different sites is considered. One analyte molecule always binds first to a specific site. Subsequently, the second analyte molecule can bind to the adjacent unoccupied site. In the second problem, two different analytes compete for one binding site on the same surface receptor. Finally, the third problem considers the case of positive cooperativity among bound molecules in the hydrogel using a simple mean-field approach. The transport in both the flow channel and the hydrogel phases of the biosensor is taken into account in this case (with few exceptions, most previous studies assume a simpler model in which the hydrogel is treated as a planar surface with the receptors). We consider simultaneously diffusion and convection through the flow channel together with diffusion and cooperativity binding on the surface and in the hydrogel. In each case, typical results for the concentration contours of the free and bound molecules in the flow channel and hydrogel regions are presented together with the time-dependent association/dissociation curves and reaction rates. For binding site competition, the analysis predicts overshoot phenomena.

Fast pressure jumps can perturb calcium and magnesium binding to troponin C F29W.

PubMed

Pearson, David S; Swartz, Darl R; Geeves, Michael A

2008-11-18

We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.

Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W

PubMed Central

Pearson, David S.; Swartz, Darl R.; Geeves, Michael A.

2009-01-01

We have used rapid pressure jump and stopped-flow fluorimetry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL.mol-1). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000 s-1 and 100 s-1. Between pCa 8-5.4 and at troponin C concentrations of 8-28 μM, the slow relaxation times were invariant indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200 - 300 μM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo. PMID:18942859

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

PubMed

Ojelabi, Ogooluwa A; Lloyd, Kenneth P; Simon, Andrew H; De Zutter, Julie K; Carruthers, Anthony

2016-12-23

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with K i (app) = 6 μm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with K i (app) = 0.3 μm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site*

PubMed Central

Ojelabi, Ogooluwa A.; Lloyd, Kenneth P.; Simon, Andrew H.; De Zutter, Julie K.; Carruthers, Anthony

2016-01-01

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 μm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 μm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites. PMID:27836974

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

Biochemical Study of Anti-Inflammatory Proteins vCCI and vMIP-II

DTIC Science & Technology

2014-07-17

protein ), where we showed that vCCI is able to bind so many different chemokines due to its general negatively charged surface , allowing it to bind...sample of these competition curves. Our conclusion from the data in Table 1 and Figure 1 is that the negatively charged surface of vCCI allows it to...Similar to our mutagenesis results, the overall data indicate that vCCI uses a negatively charged surface to bind positive charges on the chemokine

X-ray absorption spectroscopic evidence for binding of the competitive inhibitor 2-mercaptoethanol to the nickel sites of Jack bean urease. A new Ni-Ni interaction in the inhibited enzyme

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

Clark, P.A.; Wilcox, D.E.; Scott, R.A.

The enzyme Jack bean urease has been identified as the first nickel-containing metalloenzyme to catalyze the hydrolysis of urea to carbon dioxide and ammonia. Competitive inhibitors such as 2-mercaptoethanol (2-ME) have been shown to dramatically affect the ground-state electronic properties of the urease Ni(II) ions. Results of preliminary structural investigations using x-ray absorption spectroscopy of the nickel salts of urease in its native and 2-ME bound forms are presented. The binding of 2-ME to Ni(II) through the thiolate sulfur is confirmed by the results of this study. 17 refs., 2 figs., 2 tabs.

Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

PubMed

Wang, X Y; Bergdahl, K; Heijbel, A; Liljebris, C; Bleasdale, J E

2001-02-28

One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.

Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release

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

Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M.

Here, nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowedmore » us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.« less

Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release

DOE PAGES

Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M.; ...

2017-12-20

Here, nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowedmore » us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.« less

Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

PubMed

Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

2006-11-24

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

Development of machine learning models to predict inhibition of 3-dehydroquinate dehydratase.

PubMed

de Ávila, Maurício Boff; de Azevedo, Walter Filgueira

2018-04-20

In this study, we describe the development of new machine learning models to predict inhibition of the enzyme 3-dehydroquinate dehydratase (DHQD). This enzyme is the third step of the shikimate pathway and is responsible for the synthesis of chorismate, which is a natural precursor of aromatic amino acids. The enzymes of shikimate pathway are absent in humans, which make them protein targets for the design of antimicrobial drugs. We focus our study on the crystallographic structures of DHQD in complex with competitive inhibitors, for which experimental inhibition constant data is available. Application of supervised machine learning techniques was able to elaborate a robust DHQD-targeted model to predict binding affinity. Combination of high-resolution crystallographic structures and binding information indicates that the prevalence of intermolecular electrostatic interactions between DHQD and competitive inhibitors is of pivotal importance for the binding affinity against this enzyme. The present findings can be used to speed up virtual screening studies focused on the DHQD structure. © 2018 John Wiley & Sons A/S.

Linear scaffolds for multivalent targeting of melanocortin receptors.

PubMed

Dehigaspitiya, Dilani Chathurika; Anglin, Bobbi L; Smith, Kara R; Weber, Craig S; Lynch, Ronald M; Mash, Eugene A

2015-12-21

Molecules bearing one, two, three, or four copies of the tetrapeptide His-dPhe-Arg-Trp were attached to scaffolds based on ethylene glycol, glycerol, and d-mannitol by means of the copper-assisted azide-alkyne cyclization. The abilities of these compounds to block binding of a probe at the melanocortin 4 receptor were evaluated using a competitive binding assay. All of the multivalent molecules studied exhibited 30- to 40-fold higher apparent affinites when compared to a monovalent control. These results are consistent with divalent binding to receptor dimers. No evidence for tri- or tetravalent binding was obtained. Differences in the interligand spacing required for divalent binding, as opposed to tri- or tetravalent binding, may be responsible for these results.

Noni as an anxiolytic and sedative: a mechanism involving its gamma-aminobutyric acidergic effects.

PubMed

Deng, S; West, B J; Palu, A K; Zhou, B-N; Jensen, C J

2007-08-01

Noni (Morinda citrifolia) is increasing in worldwide popularity as a food or dietary supplement with versatile health benefits. The aim of this study was to investigate the effects of Noni fruit on anxiety symptoms in vitro. To this end, a competitive GABAa receptor-binding assay was developed. Our preliminary study indicates that the methanol crude extract of Noni fruit showed significant affinity to the gamma-aminobutyric acid A (GABAa) inhibitory neurotransmitter receptors, and displayed 75% binding inhibition of the agonist radioligand [3H] muscimol at a concentration of 100 microg/ml. Further experiments demonstrated that the MeOH extract, and its BuOH and H2O partitions, exhibited IC50 values of 22.8, 27.2, and 17.1 microg/ml, respectively, in the GABAa-binding assay. Experimental results with Noni fruit indicate the presence of competitive ligand(s), which may bind to the GABAa receptor as an agonist, and thus induce its anxiolytic and sedative effects. The study provides an in vitro rationale for one of Noni's versatile and traditional uses. In addition, an HPLC fingerprint profile of the methanolic extract of Noni fruit has been established for quality control purpose.

Spectroscopic and molecular docking studies on the interaction of antiviral drug nevirapine with calf thymus DNA.

PubMed

Moghadam, Neda Hosseinpour; Salehzadeh, Sadegh; Shahabadi, Nahid

2017-09-02

The interaction of calf thymus DNA with nevirapine at physiological pH was studied by using absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, salt effect studies and computational methods. The drug binds to ct-DNA in a groove binding mode, as shown by slight variation in the viscosity of ct-DNA. Furthermore, competitive fluorimetric studies with Hoechst 33258 indicate that nevirapine binds to DNA via groove binding. Moreover, the structure of nevirapine was optimized by DFT calculations and was used for the molecular docking calculations. The molecular docking results suggested that nevirapine prefers to bind on the minor groove of ct-DNA.

High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors*

PubMed Central

Drisdel, Renaldo C.; Sharp, Douglas; Henderson, Tricia; Hales, Tim G.; Green, William N.

2008-01-01

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. PMID:17702741

AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes ▿ †

PubMed Central

Waligora, Elizabeth A.; Ramsey, Deborah M.; Pryor, Edward E.; Lu, Haiping; Hollis, Thomas; Sloan, Gina P.; Deora, Rajendar; Wozniak, Daniel J.

2010-01-01

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection. PMID:20709902

Mercury(II) sorption to two Florida Everglades peat--Evidence for strong and weak binding and competition by dissolved organic matter released from the peat

USGS Publications Warehouse

Drexel, R. Todd; Haitzer, Markus; Ryan, Joseph N.; Aiken, George R.; Nagy, Kathryn L.

2002-01-01

The binding of mercury(II) to two peats from Florida Everglades sites with different rates of mercury methylation was measured at pH 6.0 and 0.01 M ionic strength. The mercury(II) sorption isotherms, measured over a total mercury(II) range of 10-7.4 to 10-3.7 M, showed the competition for mercury(II) between the peat and dissolved organic matter released from the peat and the existence of strong and weak binding sites for mercury(II). Binding was portrayed by a model accounting for strong and weak sites on both the peat and the released DOM. The conditional binding constants (for which the ligand concentration was set as the concentration of reduced sulfur in the organic matter as measured by X-ray absorption near-edge structure spectroscopy) determined for the strong sites on the two peats were similar (Kpeat,s = 1021.8±0.1and 1022.0±0.1 M-1), but less than those determined for the DOM strong sites (Kdom,s = 1022.8±0.1and 1023.2±0.1 M-1), resulting in mercury(II) binding by the DOM at low mercury(II) concentrations. The magnitude of the strong site binding constant is indicative of mercury(II) interaction with organic thiol functional groups. The conditional binding constants determined for the weak peat sites (Kpeat,w = 1011.5±0.1 and 1011.8±0.1 M-1) and weak DOM sites (Kdom,w = 108.7±3.0 and 107.3±4.5 M-1) were indicative of mercury(II) interaction with carboxyl and phenol functional groups.

The binding sites for benztropines and dopamine in the dopamine transporter overlap

PubMed Central

Bisgaard, Heidi; Larsen, M. Andreas B.; Mazier, Sonia; Beuming, Thijs; Newman, Amy Hauck; Weinstein, Harel; Shi, Lei; Loland, Claus J.; Gether, Ulrik

2013-01-01

Analogues of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational modeling together with site-directed mutagenesis to characterize the binding site for BZTs in DAT. Docking into molecular models based on the structure of the bacterial homologue LeuT supported a BZT binding site that overlaps with the substrate binding pocket. In agreement, mutations of residues within the pocket, including Val1523.46* to Ala or Ile, Ser4228.60 to Ala and Asn1573.51 to Cys or Ala, resulted in decreased affinity for BZT and the analog JHW007, as assessed in [3H]dopamine uptake inhibition assays and/or [3H]CFT competition binding assay. A putative polar interaction of one of the phenyl ring fluorine substituents in JHW007 with Asn1573.51 was used as a criterion for determining likely binding poses and establish a structural context for the mutagenesis findings. The analysis positioned the other fluorine substituted phenyl ring of JHW007 in close proximity to Ala47910.51/Ala48010.52 in transmembrane segment (TM) 10. The lack of such an interaction for BZT led to a more tilted orientation, as compared to JHW007, bringing one of the phenyl rings even closer to Ala47910.51/Ala48010.52. Mutation of Ala47910.51 and Ala48010.52 to valines supported these predictions with a larger decrease in the affinity for BZT than for JHW007. Summarized, our data suggest that BZTs display a classical competitive binding mode with binding sites overlapping those of cocaine and dopamine. PMID:20816875

The mechanistic basis for noncompetitive ibogaine inhibition of serotonin and dopamine transporters.

PubMed

Bulling, Simon; Schicker, Klaus; Zhang, Yuan-Wei; Steinkellner, Thomas; Stockner, Thomas; Gruber, Christian W; Boehm, Stefan; Freissmuth, Michael; Rudnick, Gary; Sitte, Harald H; Sandtner, Walter

2012-05-25

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.

The Mechanistic Basis for Noncompetitive Ibogaine Inhibition of Serotonin and Dopamine Transporters*

PubMed Central

Bulling, Simon; Schicker, Klaus; Zhang, Yuan-Wei; Steinkellner, Thomas; Stockner, Thomas; Gruber, Christian W.; Boehm, Stefan; Freissmuth, Michael; Rudnick, Gary; Sitte, Harald H.; Sandtner, Walter

2012-01-01

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study. PMID:22451652

Androgen Receptor Antagonism By Divalent Ethisterone Conjugates In Castrate-Resistant Prostate Cancer Cells

PubMed Central

Levine, Paul M.; Lee, Eugine; Greenfield, Alex; Bonneau, Richard; Logan, Susan K.; Garabedian, Michael J.; Kirshenbaum, Kent

2013-01-01

Sustained treatment of prostate cancer with Androgen Receptor (AR) antagonists can evoke drug resistance, leading to castrate-resistant disease. Elevated activity of the AR is often associated with this highly aggressive disease state. Therefore, new therapeutic regimens that target and modulate AR activity could prove beneficial. We previously introduced a versatile chemical platform to generate competitive and non-competitive multivalent peptoid oligomer conjugates that modulate AR activity. In particular, we identified a linear and a cyclic divalent ethisterone conjugate that exhibit potent anti-proliferative properties in LNCaP-abl cells, a model of castrate-resistant prostate cancer. Here, we characterize the mechanism of action of these compounds utilizing confocal microscopy, time-resolved fluorescence resonance energy transfer, chromatin immunoprecipitation, flow cytometry, and microarray analysis. The linear conjugate competitively blocks AR action by inhibiting DNA binding. In addition, the linear conjugate does not promote AR nuclear localization or co-activator binding. In contrast, the cyclic conjugate promotes AR nuclear localization and induces cell-cycle arrest, despite its inability to compete against endogenous ligand for binding to AR in vitro. Genome-wide expression analysis reveals that gene transcripts are differentially affected by treatment with the linear or cyclic conjugate. Although the divalent ethisterone conjugates share extensive chemical similarities, we illustrate that they can antagonize the AR via distinct mechanisms of action, establishing new therapeutic strategies for potential applications in AR pharmacology. PMID:22871957

Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding.

PubMed

Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael; Posern, Guido

2016-05-15

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin-MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin-RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Binding Specificity of Two PBPs in the Yellow Peach Moth Conogethes punctiferalis (Guenée)

PubMed Central

Ge, Xing; Ahmed, Tofael; Zhang, Tiantao; Wang, Zhenying; He, Kanglai; Bai, Shuxiong

2018-01-01

Pheromone binding proteins (PBPs) play an important role in olfaction of insects by transporting sex pheromones across the sensillum lymph to odorant receptors. To obtain a better understanding of the molecular basis between PBPs and semiochemicals, we have cloned, expressed, and purified two PBPs (CpunPBP2 and CpunPBP5) from the antennae of Conogethes punctiferalis. Fluorescence competitive binding assays were used to investigate binding affinities of CpunPBP2 and CpunPBP5 to sex pheromone and volatiles. Results indicate both CpunPBP2 and CpunPBP5 bind sex pheromones E10-16:Ald, Z10-16:Ald and hexadecanal with higher affinities. In addition, CpunPBP2 and CpunPBP5 also could bind some odorants, such as 1-tetradecanol, trans-caryopyllene, farnesene, and β-farnesene. Homology modeling to predict 3D structure and molecular docking to predict key binding sites were used, to better understand interactions of CpunPBP2 and CpunPBP5 with sex pheromones E10-16:Ald and Z10-16:Ald. According to the results, Phe9, Phe33, Ser53, and Phe115 were key binding sites predicted for CpunPBP2, as were Ser9, Phe12, Val115, and Arg120 for CpunPBP5. Binding affinities of four mutants of CpunPBP2 and four mutants of CpunPBP5 with the two sex pheromones were investigated by fluorescence competitive binding assays. Results indicate that single nucleotides mutation may affect interactions between PBPs and sex pheromones. Expression levels of CpunPBP2 and CpunPBP5 in different tissues were evaluated using qPCR. Results show that CpunPBP2 and CpunPBP5 were largely amplified in the antennae, with low expression levels in other tissues. CpunPBP2 was expressed mainly in male antennae, whereas CpunPBP5 was expressed mainly in female antennae. These results provide new insights into understanding the recognition between PBPs and ligands. PMID:29666585

STUDIES OF VERAPAMIL BINDING TO HUMAN SERUM ALBUMIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Chen, Sike; Hage, David S.

2008-01-01

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104 M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104 M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body. PMID:18980867

Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis.

PubMed

Duffy, Noah H; Lester, Henry A; Dougherty, Dennis A

2012-10-19

The serotonin type 3 receptor (5-HT(3)R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT(3)R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT(3)A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

PubMed Central

Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

2012-01-01

The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET

PubMed Central

Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2013-01-01

The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5′-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic. PMID:23303779

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

PubMed

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

2012-12-04

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Interaction of Diuron and Related Substituted Phenylureas with the Ah Receptor Pathway

PubMed Central

Zhao, Bin; Baston, David S.; Hammock, Bruce; Denison, Michael S.

2011-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals, including the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we have examined the ability of diuron, a widely used herbicide, and several structurally related substituted phenylureas to bind to and activate/inhibit the AhR and AhR signal transduction. Diuron induced CYP1A1 mRNA levels in mouse hepatoma (Hepa1c1c7) cells and AhR-dependent luciferase reporter gene expression in stably transfected mouse, rat, guinea pig, and human cell lines. In addition, ligand binding and gel retardation analysis demonstrated the ability of diuron to competitively bind to and stimulate AhR transformation and DNA binding in vitro and in intact cells. Several structurally related substituted phenylureas competitively bound to the guinea pig hepatic cytosolic AhR, inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced AhR-dependent luciferase reporter gene expression in a species-specific manner and stimulated AhR transformation and DNA binding, consistent with their role as partial AhR agonists. These results demonstrate not only that diuron and related substituted phenylureas are AhR ligands but also that exposure to these chemicals could induce/inhibit AhR-dependent biological effects. PMID:16788953

Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

PubMed Central

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

2014-01-01

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021

Inclusion complex formation of ionic liquids with 4-sulfonatocalixarenes studied by competitive binding of berberine alkaloid fluorescent probe

NASA Astrophysics Data System (ADS)

Miskolczy, Zsombor; Biczók, László

2009-07-01

A clinically important natural isoquinoline alkaloid, berberine, was used as a fluorescent probe to study the encapsulation of 1-alkyl-3-methylimidazolium (C nMIm +) type ionic liquids in 4-sulfonato-substituted calix[4]arene (SCX4) and calix[6]arene (SCX6) at pH 2. Addition of ionic liquids to the aqueous solution of berberine-SCXn inclusion complexes brought about considerable fluorescence intensity diminution due to the extrusion of berberine from the macrocycle into the aqueous phase by the competitive inclusion of C nMIm + cation. The lengthening of the aliphatic side chain of the imidazolium moiety diminished the equilibrium constant of complexation with SCX4, but enhanced the stability of SCX6 complexes. Larger binding strength was found for SCX4.

Benzophenone based fluorophore for selective detection of Sn2+ ion: Experimental and theoretical study.

PubMed

Jadhav, Amol G; Shinde, Suvidha S; Lanke, Sandip K; Sekar, Nagaiyan

2017-03-05

Synthesis of novel benzophenone-based chemosensor is presented for the selective sensing of Sn 2+ ion. Screening of competitive metal ions was performed by competitive experiments. The specific cation recognition ability of chemosensor towards Sn 2+ was investigated by experimental (UV-visible, fluorescence spectroscopy, 1 H NMR, 13 C NMR, FTIR and HRMS) methods and further supported by Density Functional Theory study. The stoichiometric binding ratio and binding constant (K a ) for complex is found to be 1:1 and 1.50×10 4 , respectively. The detection limit of Sn 2+ towards chemosensor was found to be 0.3898ppb. Specific selectivity and superiority of chemosensor over another recently reported chemosensor is presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Anion complexation and the Hofmeister effect.

PubMed

Carnegie, Ryan S; Gibb, Corinne L D; Gibb, Bruce C

2014-10-20

The (1)H NMR spectroscopic analysis of the binding of the ClO4(-) anion to the hydrophobic, concave binding site of a deep-cavity cavitand is presented. The strength of association between the host and the ClO4(-) anion is controlled by both the nature and concentration of co-salts in a manner that follows the Hofmeister series. A model that partitions this trend into the competitive binding of the co-salt anion to the hydrophobic pocket of the host and counterion binding to its external carboxylate groups successfully accounts for the observed changes in ClO4(-) affinity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of Cyclin A Binders with a Fluorescent Peptide Sensor.

PubMed

Pazos, Elena; Mascareñas, José L; Vázquez, M Eugenio

2016-01-01

A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

PubMed

Schöne, Tobias; Grimm, Lena Lisbeth; Sakai, Naoki; Zhang, Linlin; Hilgenfeld, Rolf; Peters, Thomas

2017-10-01

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3 pro , we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3 pro . Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC 50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3 pro . To identify the matching protein binding site, chemical shift perturbation studies employing 1 H, 15 N-TROSY-HSQC experiments with uniformly 2 H, 15 N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 pro inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis and in vitro characterization of a P2X7 radioligand [123I]TZ6019 and its response to neuroinflammation in a mouse model of Alzheimer disease.

PubMed

Jin, Hongjun; Han, Junbin; Resing, Derek; Liu, Hui; Yue, Xuyi; Miller, Rebecca L; Schoch, Kathleen M; Miller, Timothy M; Perlmutter, Joel S; Egan, Terrance M; Tu, Zhude

2018-02-05

The purinergic receptor P2X ligand-gated ion channel 7 (P2X7 receptor) is a promising imaging target to detect neuroinflammation. Herein, we report development of a potent iodinated radiotracer for P2X7 receptor, [ 123 I]TZ6019. The radiosynthesis of [ 123 I]TZ6019 was accomplished by allylic-tin precursor iodination using [ 123 I]NaI with good radiochemical yield of 85% and high radiochemical purity of > 99%. Human embryonic kidney 293 (HEK-293) cell line stably transfected with the human P2X7 receptor was used to characterize the binding affinity of TZ6019 by fluorescence, radioactive competitive, and saturation binding assays. A radioligand competitive binding assay with [ 123 I]TZ6019 demonstrated that the nonradioactive compound TZ6019 has an IC 50 value of 9.49 ± 1.4nM, and the known P2X7 receptor compound GSK1482160 has an IC 50 value of 4.30 ± 0.86nM, consistent with previous reports. The radioligand saturation binding assay and competitive assay revealed that [ 123 I]TZ6019 specifically bound to the human P2X7 receptor with high affinity (K i = 6.3 ± 0.9nM). In vitro autoradiography quantification with brain slices collected from 9-month old P301S tau transgenic mice along with wild type controls, revealed higher binding of [ 123 I]TZ6019 (35% increase) in the brain of P301S transgenic mice (n = 3, p = 0.04) compared to wild type controls. The immunofluorescence microscopy confirmed that expression of P2X7 receptor was colocalized with astrocytes in the tauopathy P301S transgenic mice. [ 123 I]TZ6019 has specific binding for P2X7 receptor and has great potential to be a radiotracer for screening new compounds and quantifying expression of P2X7 receptor in neuroinflammation related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Segregation of O2 and CO on the surface of dust grains determines the desorption energy of O2

NASA Astrophysics Data System (ADS)

Noble, J. A.; Diana, S.; Dulieu, F.

2015-12-01

Selective depletion towards pre-stellar cores is still not understood. The exchange between the solid and gas phases is central to this mystery. The aim of this paper is to show that the thermal desorption of O2 and CO from a submonolayer mixture is greatly affected by the composition of the initial surface population. We have performed thermally programmed desorption (TPD) experiments on various submonolayer mixtures of O2 and CO. Pure O2 and CO exhibit almost the same desorption behaviour, but their desorption differs strongly when mixed. Pure O2 is slightly less volatile than CO, while in mixtures, O2 desorbs earlier than CO. We analyse our data using a desorption law linking competition for binding sites with desorption, based on the assumption that the binding energy distribution of both molecules is the same. We apply Fermi-Dirac statistics in order to calculate the adsorption site population distribution, and derive the desorbing fluxes. Despite its simplicity, the model reproduces the observed desorption profiles, indicating that competition for adsorption sites is the reason for lower temperature O2 desorption. CO molecules push-out or `dislodge' O2 molecules from the most favourable binding sites, ultimately forcing their early desorption. It is crucial to consider the surface coverage of dust grains in any description of desorption. Competition for access to binding sites results in some important discrepancies between similar kinds of molecules, such as CO and O2. This is an important phenomenon to be investigated in order to develop a better understanding of the apparently selective depletion observed in dark molecular clouds.

Attachment of UDP-hexosamines to the ribosomes isolated from rat liver

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

Kopacz-Jodczyk, T.; Paszkiewicz-Gadek, A.; Galasinski, W.

1988-06-01

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.

The attachment of UDP-hexosamines to the ribosomes isolated from rat liver.

PubMed

Kopacz-Jodczyk, T; Paszkiewicz-Gadek, A; Gałasiński, W

1988-06-01

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.

Molecularly imprinted composite cryogel for albumin depletion from human serum.

PubMed

Andaç, Müge; Baydemir, Gözde; Yavuz, Handan; Denizli, Adil

2012-11-01

A new composite protein-imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl-methacylate-based molecularly imprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform infrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity. Copyright © 2012 John Wiley & Sons, Ltd.

(3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

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

Norman, A.B.; Battaglia, G.; Creese, I.

1985-12-01

In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

Structural basis for dual roles of Aar2p in U5 snRNP assembly

PubMed Central

Weber, Gert; Cristão, Vanessa F.; Santos, Karine F.; Jovin, Sina Mozaffari; Heroven, Anna C.; Holton, Nicole; Lührmann, Reinhard; Beggs, Jean D.; Wahl, Markus C.

2013-01-01

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains. To elucidate the molecular basis for this competition, we determined the crystal structure of Aar2p in complex with the Prp8p RNase H and Jab1/MPN domains. Aar2p binds on one side of the RNase H domain and extends its C terminus to the other side, where the Jab1/MPN domain is docked onto a composite Aar2p–RNase H platform. Known Brr2p interaction sites of the Jab1/MPN domain remain available, suggesting that Aar2p-mediated compaction of the Prp8p domains sterically interferes with Brr2p binding. Moreover, Aar2p occupies known RNA-binding sites of the RNase H domain, and Aar2p interferes with binding of U4/U6 di-snRNA to the Prp8p C-terminal region. Structural and functional analyses of phospho-mimetic mutations reveal how phosphorylation reduces affinity of Aar2p for Prp8p and allows Brr2p and U4/U6 binding. Our results show how Aar2p regulates both protein and RNA binding to Prp8p during U5 snRNP assembly. PMID:23442228

Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase.

PubMed Central

Sok, D E; Kim, Y B; Choi, S J; Jung, C H; Cha, S H

1994-01-01

Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites. PMID:8053896

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

PubMed Central

Liu, Xinwei; Ridgway, Neale D.

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus. PMID:25255026

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

PubMed

Liu, Xinwei; Ridgway, Neale D

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

Insights into the interaction of methotrexate and human serum albumin: A spectroscopic and molecular modeling approach.

PubMed

Cheng, Li-Yang; Fang, Min; Bai, Ai-Min; Ouyang, Yu; Hu, Yan-Jun

2017-08-01

In this study, fluorescence spectroscopy and molecular modeling approaches were employed to investigate the binding of methotrexate to human serum albumin (HSA) under physiological conditions. From the mechanism, it was demonstrated that fluorescence quenching of HSA by methotrexate results from the formation of a methotrexate/HSA complex. Binding parameters calculated using the Stern-Volmer method and the Scatchard method showed that methotrexate binds to HSA with binding affinities in the order 10 4  L·mol -1 . Thermodynamic parameter studies revealed that the binding reaction is spontaneous, and that hydrogen bonds and van der Waals interactions play a major role in the reaction. Site marker competitive displacement experiments and a molecular modeling approach demonstrated that methotrexate binds with appropriate affinity to site I (subdomain IIA) of HSA. Furthermore, we discuss some factors that influence methotrexate binding to HSA. Copyright © 2017 John Wiley & Sons, Ltd.

The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators

PubMed Central

Martínez-Martínez, Sara; Genescà, Lali; Rodríguez, Antonio; Raya, Alicia; Salichs, Eulàlia; Were, Felipe; López-Maderuelo, María Dolores; Redondo, Juan Miguel; de la Luna, Susana

2009-01-01

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca2+-calcineurin signaling. PMID:19332797

Intercalation of a Zn(II) complex containing ciprofloxacin drug between DNA base pairs.

PubMed

Shahabadi, Nahid; Asadian, Ali Ashraf; Mahdavi, Mryam

2017-11-02

In this study, an attempt has been made to study the interaction of a Zn(II) complex containing an antibiotic drug, ciprofloxacin, with calf thymus DNA using spectroscopic methods. It was found that Zn(II) complex could bind with DNA via intercalation mode as evidenced by: hyperchromism in UV-Vis spectrum; these spectral characteristics suggest that the Zn(II) complex interacts with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. DNA binding constant (K b = 1.4 × 10 4 M -1 ) from spectrophotometric studies of the interaction of Zn(II) complex with DNA is comparable to those of some DNA intercalative polypyridyl Ru(II) complexes 1.0 -4.8 × 10 4 M -1 . CD study showed stabilization of the right-handed B form of DNA in the presence of Zn(II) complex as observed for the classical intercalator methylene blue. Thermodynamic parameters (ΔH < 0 and ΔS < 0) indicated that hydrogen bond and Van der Waals play main roles in this binding prose. Competitive fluorimetric studies with methylene blue (MB) dye have shown that Zn(II) complex exhibits the ability of this complex to displace with DNA-MB, indicating that it binds to DNA in strong competition with MB for the intercalation.

The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

PubMed Central

Reed, Ken C.; Bygrave, Fyfe L.

1974-01-01

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of 45Ca2+ transport by rat liver mitochondria. This method was used in conjunction with studies of Ca2+-stimulated respiration to examine the mechanisms of inhibition of Ca2+ transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca2+ transport non-competitively with Ki 3×10−8m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La3+ is competitive (Ki=2×10−8m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the `low-affinity' Ca2+-binding sites). La3+ competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La3+. The results are discussed briefly in relation to possible models for a Ca2+ carrier. PMID:4375957

Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.

PubMed

Van Dolah, F M; Finley, E L; Haynes, B L; Doucette, G J; Moeller, P D; Ramsdell, J S

1994-01-01

The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.

Staufen-mediated mRNA decay

PubMed Central

Park, Eonyoung; Maquat, Lynne E.

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

PubMed

Kikkawa, Yamato; Ogawa, Takaho; Sudo, Ryo; Yamada, Yuji; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi; Miner, Jeffrey H

2013-10-25

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Cooperative interplay of let-7 mimic and HuR with MYC RNA.

PubMed

Gunzburg, Menachem J; Sivakumaran, Andrew; Pendini, Nicole R; Yoon, Je-Hyun; Gorospe, Myriam; Wilce, Matthew C J; Wilce, Jacqueline A

2015-01-01

Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites.

Experimental and computational studies on the effects of valganciclovir as an antiviral drug on calf thymus DNA.

PubMed

Shahabadi, Nahid; Pourfoulad, Mehdi; Moghadam, Neda Hosseinpour

2017-01-02

DNA-binding properties of an antiviral drug, valganciclovir (valcyte) was studied by using emission, absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, and computational studies. The drug bound to calf thymus DNA (ct-DNA) in a groove-binding mode. The calculated binding constant of UV-vis, K a , is comparable to groove-binding drugs. Competitive fluorimetric studies with Hoechst 33258 showed that valcyte could displace the DNA-bound Hoechst 33258. The drug could not displace intercalated methylene blue from DNA double helix. Furthermore, the induced detectable changes in the CD spectrum of ct-DNA as well as changes in its viscosity confirm the groove-binding mode. In addition, an integrated molecular docking was employed to further investigate the binding interactions between valcyte and calf thymus DNA.

Competitive interactions affect working memory performance for both simultaneous and sequential stimulus presentation.

PubMed

Ahmad, Jumana; Swan, Garrett; Bowman, Howard; Wyble, Brad; Nobre, Anna C; Shapiro, Kimron L; McNab, Fiona

2017-07-06

Competition between simultaneously presented visual stimuli lengthens reaction time and reduces both the BOLD response and neural firing. In contrast, conditions of sequential presentation have been assumed to be free from competition. Here we manipulated the spatial proximity of stimuli (Near versus Far conditions) to examine the effects of simultaneous and sequential competition on different measures of working memory (WM) for colour. With simultaneous presentation, the measure of WM precision was significantly lower for Near items, and participants reported the colour of the wrong item more often. These effects were preserved when the second stimulus immediately followed the first, disappeared when they were separated by 500 ms, and were partly recovered (evident for our measure of mis-binding but not WM precision) when the task was altered to encourage participants to maintain the sequentially presented items together in WM. Our results show, for the first time, that competition affects the measure of WM precision, and challenge the assumption that sequential presentation removes competition.

From glue to gasoline: how competition turns perspective takers unethical.

PubMed

Pierce, Jason R; Kilduff, Gavin J; Galinsky, Adam D; Sivanathan, Niro

2013-10-01

Perspective taking is often the glue that binds people together. However, we propose that in competitive contexts, perspective taking is akin to adding gasoline to a fire: It inflames already-aroused competitive impulses and leads people to protect themselves from the potentially insidious actions of their competitors. Overall, we suggest that perspective taking functions as a relational amplifier. In cooperative contexts, it creates the foundation for prosocial impulses, but in competitive contexts, it triggers hypercompetition, leading people to prophylactically engage in unethical behavior to prevent themselves from being exploited. The experiments reported here establish that perspective taking interacts with the relational context--cooperative or competitive--to predict unethical behavior, from using insidious negotiation tactics to materially deceiving one's partner to cheating on an anagram task. In the context of competition, perspective taking can pervert the age-old axiom "do unto others as you would have them do unto you" into "do unto others as you think they will try to do unto you."

Local anesthetics QX 572 and benzocaine act at separate sites on the batrachotoxin-activated sodium channel

PubMed Central

1981-01-01

We have studied the effect of local anesthetics QX 572, which is permanently charged, and benzocaine, which is neutral, on batrachotoxin- activated sodium channels in mouse neuroblastoma N18 cells. The dose- response curves for each drug suggest that QX 752 and benzocaine each act on a single class of binding sites. The dissociation constants are 3.15 X 10(-5) M for QX 572 and 2.65 X 10(-4) M for benzocaine. Equilibrium and kinetic experiments indicate that both drugs are competitive inhibitors of batrachotoxin. When benzocaine and QX 572 are present with batrachotoxin, they are much more effective at inhibiting Na+ flux than would be predicted by a one-site model. Our results indicate that QX 572 and benzocaine bind to separate sites, each of which interacts competitively with batrachotoxin. PMID:6267160

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors.

PubMed

Austin, Carol; Pettit, Simon N; Magnolo, Sharon K; Sanvoisin, Jonathan; Chen, Wenjie; Wood, Stephen P; Freeman, Lauren D; Pengelly, Reuben J; Hughes, Dallas E

2012-08-01

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.

Dual-Color Luciferase Complementation for Chemokine Receptor Signaling.

PubMed

Luker, Kathryn E; Luker, Gary D

2016-01-01

Chemokine receptors may share common ligands, setting up potential competition for ligand binding, and association of activated receptors with downstream signaling molecules such as β-arrestin. Determining the "winner" of competition for shared effector molecules is essential for understanding integrated functions of chemokine receptor signaling in normal physiology, disease, and response to therapy. We describe a dual-color click beetle luciferase complementation assay for cell-based analysis of interactions of two different chemokine receptors, CXCR4 and ACKR3, with the intracellular scaffolding protein β-arrestin 2. This assay provides real-time quantification of receptor activation and signaling in response to chemokine CXCL12. More broadly, this general imaging strategy can be applied to quantify interactions of any set of two proteins that interact with a common binding partner. © 2016 Elsevier Inc. All rights reserved.

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms

PubMed Central

Nazim, Mohammad; Masuda, Akio; Rahman, Mohammad Alinoor; Nasrin, Farhana; Takeda, Jun-ichi; Ohe, Kenji; Ohkawara, Bisei; Ito, Mikako

2017-01-01

Abstract Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation. PMID:28180311

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms.

PubMed

Nazim, Mohammad; Masuda, Akio; Rahman, Mohammad Alinoor; Nasrin, Farhana; Takeda, Jun-Ichi; Ohe, Kenji; Ohkawara, Bisei; Ito, Mikako; Ohno, Kinji

2017-02-17

Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

Glucose Sensors Based on Microcapsules Containing an Orange/Red Competitive Binding Resonance Energy Transfer Assay

PubMed Central

CHINNAYELKA, SWETHA; McSHANE, and MICHAEL J.

2015-01-01

Fluorescent sensing systems offer the potential for noninvasive monitoring with implantable devices, but they require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility while maintaining adequate response characteristics. A recent development towards this goal is a highly specific and sensitive competitive binding assay for glucose using apo-glucose oxidase (apo-GOx) as the recognition element and dextran as the competing ligand; this has been demonstrated as a glucose sensor system by encapsulating the competitive binding assay in semipermeable microcapsule carriers. This paper describes the extension of this sensor design to longer wavelengths in an attempt to increase the applicability to in vivo monitoring. The glucose sensitivity of the tetramethylrhodamine isothiocyanate-dextran (TD) and cyanine Cy5-apo-GOx (CAG) complexes showed five to 10 times greater specificity for β-D-glucose over other sugars. Microcapsules loaded with TD/CAG complexes exhibited a linear, totally reversible response in the range of 0–720 mg/dL, with a sensitivity (percent change in intensity ratio) of 0.06%/(mg/dL). The decrease in sensitivity observed with the use of longer-wavelength dyes is most likely to be compensated with the deeper penetration of light and reduced tissue scattering. These findings imply that the encapsulation of sensing assay elements in microcapsules is a simple and translatable method for the fabrication of stable biosensors, and optimization of resonance energy transfer pairs and assay component preparation will further improve the response to approach clinically relevant performance. PMID:16800748

Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane

PubMed Central

2017-01-01

The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests. PMID:28123906

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

PubMed Central

Naguib, Fardos N. M.; Rais, Reem H.; Al Safarjalani, Omar N.; el Kouni, Mahmoud H.

2015-01-01

Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ± 0.0002 mM, KATP = 0.05 ± 0.008 mM, and Vmax = 920 ± 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5′-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii. PMID:26112826

Characterization of Interactions between Heparin/Glycosaminoglycan and Adeno-associated Virus

PubMed Central

Zhang, Fuming; Aguilera, Javier; Beaudet, Julie M.; Xie, Qing; Lerch, Thomas F.; Davulcu, Omar; Colón, Wilfredo; Chapman, Michael S.; Linhardt, Robert J.

2013-01-01

Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this report, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans. Surface plasmon resonance results revealed that heparin binds to AAV with extremely high affinity. Solution competition studies shows that AAV binding to heparin is chain length dependent. AAV prefers to bind full chain heparin. All sulfo groups (especially N-sulfo and 6-O-sulfo groups) on heparin are important for the AAV- heparin interaction. Higher levels of sulfo group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 complexed with heparin. PMID:23952613

The novel fluorescent CDP-analogue (Pbeta)MABA-CDP is a specific probe for the NMP binding site of UMP/CMP kinase.

PubMed

Rudolph, M G; Veit, T J; Reinstein, J

1999-12-01

Direct thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N-methylanthraniloyl- (mant) group to the beta-phosphate of CDP via a butyl linker, a CDP analogue [(Pbeta)MABA-CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments.

The novel fluorescent CDP-analogue (Pbeta)MABA-CDP is a specific probe for the NMP binding site of UMP/CMP kinase.

PubMed Central

Rudolph, M. G.; Veit, T. J.; Reinstein, J.

1999-01-01

Direct thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N-methylanthraniloyl- (mant) group to the beta-phosphate of CDP via a butyl linker, a CDP analogue [(Pbeta)MABA-CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments. PMID:10631985

Biosorption of uranium by Pseudomonas aeruginosa strain CSU: Characterization and comparison studies

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

Hu, M.Z.C.; Norman, J.M.; Faison, B.D.

1996-07-20

Pseudomonas aeruginosa strain CSU, a nongenetically engineered bacterial strain known to bind dissolved hexavalent uranium (as UO{sub 2}{sup 2+} and/or its cationic hydroxo complexes) was characterized with respect to its sorptive activity. The uranium biosorption equilibrium could be described by the Langmuir isotherm. The rate of uranium adsorption increased following permeabilization of the outer and/or cytoplasmic membrane by organic solvents such as acetone. P. aeruginosa CSU biomass was significantly more sorptive toward uranium than certain novel, patented biosorbents derived from algal or fungal biomass sources. P. aeruginosa CSU biomass was also competitive with commercial cation-exchange resins, particularly in the presencemore » of dissolved transition metals. Uranium binding by P. aeruginosa CSU was clearly pH dependent. Uranium loading capacity increased with increasing pH under acidic conditions, presumably as a function of uranium speciation and due to the H{sup +} competition at some binding sites. Nevertheless, preliminary evidence suggests that this microorganism is also capable of binding anionic hexavalent uranium complexes. Ferric iron was a strong inhibitor of uranium binding to P. aeruginosa CSU biomass, and the presence of uranium also decreased the Fe{sup 3+} loading when the biomass was not saturated with Fe{sup 3+}. Thus, a two-state process in which iron and uranium are removed in consecutive steps was proposed for efficient use of the biomass as a biosorbent in uranium removal from mine wastewater, especially acidic leachates.« less

A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent, pinostrobin

PubMed Central

FEROZ, Shevin R.; SUMI, Rumana A.; MALEK, Sri N.A.; TAYYAB, Saad

2014-01-01

The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, Ka in the range of 1.49 – 6.12 × 104 M−1, with 1:1 binding stoichiometry. Based on the PS–albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics. PMID:25519455

Molecular characterization and functional analysis of pheromone binding protein 1 from Cydia pomonella (L.).

PubMed

Tian, Z; Zhang, Y

2016-12-01

A full-length cDNA encoding Cydia pomonella pheromone binding protein 1 (CpomPBP1) was cloned and characterized. CpomPBP1, possessing the typical characteristics of lepidopteran odorant binding proteins, was detected to be specifically expressed in the antennae of male and female moths at the mRNA and protein level. Soluble recombinant CpomPBP1 was subjected to in vitro binding to analyse its binding properties and to search for potentially active semiochemicals. A competitive binding assay showed that three 12-carbon ligands, codlemone, 1-dodecanol and E,E-2,4-dodecadienal, were able to bind to CpomPBP1 in decreasing order of affinity. Moreover, unlike the wild-type CpomPBP1, the C-terminus truncated CpomPBP1 exhibited high affinity to ligands even in an acidic environment, suggesting that the C-terminus plays a role in preventing ligands from binding to CpomPBP1 in a lower pH environment. © 2016 The Royal Entomological Society.

Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase

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

Maley, F.; Maley, G.F.

1983-01-01

It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A preventsmore » the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte(/sup 14/C)GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH/sub 2/H/sub 4/PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two /sup 14/C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.« less

Binding of ATP by pertussis toxin and isolated toxin subunits

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

Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

1990-07-03

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner;more » however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.« less

Predicting MHC-II binding affinity using multiple instance regression

PubMed Central

EL-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

2011-01-01

Reliably predicting the ability of antigen peptides to bind to major histocompatibility complex class II (MHC-II) molecules is an essential step in developing new vaccines. Uncovering the amino acid sequence correlates of the binding affinity of MHC-II binding peptides is important for understanding pathogenesis and immune response. The task of predicting MHC-II binding peptides is complicated by the significant variability in their length. Most existing computational methods for predicting MHC-II binding peptides focus on identifying a nine amino acids core region in each binding peptide. We formulate the problems of qualitatively and quantitatively predicting flexible length MHC-II peptides as multiple instance learning and multiple instance regression problems, respectively. Based on this formulation, we introduce MHCMIR, a novel method for predicting MHC-II binding affinity using multiple instance regression. We present results of experiments using several benchmark datasets that show that MHCMIR is competitive with the state-of-the-art methods for predicting MHC-II binding peptides. An online web server that implements the MHCMIR method for MHC-II binding affinity prediction is freely accessible at http://ailab.cs.iastate.edu/mhcmir. PMID:20855923

Cooperative interplay of let-7 mimic and HuR with MYC RNA

PubMed Central

Gunzburg, Menachem J; Sivakumaran, Andrew; Pendini, Nicole R; Yoon, Je-Hyun; Gorospe, Myriam; Wilce, Matthew Cj; Wilce, Jacqueline A

2015-01-01

Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites. PMID:26177105

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

PubMed Central

Pinkney, M; Hoggett, J G

1988-01-01

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase. PMID:2839152

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

PubMed

Pinkney, M; Hoggett, J G

1988-03-15

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

Interaction studies of resistomycin from Streptomyces aurantiacus AAA5 with calf thymus DNA and bovine serum albumin

NASA Astrophysics Data System (ADS)

Vijayabharathi, R.; Sathyadevi, P.; Krishnamoorthy, P.; Senthilraja, D.; Brunthadevi, P.; Sathyabama, S.; Priyadarisini, V. Brindha

2012-04-01

Resistomycin, a secondary metabolite produced by Streptomyces aurantiacus AAA5. The binding interaction of resistomycin with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorimetry, circular dichroism (CD) and synchronous fluorescence techniques under physiological conditions in vitro. Absorption spectral studies along with the fluorescence competition with ethidium bromide measurements and circular dichroism clearly suggest that the resistomycin bind with CT DNA relatively strong via groove binding. BSA interaction results revealed that the drug was found to quench the fluorescence intensity of the protein through a static quenching mechanism. The number of binding sites 'n' and apparent binding constant 'K' calculated according to the Scatchard equation exhibit a good binding property to bovine serum albumin protein. In addition, the results observed from synchronous fluorescence measurements clearly demonstrate the occurrence of conformational changes of BSA upon addition of the test compound.

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

PubMed

Bel, Yolanda; Sheets, Joel J; Tan, Sek Yee; Narva, Kenneth E; Escriche, Baltasar

2017-06-01

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens ) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125 -iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. Copyright © 2017 Bel et al.

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

PubMed

Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

2016-01-01

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay

PubMed Central

Lakshmipriya, Thangavel; Gopinath, Subash C. B.; Tang, Thean-Hock

2016-01-01

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors. PMID:26954237

Detection of fumonisin b1 and ochratoxin a in grain products using microsphere-based fluid array immunoassays.

PubMed

Anderson, George P; Kowtha, Vasudha A; Taitt, Chris R

2010-02-01

Grain products are a staple of diets worldwide and therefore, the ability to accurately and efficiently detect foodborne contaminants such as mycotoxins is of importance to everyone. Here we describe an indirect competitive fluid array fluoroimmunoassay to quantify the mycotoxins, fumonisin B1 and ochratoxin A. Both toxins were immobilized to the surface of microspheres using a variety of intermediate molecules and binding of biotinylated "tracer" antibody tracers determined through flow cytometry using streptavidin-phycoerythrin conjugates and the Luminex100 flow cytometer. Competitive assays were developed where the binding of biotinylated monoclonal antibodies to fumonisin B and ochratoxin A was competitively inhibited by different concentrations of those toxins in solution. Concentrations of fumonisin giving 50% inhibition were 300 pg/mL in buffer, 100 ng/g in spiked oats, and 1 μg/g in spiked cornmeal; analogous concentrations for ochratoxin A were 30 ng/mL in buffer, 30 ng/g in spiked oats, and 10 ng/g in spiked corn. The future challenge will be to expand the number of mycotoxins tested both individually and in multiplexed format using this platform.

The Role of Nitric Oxide in Modulating Retinal, Choroidal, and Anterior Uveal Blood Flows in the Domestic Piglet

DTIC Science & Technology

1993-11-17

that are substituted at the quanidino nitrogens are competitive Inhibitors of nitric ox!de synthase in a dOS&<lependent and enantiomerically specific...by nitric oxide. We were able to reduce basal chorc»dal and ante nor wea blood 99 flOYI by 47% and 43%, respectively, by enantiomeric specific...Atthough competitive blockade of NOS by L-NAME is enantiomerically specKle, It Is possible that there Is an allosteric binding site for these arginine

Involvement of sulfates from cruzipain, a major antigen of Trypanosoma cruzi, in the interaction with immunomodulatory molecule Siglec-E.

PubMed

Ferrero, Maximiliano R; Heins, Anja M; Soprano, Luciana L; Acosta, Diana M; Esteva, Mónica I; Jacobs, Thomas; Duschak, Vilma G

2016-02-01

In order to investigate the involvement of sulfated groups in the Trypanosoma cruzi host-parasite relationship, we studied the interaction between the major cysteine proteinase of T. cruzi, cruzipain (Cz), a sulfate-containing sialylated molecule and the sialic acid-binding immunoglobulin like lectin-E (Siglec-E). To this aim, ELISA, indirect immunofluorescence assays and flow cytometry, using mouse Siglec-E-Fc fusion molecules and glycoproteins of parasites, were performed. Competition assays verified that the lectins, Maackia amurensis II (Mal II) and Siglec-E-Fc, compete for the same binding sites. Taking into account that Mal II binding remains unaltered by sulfation, we established this lectin as sialylation degree control. Proteins of an enriched microsomal fraction showed the highest binding to Siglec-E as compared with those from the other parasite subcellular fractions. ELISA assays and the affinity purification of Cz by a Siglec-E column confirmed the interaction between both molecules. The significant decrease in binding of Siglec-E-Fc to Cz and to its C-terminal domain (C-T) after desulfation of these molecules suggests that sulfates contribute to the interaction between Siglec-E-Fc and these glycoproteins. Competitive ELISA assays confirmed the involvement of sulfated epitopes in the affinity between Siglec-E and Cz, probably modified by natural protein environment. Interestingly, data from flow cytometry of untreated and chlorate-treated parasites suggested that sulfates are not primary receptors, but enhance the binding of Siglec-E to trypomastigotic forms. Altogether, our findings support the notion that sulfate-containing sialylated glycoproteins interact with Siglec-E, an ortholog protein of human Siglec-9, and might modulate the immune response of the host, favoring parasitemia and persistence of the parasite.

Supramolecular interaction of 6-shogaol, a therapeutic agent of Zingiber officinale with human serum albumin as elucidated by spectroscopic, calorimetric and molecular docking methods.

PubMed

Feroz, S R; Mohamad, S B; Lee, G S; Malek, S N A; Tayyab, S

2015-06-01

6-Shogaol, one of the main bioactive constituents of Zingiber officinale has been shown to possess various therapeutic properties. Interaction of a therapeutic compound with plasma proteins greatly affects its pharmacokinetic and pharmacodynamic properties. The present investigation was undertaken to characterize the interaction between 6-shogaol and the main in vivo transporter, human serum albumin (HSA). Various binding characteristics of 6-shogaol-HSA interaction were studied using fluorescence spectroscopy. Thermal stability of 6-shogaol-HSA system was determined by circular dichroism (CD) and differential scanning calorimetric (DSC) techniques. Identification of the 6-shogaol binding site on HSA was made by competitive drug displacement and molecular docking experiments. Fluorescence quench titration results revealed the association constant, Ka of 6-shogaol-HSA interaction as 6.29 ± 0.33 × 10(4) M(-1) at 25 ºC. Values of the enthalpy change (-11.76 kJ mol(-1)) and the entropy change (52.52 J mol(-1) K(-1)), obtained for the binding reaction suggested involvement of hydrophobic and van der Waals forces along with hydrogen bonds in the complex formation. Higher thermal stability of HSA was noticed in the presence of 6-shogaol, as revealed by DSC and thermal denaturation profiles. Competitive ligand displacement experiments along with molecular docking results suggested the binding preference of 6-shogaol for Sudlow's site I of HSA. All these results suggest that 6-shogaol binds to Sudlow's site I of HSA through moderate binding affinity and involves hydrophobic and van der Waals forces along with hydrogen bonds. Copyright © 2015 Elsevier GmbH. All rights reserved.

Quercetin inhibits glucose transport by binding to an exofacial site on GLUT1.

PubMed

Hamilton, Kathryn E; Rekman, Janelle F; Gunnink, Leesha K; Busscher, Brianna M; Scott, Jordan L; Tidball, Andrew M; Stehouwer, Nathan R; Johnecheck, Grace N; Looyenga, Brendan D; Louters, Larry L

2018-05-29

Quercetin, a common dietary flavone, is a competitive inhibitor of glucose uptake and is also thought to be transported into cells by GLUT1. In this study, we confirm that quercetin is a competitive inhibitor of GLUT1 and also demonstrate that newly synthesized compounds, WZB-117 and BAY-876 are robust inhibitors of GLUT1 in L929 cells. To measure quercetin interaction with L929 cells, we develop a new fluorescent assay using flow cytometry. The binding of quercetin and its inhibitory effects on 2-deoxyglucose (2DG) uptake showed nearly identical dose dependent effects, with both having maximum effects between 50 and 100 μM and similar half maximum effects at 8.9 and 8.5 μM respectively. The interaction of quercetin was rapid with t 1/2 of 54 s and the onset and loss of its inhibitory effects on 2DG uptake were equally fast. This suggests that either quercetin is simply binding to surface GLUT1 or its transport in and out of the cell reaches equilibrium very quickly. If quercetin is transported, the co-incubation of quercetin with other glucose inhibitors should block quercetin uptake. However, we observed that WZB-117, an exofacial binding inhibitor of GLUT1 reduced quercetin interaction, while cytochalasin B, an endofacial binding inhibitor, enhanced quercetin interaction, and BAY-876 had no effect on quercetin interaction. Taken together, these data are more consistent with quercetin simply binding to GLUT1, but not actually being transported into L929 cells via the glucose channel in GLUT1. Copyright © 2018. Published by Elsevier B.V.

Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

PubMed Central

Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

2009-01-01

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

The role of competitive binding to human serum albumin on efavirenz-warfarin interaction: a nuclear magnetic resonance study.

PubMed

Wanke, Riccardo; Harjivan, Shrika G; Pereira, Sofia A; Marques, M Matilde; Antunes, Alexandra M M

2013-11-01

The potential for co-prescription of the anti-human immunodeficiency virus (anti-HIV) drug efavirenz (EFV) and the oral anticoagulant warfarin (WAR) is currently high as EFV is a drug of choice for HIV type 1 infection and because cardiovascular disease is increasing among HIV-infected individuals. However, clinical reports of EFV-WAR interaction, leading to WAR overdosing, call for elucidation of the mechanisms involved in this drug-drug interaction. Here we present the first report demonstrating competition of the two drugs for the same binding site of human serum albumin. Using ligand-based nuclear magnetic resonance experiments, this study proves that EFV has an effect on the concentration of free WAR. This previously unidentified EFV-WAR interaction represents a potential risk factor that should be taken into account when considering treatment options. Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Quantification of the interceptor action of caffeine on the in vitro biological effect of the anti-tumour agent topotecan.

PubMed

Evstigneev, M P; Mosunov, A A; Evstigneev, V P; Parkes, H G; Davies, D B

2011-08-01

Using published in vitro data on the dependence of the percentage of apoptosis induced by the anti-cancer drug topotecan in a leukaemia cell line on the concentration of added caffeine, and a general model of competitive binding in a system containing two aromatic drugs and DNA, it has been shown to be possible to quantify the relative change in the biological effect just using a set of component concentrations and equilibrium constants of the complexation of the drugs. It is also proposed that a general model of competitive binding and parameterization of that model may potentially be applied to any system of DNA-targeting aromatic drugs under in vitro conditions. The main reasons underpinning the proposal are the general feature of the complexation of aromatic drugs with DNA and their interaction in physiological media via hetero-association.

Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.

PubMed

Mo, Zhi-Zhun; Wang, Xiu-Fen; Zhang, Xie; Su, Ji-Yan; Chen, Hai-Ming; Liu, Yu-Hong; Zhang, Zhen-Biao; Xie, Jian-Hui; Su, Zi-Ren

2015-07-16

The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C. The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. The IC50 of ASB against JBU and HPU was 3.28±0.13 mM and 3.17±0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki=2.86×10(-3) mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki*=1.33×10(-4) mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap. ASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

The structural basis for function in diamond-like carbon binding peptides.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Linder, Markus B

2014-07-29

The molecular structural basis for the function of specific peptides that bind to diamond-like carbon (DLC) surfaces was investigated. For this, a competition assay that provided a robust way of comparing relative affinities of peptide variants was set up. Point mutations of specific residues resulted in significant effects, but it was shown that the chemical composition of the peptide was not sufficient to explain peptide affinity. More significantly, rearrangements in the sequence indicated that the binding is a complex recognition event that is dependent on the overall structure of the peptide. The work demonstrates the unique properties of peptides for creating functionality at interfaces via noncovalent binding for potential applications in, for example, nanomaterials, biomedical materials, and sensors.

In vitro assessment of phthalate acid esters-trypsin complex formation.

PubMed

Chi, Zhenxing; Zhao, Jing; Li, Weiguo; Araghi, Arash; Tan, Songwen

2017-10-01

In this work, interactions of three phthalate acid esters (PAEs), including dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP), with trypsin have been studied in vitro, under simulated physiological conditions using multi-spectroscopic techniques and molecular modeling. The results show that these PAEs can bind to the trypsin, forming trypsin-PAEs complexes, mainly via hydrophobic interactions, with the affinity order of DMP > DEP > DBP. Binding to the PAEs is found to result in molecular deformation of trypsin. The modeling results suggest that only DBP can bind with the amino acid residues of the catalytic triad and S1 binding pocket of trypsin, leading to potential competitive enzyme inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Flow cytometer measurement of binding assays

DOEpatents

Saunders, George C.

1987-01-01

A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

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

McCabe, R.T.; Mahan, D.R.; Smith, R.B.

1990-10-01

The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptormore » sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.« less

Screening a fragment cocktail library using ultrafiltration

PubMed Central

Shibata, Sayaka; Zhang, Zhongsheng; Korotkov, Konstantin V.; Delarosa, Jaclyn; Napuli, Alberto; Kelley, Angela M.; Mueller, Natasha; Ross, Jennifer; Zucker, Frank H.; Buckner, Frederick S.; Merritt, Ethan A.; Verlinde, Christophe L. M. J.; Van Voorhis, Wesley C.; Hol, Wim G. J.; Fan, Erkang

2011-01-01

Ultrafiltration provides a generic method to discover ligands for protein drug targets with millimolar to micromolar Kd, the typical range of fragment-based drug discovery. This method was tailored to a 96-well format, and cocktails of fragment-sized molecules, with molecular masses between 150 and 300 Da, were screened against medical structural genomics target proteins. The validity of the method was confirmed through competitive binding assays in the presence of ligands known to bind the target proteins. PMID:21750879

The mechanism of interactions between tea polyphenols and porcine pancreatic alpha‐amylase: Analysis by inhibition kinetics, fluorescence quenching, differential scanning calorimetry and isothermal titration calorimetry

PubMed Central

Sun, Lijun; Gidley, Michael J.

2017-01-01

Scope This study aims to use a combination of biochemical and biophysical methods to derive greater mechanistic understanding of the interactions between tea polyphenols and porcine pancreatic α‐amylase (PPA). Methods and results The interaction mechanism was studied through fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) and compared with inhibition kinetics. The results showed that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red‐shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also suggested by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between the reciprocal of competitive inhibition constant (1/K ic), quenching constant (K FQ) and binding constant (K itc). Conclusion The combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA. PMID:28618113

Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action.

PubMed

Bradley, M E; Dombrecht, B; Manini, J; Willis, J; Vlerick, D; De Taeye, S; Van den Heede, K; Roobrouck, A; Grot, E; Kent, T C; Laeremans, T; Steffensen, S; Van Heeke, G; Brown, Z; Charlton, S J; Cromie, K D

2015-02-01

Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ

PubMed Central

FAULHAMMER, DIRK; ESCHGFÄLLER, BERND; STARK, SANDRA; BURGSTALLER, PETRA; ENGLBERGER, WERNER; ERFURTH, JEANNETTE; KLEINJUNG, FRANK; RUPP, JOHANNA; VULCU, SEBASTIAN DAN; SCHRÖDER, WERNER; VONHOFF, STEFAN; NAWRATH, HERMANN; GILLEN, CLEMENS; KLUSSMANN, SVEN

2004-01-01

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are l-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to d-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC50 values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPγS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K+ channels. PMID:14970396

Deciphering the mechanism of interaction of edifenphos with calf thymus DNA

NASA Astrophysics Data System (ADS)

Ahmad, Ajaz; Ahmad, Masood

2018-01-01

Edifenphos is an important organophosphate pesticide with many antifungal and anti-insecticidal properties but it may cause potential hazards to human health. In this work, we have tried to explore the binding mode of action and mechanism of edifenphos to calf thymus DNA (CT-DNA). Several experiments such as ultraviolet-visible absorption spectra and emission spectroscopy showed complex formation between edifenphos and CT-DNA and low binding constant values supporting groove binding mode. These results were further confirmed by circular dichroism (CD), CT-DNA melting studies, viscosity measurements, density functional theory and molecular docking. CD study suggests that edifenphos does not alter native structure of CT-DNA. Isothermal calorimetry reveals that binding of edifenphos with CT-DNA is enthalpy driven process. Competitive binding assay and effect of ionic strength showed that edifenphos binds to CT-DNA via groove binding manner. Hence, edifenphos is a minor groove binder preferably interacting with A-T regions with docking score - 6.84 kJ/mol.

Binding of alkylphenols and alkylated non-phenolics to the rainbow trout (Oncorhynchus mykiss) plasma sex steroid-binding protein.

PubMed

Tollefsen, K-E

2007-09-01

Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). Although the estrogenic properties of the alkylphenols are well documented, alternative mechanisms of action are poorly described. In the present work, the interaction of a range of alkyl-substituted phenols and alkyl-substituted non-phenolics with the rainbow trout (Oncorhynchus mykiss) sex steroid-binding protein (rtSBP) were determined by competitive ligand-binding studies. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure were assessed. The results showed that the rtSBP binds to most chemical structures tested, although the highest affinity was obtained for mono-substituted alkylphenols with a chain length of four to eight methyl groups. Interestingly, rtSBP binding was also observed for non-phenolic compounds such as 4-t-butylcyclohexanol and 4-t-butylnitrobenzene suggesting that the rtSBP has a broad binding specificity for alkylphenols and alkylated non-phenolics.

The coastal environment affects lead and sodium uptake by the moss Hypnum cupressiforme used as an air pollution biomonitor.

PubMed

Renaudin, Marie; Leblond, Sébastien; Meyer, Caroline; Rose, Christophe; Lequy, Emeline

2018-02-01

Several studies suggest that potential competition exists between marine cations and heavy metals for binding sites on the cell wall of mosses. This competition would impact the heavy metal concentration measured in mosses by biomonitoring programs, which may underestimate air pollution by heavy metals in a coastal environment. In the present study, we aim to identify possible mechanisms affecting lead uptake by mosses in a coastal environment, specifically, the competition between lead (Pb 2+ ) and sodium (Na + ) for binding sites in Hypnum cupressiforme (Hc). We also compared the response of continental and coastal Hc populations to Pb 2+ exposure by immersing the moss samples in artificial solutions that comprised six experimental treatments and subsequently locating and quantifying Pb 2+ and Na + using the sequential elution technique and X-ray microanalyses with a scanning electron microscope. We demonstrated that high concentrations of Pb 2+ prevented Na + from binding to the cell wall. We also examined the effect of the salt acclimation of Hc on Pb 2+ and Na + accumulation. Coastal Hc populations accumulated more Na and less Pb than continental Hc populations in all treatments. Moreover, our results showed treatment effects on the intra/extracellular distribution of Na + , as well as site. This feedback on the influence of salt stress tolerance on Pb 2+ uptake by mosses requires further study and can be investigated for other heavy metals, leading to a better use of mosses as biomonitoring tools. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Raf-competitive K-Ras binder can fail to functionally antagonize signaling.

PubMed

Kauke, Monique J; Tisdale, Alison W; Kelly, Ryan L; Braun, Christian J; Hemann, Michael T; Wittrup, K Dane

2018-05-02

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically-encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacological efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Copyright ©2018, American Association for Cancer Research.

Macromolecular Competition Titration Method: Accessing Thermodynamics of the Unmodified Macromolecule–Ligand Interactions Through Spectroscopic Titrations of Fluorescent Analogs

PubMed Central

Bujalowski, Wlodzimierz; Jezewska, Maria J.

2011-01-01

Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligand–macromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of protein–nucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the protein–nucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying protein–nucleic acid interactions, it can generally be applied to any ligand–macromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined. PMID:21195223

Surface structural ion adsorption modeling of competitive binding of oxyanions by metal (hydr)oxides

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

Hiemstra, T.; Riemsdijk, W.H. van

1999-02-01

An important challenge in surface complexation models (SCM) is to connect the molecular microscopic reality to macroscopic adsorption phenomena. This study elucidates the primary factor controlling the adsorption process by analyzing the adsorption and competition of PO{sub 4}, AsO{sub 4}, and SeO{sub 3}. The authors show that the structure of the surface-complex acting in the dominant electrostatic field can be ascertained as the primary controlling adsorption factor. The surface species of arsenate are identical with those of phosphate and the adsorption behavior is very similar. On the basis of the selenite adsorption, The authors show that the commonly used 1pKmore » models are incapable to incorporate in the adsorption modeling the correct bidentate binding mechanism found by spectroscopy. The use of the bidentate mechanism leads to a proton-oxyanion ratio and corresponding pH dependence that are too large. The inappropriate intrinsic charge attribution to the primary surface groups and the condensation of the inner sphere surface complex to a point charge are responsible for this behavior of commonly used 2pK models. Both key factors are differently defined in the charge distributed multi-site complexation (CD-MUSIC) model and are based in this model on a surface structural approach. The CD-MUSIC model can successfully describe the macroscopic adsorption phenomena using the surface speciation and binding mechanisms as found by spectroscopy. The model is also able to predict the anion competition well. The charge distribution in the interface is in agreement with the observed structure of surface complexes.« less

A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide.

PubMed

Wessels, M R; Muñoz, A; Kasper, D L

1987-12-01

We recently reported that the single repeating-unit pentasaccharide of type III group B Streptococcus (GBS) capsular polysaccharide is only weakly reactive with type III GBS antiserum. To further elucidate the relationship between antigen-chain length and antigenicity, tritiated oligosaccharides derived from type III capsular polysaccharide were used to generate detailed saturation binding curves with a fixed concentration of rabbit antiserum in a radioactive antigen-binding assay. A graded increase in affinity of antigen-antibody binding was seen as oligosaccharide size increased from 2.6 repeating units to 92 repeating units. These differences in affinity of antibody binding to oligosaccharides of different molecular size were confirmed by immunoprecipitation and competitive ELISA, two independent assays of antigen-antibody binding. Analysis of the saturation binding experiment indicated a difference of 300-fold in antibody-binding affinity for the largest versus the smallest tested oligosaccharides. Unexpectedly, the saturation binding values approached by the individual curves were inversely related to oligosaccharide chain length on a molar basis but equivalent on a weight basis. This observation is compatible with a model in which binding of an immunoglobulin molecule to an antigenic site on the polysaccharide facilitates subsequent binding of antibody to that antigen.

Staufen-mediated mRNA decay.

PubMed

Park, Eonyoung; Maquat, Lynne E

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. Copyright © 2013 John Wiley & Sons, Ltd.

Interactions of L-3,5,3'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes

PubMed Central

Westergard, Thomas; Salari, Reza; Martin, Joseph V.; Brannigan, Grace

2015-01-01

Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3’-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone. PMID:26421724

Interactions of L-3,5,3'-Triiodothyronine [corrected], Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

PubMed

Westergard, Thomas; Salari, Reza; Martin, Joseph V; Brannigan, Grace

2015-01-01

Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

Surface-enhanced Raman spectroscopy competitive binding biosensor development utilizing surface modification of silver nanocubes and a citrulline aptamer

NASA Astrophysics Data System (ADS)

Walton, Brian M.; Jackson, George W.; Deutz, Nicolaas; Cote, Gerard

2017-07-01

A point-of-care (PoC) device with the ability to detect biomarkers at low concentrations in bodily fluids would have an enormous potential for medical diagnostics outside the central laboratory. One method to monitor analytes at low concentrations is by using surface-enhanced Raman spectroscopy (SERS). In this preliminary study toward using SERS for PoC biosensing, the surface of colloidal silver (Ag) nanocubes has been modified to test the feasibility of a competitive binding SERS assay utilizing aptamers against citrulline. Specifically, Ag nanocubes were functionalized with mercaptobenzoic acid, as well as a heterobifunctional polyethylene glycol linker that forms an amide bond with the amino acid citrulline. After the functionalization, the nanocubes were characterized by zeta-potential, transmission electron microscopy images, ultraviolet/visible spectroscopy, and by SERS. The citrulline aptamers were developed and tested using backscattering interferometry. The data show that our surface modification method does work and that the functionalized nanoparticles can be detected using SERS down to a 24.5 picomolar level. Last, we used microscale thermophoresis to show that the aptamers bind to citrulline with at least a 50 times stronger affinity than other amino acids.

Z-DNA binding protein from chicken blood nuclei

NASA Technical Reports Server (NTRS)

Herbert, A. G.; Spitzner, J. R.; Lowenhaupt, K.; Rich, A.

1993-01-01

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor.

PubMed

Cederblad, Linda; Rosengren, Birgitta; Ryberg, Erik; Hermansson, Nils-Olov

2016-03-01

The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [(35)S]GTPγS (guanosine 5'-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a β-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of (125)I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and β-arrestin recruitment in a biased way. © 2016 Authors.

Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-{alpha}-induced NF-{kappa}B activity by binding to TNFR1

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

Hwang, Jae Ryoung; Huh, Jae Ho; Lee, Yoonna

2011-02-25

Research highlights: {yields} Binding assays demonstrated that secreted- and cellular-IGFBP-5 interacted with TNFR1. {yields} The interaction between IGFBP-5 and TNFR1 was inhibited by TNF-{alpha} and was blocked TNF-{alpha}-activated NF-{kappa}B activity. {yields} IGFBP-5 interacted with TNFR1 through its N- and L-domains but the binding of L-domain to TNFR1 was blocked by TNF-{alpha}. {yields} Competition between the L-domain of IGFBP-5 and TNF-{alpha} blocked TNF-{alpha}-induced NF-{kappa}B activity. {yields} This study suggests that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor. -- Abstract: IGFBP-5 is known to be involved in various cell phenomena such as proliferation,more » differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-{alpha}, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-{kappa}B activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-{alpha} in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-{alpha} resulted in inhibition of TNF-{alpha}-induced NF-{kappa}{Beta} activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor.« less

Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase.

PubMed

Townley, Robert; Shapiro, Lawrence

2007-03-23

The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular ATP (adenosine triphosphate) and AMP concentrations. Here, we report crystal structures at 2.9 and 2.6 A resolution for ATP- and AMP-bound forms of a core alphabetagamma adenylate-binding domain from the fission yeast AMPK homolog. ATP and AMP bind competitively to a single site in the gamma subunit, with their respective phosphate groups positioned near function-impairing mutants. Unexpectedly, ATP binds without counterions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

Comparative theoretical study of the binding of luciferyl-adenylate and dehydroluciferyl-adenylate to firefly luciferase

NASA Astrophysics Data System (ADS)

Pinto da Silva, Luís; Vieira, João; Esteves da Silva, Joaquim C. G.

2012-08-01

This is the first report of a study employing a computational approach to study the binding of (D/L)-luciferyl-adenlyates and dehydroluciferyl-adenylate to firefly luciferase. A semi-empirical/molecular mechanics methodology was used to study the interaction between these ligands and active site molecules. All adenylates are complexed with the enzyme, mostly due to electrostatic interactions with cationic residues. Dehydroluciferyl-adenylate is expected to be a competitive inhibitor of luciferyl-adenylate, as their binding mechanism and affinity to luciferase are very similar. Both luciferyl-adenylates adopt the L-orientation in the active site of luciferase.

Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

PubMed

Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K

1992-05-01

Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

Comparative effectiveness of Calabadion and sugammadex to reverse non-depolarizing neuromuscular blocking agents

PubMed Central

Haerter, Friederike; Simons, Jeroen Cedric Peter; Foerster, Urs; Duarte, Ingrid Moreno; Diaz-Gil, Daniel; Ganapati, Shweta; Eikermann-Haerter, Katharina; Ayata, Cenk; Zhang, Ben; Blobner, Manfred; Isaacs, Lyle; Eikermann, Matthias

2015-01-01

Background We evaluated the comparative effectiveness of calabadion 2 to reverse non-depolarizing neuromuscular blocking agents (NMBAs) by binding and inactivation. Methods The dose-response relationship of drugs to reverse vecuronium, rocuronium, and cisatracurium-induced neuromuscular block (NMB) was evaluated in vitro (competition binding assays and urine analysis), ex vivo (n=34; phrenic nerve hemidiaphragm preparation) and in vivo (n=108; quadriceps femoris muscle of the rat). Cumulative dose-response curves of calabadions, neostigmine, or sugammadex were created ex vivo at steady-state deep NMB. In living rats, we studied the dose-response relationship of the test drugs to reverse deep block under physiological conditions and we measured the amount of calabadion 2 excreted in the urine. Results In vitro experiments showed that calabadion 2 binds rocuronium with 89 times the affinity of sugammadex (Ka = 3.4 × 109 M−1 and Ka = 3.8 × 107 M−1). Urine analysis (proton nuclear magnetic resonance), competition binding assays and ex vivo study results obtained in the absence of metabolic deactivation are in accordance with an 1:1 binding ratio of sugammadex and calabadion 2 toward rocuronium. In living rats, calabadion 2 dose-dependently and rapidly reversed all NMBAs tested. The molar potency of calabadion 2 to reverse vecuronium and rocuronium was higher compared to sugammadex. Calabadion 2 was eliminated renally, and did not affect blood pressure or heart rate. Conclusion Calabadion 2 reverses NMB-induced by benzylisoquinolines and steroidal NMBAs in rats more effectively, i.e. faster, than sugammadex. Calabadion 2 is eliminated in the urine and well tolerated in rats. PMID:26418697

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

PubMed

Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

2015-03-31

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples. Copyright © 2015 Elsevier B.V. All rights reserved.

[High non-specific binding of the beta(1) -selective radioligand 2-(125)I-ICI-H].

PubMed

Riemann, B; Law, M P; Kopka, K; Wagner, St; Luthra, S; Pike, V W; Neumann, J; Kirchhefer, U; Schmitz, W; Schober, O; Schäfers, M

2003-08-01

As results of cardiac biopsies suggest, myocardial beta(1) -adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac beta(2)-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess beta-adrenoceptors non-invasively. Among the novel racemic analogues of the established beta(1)-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to beta(1)-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative sub-type selective beta(1)-adrenergic radioligand in cardiac imaging. Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac beta-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-(125)I-ICI-H from the silylated precursor 2-SiMe(3)-ICI-H was established. The specific activity was 80 GBq/ micro mol, the radiochemical yield ranged from 70 to 80%. The unlabelled compound 2-I-ICI-H showed high beta(1)-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac beta-adrenoceptors. The radiolabelled counterpart 2-(125)I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac beta(1)-adrenoceptors in vivo. Because of its high non-specific binding 2-(125)I-ICI-H is no suitable radiotracer for imaging in vivo.

SNHG16 contributes to breast cancer cell migration by competitively binding miR-98 with E2F5.

PubMed

Cai, Chang; Huo, Qiang; Wang, Xiaolong; Chen, Bing; Yang, Qifeng

2017-04-01

Long noncoding RNAs (lncRNAs) have been proved to play important roles in cellular processes of cancer, including the development, proliferation, and migration of cancer cells. In the present study, we demonstrated small nucleolar RNA host gene 16 (SNHG16) as an oncogene on cell migration in breast cancer. Expression levels of SNHG16 were found to be frequently higher in breast cancer tissues than in the paired noncancerous tissues. Gain- and loss-of-function studies proved that SNHG16 significantly promoted breast cancer cell migration. We predicted SNHG16 as a competitive endogenous RNA (ceRNA) of E2F transcription factor 5 protein (E2F5) via competition for the shared miR-98 through bioinformatics analysis, and proved this regulation using relative quantitative real-time PCR (qRT-PCR), western blot, RNA immunoprecipitation (RIP) assay and luciferase reporter assay. In addition, we identified a positive correlation between SNHG16 and E2F5 in breast cancer tissues. Furthermore, we demonstrated that forced expression of miR-98 could partially abrogate SNHG16-mediated increase of breast cancer cells migration, suggesting that SNHG16 promoted cell migration in a miR-98 dependent manner. Taken together, our findings indicated that SNHG16 induces breast cancer cell migration by competitively binding miR-98 with E2F5, and SNHG16 can serve as a potential therapeutic target for breast cancer treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Tiberi, M.; Magnan, J.

The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid (3H)D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid (3H)D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid (3H)U69593 (Kd = 3.31 nM, Rmore » = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by (3H)U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan (3H)ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, (3H)ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of (3H)ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with (3H)U69593. Saturation studies using the nonselective opioid (3H)bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue).« less

Complexes of lutein with bovine and caprine caseins and their impact on lutein chemical stability in emulsion systems: Effect of arabinogalactan.

PubMed

Mora-Gutierrez, A; Attaie, R; Núñez de González, M T; Jung, Y; Woldesenbet, S; Marquez, S A

2018-01-01

Lutein is an important xanthophyll carotenoid with many benefits to human health. Factors affecting the application of lutein as a functional ingredient in low-fat dairy-like beverages (pH 6.0-7.0) are not well understood. The interactions of bovine and caprine caseins with hydrophobic lutein were studied using UV/visible spectroscopy as well as fluorescence. Our studies confirmed that the aqueous solubility of lutein is improved after binding with bovine and caprine caseins. The rates of lutein solubilization by the binding to bovine and caprine caseins were as follows: caprine α S1 -II-casein 34%, caprine α S1 -I-casein 10%, and bovine casein 7% at 100 μM lutein. Fluorescence of the protein was quenched on binding supporting complex formation. The fluorescence experiments showed that the binding involves tryptophan residues and some nonspecific interactions. Scatchard plots of lutein binding to the caseins demonstrated competitive binding between the caseins and their sites of interaction with lutein. Competition experiments suggest that caprine α S1 -II casein will bind a larger number of lutein molecules with higher affinity than other caseins. The chemical stability of lutein was largely dependent on casein type and significant increases occurred in the chemical stability of lutein with the following pattern: caprine α S1 -II-casein > caprine α S1 -I-casein > bovine casein. Addition of arabinogalactan to lutein-enriched emulsions increases the chemical stability of lutein-casein complexes during storage under accelerated photo-oxidation conditions at 25°C. Therefore, caprine α S1 -II-casein alone and in combination with arabinogalactan can have important applications in the beverage industry as carrier of this xanthophyll carotenoid (lutein). Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Competition-cooperation relationship networks characterize the competition and cooperation between proteins

PubMed Central

Li, Hong; Zhou, Yuan; Zhang, Ziding

2015-01-01

By analyzing protein-protein interaction (PPI) networks, one can find that a protein may have multiple binding partners. However, it is difficult to determine whether the interactions with these partners occur simultaneously from binary PPIs alone. Here, we construct the yeast and human competition-cooperation relationship networks (CCRNs) based on protein structural interactomes to clearly exhibit the relationship (competition or cooperation) between two partners of the same protein. If two partners compete for the same interaction interface, they would be connected by a competitive edge; otherwise, they would be connected by a cooperative edge. The properties of three kinds of hubs (i.e., competitive, modest, and cooperative hubs) are analyzed in the CCRNs. Our results show that competitive hubs have higher clustering coefficients and form clusters in the human CCRN, but these tendencies are not observed in the yeast CCRN. We find that the human-specific proteins contribute significantly to these differences. Subsequently, we conduct a series of computational experiments to investigate the regulatory mechanisms that avoid competition between proteins. Our comprehensive analyses reveal that for most yeast and human protein competitors, transcriptional regulation plays an important role. Moreover, the human-specific proteins have a particular preference for other regulatory mechanisms, such as alternative splicing. PMID:26108281

3H[2-(2-benzofuranyl)-2-imidazoline] (BFI) binding in human platelets: modulation by tranylcypromine.

PubMed

Wiest, S A; Steinberg, M I

1999-08-01

2-(2-Benzofuranyl)-2-imidazoline (BFI) is a highly selective ligand for imidazoline-type 2 (I2) binding sites that are known to be associated with monoamine oxidase (MAO). Recently we demonstrated a potentiation of 3H-BFI binding in human but not in rat brain by the nonselective MAO inhibitor tranylcypromine. In the present studies, we evaluated the effect of tranylcypromine on the binding of 3H-BFI to human platelet inner membranes. Membranes were incubated with 3H-BFI at 22 degrees C in 50 mM Tris, 1.5 mM EDTA, pH 7.5. Saturation experiments with 3H-BFI (0.5-80 nM) were analyzed using non-linear curve fitting. Addition of tranylcypromine (0.1 mM) increased the number of 3H-BFI binding sites (Bmax=0.35+/-0.06 vs. 1.87+/-0.15 pmol/mg protein for vehicle and tranylcypromine, respectively) and increased 3H-BFI affinity slightly (KD =16.0+/-4.1 vs. 6.5+/-0.3 nM for vehicle and tranylcypromine, respectively). In competitive binding experiments using the less selective I2 ligand, 3H-idazoxan, tranylcypromine only weakly inhibited binding. Preincubation of platelet membranes with tranylcypromine (1 nM-10 microM) enhanced the Bmax of 3H-BFI binding in a concentration-dependent manner peaking at 1 microM (13 x control) and returning to near baseline at 100 microM. 3H-BFI binding was displaced monophasically (in order of decreasing potency) by BFI > or = 2-(4,5-dihydroimidazol-2-yl)quinoline (BU224) > or = cirazoline >idazoxan >(1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline (RX821002)= moxonidine. Amiloride, clorgyline, guanabenz and clonidine displayed biphasic curves with nanomolar high affinity components. Tranylcypromine altered the competition curves for all ligands (except BFI) by increasing the affinities for clonidine and RX821002 and decreasing affinities for BU224, cirazoline, guanabenz, idazoxan, clorgyline, moxonidine, and amiloride. Thus, in human platelets tranylcypromine exposes a high capacity 3H-BFI binding site distinct from previously described I2 sites that retains high affintiy for BFI but not other I2 ligands. Our results suggest that 3H-BFI and 3H-idazoxan may not be considered as interchangeable probes for the I2 binding site.

Pharmacological characterization of CCKB receptors in human brain: no evidence for receptor heterogeneity.

PubMed

Kinze, S; Schöneberg, T; Meyer, R; Martin, H; Kaufmann, R

1996-10-11

In this paper, cholecystokinin (CCK) B-type binding sites were characterized with receptor binding studies in different human brain regions (various parts of cerebral cortex, basal ganglia, hippocampus, thalamus, cerebellar cortex) collected from 22 human postmortem brains. With the exception of the thalamus, where no specific CCK binding sites were found, a pharmacological characterization demonstrated a single class of high affinity CCK sites in all brain areas investigated. Receptor densities ranged from 0.5 fmol/mg protein (hippocampus) to 8.4 fmol/mg protein (nucleus caudatus). These CCK binding sites displayed a typical CCKA binding profile as shown in competition studies by using different CCK-related compounds and non peptide CCK antagonists discriminating between CCKA and CCKB sites. The rank order of agonist or antagonist potency in inhibiting specific sulphated [propionyl-3H]cholecystokinin octapeptide binding was similar and highly correlated for the brain regions investigated as demonstrated by a computer-assisted analysis. Therefore it is concluded that CCKB binding sites in human cerebral cortex, basal ganglia, cerebellar cortex share identical ligand binding characteristics.

Binding modes of environmental endocrine disruptors to human serum albumin: insights from STD-NMR, ITC, spectroscopic and molecular docking studies.

PubMed

Yang, Hongqin; Huang, Yanmei; Liu, Jiuyang; Tang, Peixiao; Sun, Qiaomei; Xiong, Xinnuo; Tang, Bin; He, Jiawei; Li, Hui

2017-09-11

Given that bisphenols have an endocrine-disrupting effect on human bodies, thoroughly exposing their potential effects at the molecular level is important. Saturation transfer difference (STD) NMR-based binding studies were performed to investigate the binding potential of two bisphenol representatives, namely, bisphenol B (BPB) and bisphenol E (BPE), toward human serum albumin (HSA). The relative STD (%) suggested that BPB and BPE show similar binding modes and orientations, in which the phenolic rings were spatially close to HSA binding site. ITC analysis results showed that BPB and BPE were bound to HSA with moderately strong binding affinity through electrostatic interactions and hydrogen bonds. The order of binding affinity of HSA for two test bisphenols is as follows: BPE > BPB. The results of fluorescence competitive experiments using 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine as competitors, combined with molecular docking indicated that both bisphenols are prone to attach to the binding site II in HSA. Spectroscopic results (FT-IR, CD, synchronous and 3D fluorescence spectra) showed that BPB/BPE induces different degrees of microenvironmental and conformational changes to HSA.

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

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

Morrison, W.J.; Offner, H.; Vandenbark, A.A.

1989-01-01

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphoctyes. Saturation binding by monosialylated ({sub 3}H)-GM{sub 1} to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (K{sub D}) of 2.2 {plus minus} 1.4 {mu}M and a binding capacity near 2 fmoles/cell. Competitive inhibition of ({sup 3}H)- GM{sub 1} binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM{sub 1}-congeneric gangliosides. A comparison between the results of these binding studies and gangliosides-induced decrease of CD4 expression demonstrated that every aspect of ({supmore » 3}H)-GM{sub 1} binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of gangliosides binding to specific sites on CD4{sup {double dagger}} T-helper lymphocytes.« less

Targeted binding of the M13 bacteriophage to thiamethoxam organic crystals.

PubMed

Cho, Whirang; Fowler, Jeffrey D; Furst, Eric M

2012-04-10

Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity. © 2012 American Chemical Society

Complex high affinity interactions occur between MHCI and superantigens

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

1998-01-01

Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

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

Unterberger, Claudia; Hanson, Steven; Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 tomore » be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.« less

Distribution and density of substance P receptors in the feline gastrointestinal tract using autoradiography

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

Rothstein, R.D.; Johnson, E.; Ouyang, A.

1991-06-01

Autoradiography was used to localize and quantify substance P receptors in the feline gastrointestinal tract. The specific binding of {sup 125}I-Bolton Hunter substance P was determined in the esophagus, lower esophageal sphincter, antrum, pylorus, duodenum, jejunum, ileum, ileocecal sphincter, and colon. Competitive binding studies indicated that substance P binding sites or NK-1 receptor sites were demonstrated. The concentration of NK-1 receptors was greatest in the distal half of the gastrointestinal tract, with the highest concentrations in the proximal colon. The circular muscle layer contained the greatest amount of substance P binding. The location and density of binding sites for substancemore » P may be important in understanding the relative importance of both the pharmacological responses to this neuropeptide and the immunohistochemical evidence of the peptide at different sites in the intestine.« less

Binding of /sup 125/I-hCG to rainbow trout (Salmo gairdneri) testis in vitro. [Human Chorionic Gonadotropin

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

Schlaghecke, R.

1983-02-01

Homogenates of maturing rainbow trout testes show specific binding sites for /sup 125/I-labeled hCG (. /sup 125/I-labeled hCG). The binding is competitively inhibited by unlabeled hCG and by a hypophyseal extract of rainbow trout. It could be demonstrated that the tissue /sup 125/I-hCG binding specificity is restricted to the gonadal preparation. The trout testis was characterized by determining affinity and capacity from Scatchard plot analysis giving a high constant of dissociation Kd 3.65 x 10(-10)/M and a low binding capacity of 0.88 x 10(-15) M/mg tissue. The test system is markedly dependent on temperature, incubation-time, and pH. The maximum bindingmore » was found at 37 degrees during 2 hr of incubation in a buffer of pH 7.5.« less

Determination of the binding properties of p-cresyl glucuronide to human serum albumin.

PubMed

Yi, Dan; Monteiro, Elisa Bernardes; Chambert, Stéphane; Soula, Hédi A; Daleprane, Julio B; Soulage, Christophe O

2018-04-26

p-Cresyl glucuronide (p-CG) is a by-product of tyrosine metabolism that accumulates in patients with end-stage renal disease. p-CG binding to human serum albumin in physiological conditions (37°C, pH 7.40) was studied by ultrafiltration (MWCO 10 kDa) and data were analyzed assuming one binding site. The estimated value of the association constant was 2.77×10 3  M -1 and a maximal stoichiometry of 3.80 mol per mole. At a concentration relevant for end-stage renal patients, p-CG was 23% bound to albumin. Competition experiments, using fluorescent probes, demonstrated that p-CG did not bind to Sudlow's site I or site II. The p-CG did not interfere with the binding of p-cresyl-sulfate or indoxyl sulfate to serum albumin. Copyright © 2018. Published by Elsevier B.V.

Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence*

PubMed Central

Tsujii, Akira; Miyamoto, Yoichi; Moriyama, Tetsuji; Tsuchiya, Yuko; Obuse, Chikashi; Mizuguchi, Kenji; Oka, Masahiro; Yoneda, Yoshihiro

2015-01-01

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence. PMID:26491019

(/sup 3/H)Batrachotoxinin A 20 alpha-benzoate binding to voltage-sensitive sodium channels: a rapid and quantitative assay for local anesthetic activity in a variety of drugs

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

McNeal, E.T.; Lewandowski, G.A.; Daly, J.W.

1985-03-01

(/sup 3/H)Batrachotoxinin A benzoate ((/sup 3/H)BTX-B) binds with high affinity to sites on voltage-dependent sodium channels in a vesicular preparation from guinea pig cerebral cortex. In this preparation, local anesthetics competitively antagonize the binding of (/sup 3/H)BTX-B. The potencies of some 40 classical local anesthetics and a variety of catecholamine, histamine, serotonin, adenosine, GABA, glycine, acetylcholine, and calcium antagonists, tranquilizers, antidepressants, barbiturates, anticonvulsants, steroids, vasodilators, antiinflammatories, anticoagulants, analgesics, and other agents have been determined. An excellent correlation with the known local anesthetic activity of many of these agents indicate that antagonism of binding of (/sup 3/H)BTX-B binding provides a rapid,more » quantitative, and facile method for the screening and investigation of local anesthetic activity.« less

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

PubMed Central

Marciniak, R A; Garcia-Blanco, M A; Sharp, P A

1990-01-01

Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo. Images PMID:2333305

Ab initio molecular dynamics determination of competitive O₂ vs. N₂ adsorption at open metal sites of M₂(dobdc).

PubMed

Parkes, Marie V; Greathouse, Jeffery A; Hart, David B; Gallis, Dorina F Sava; Nenoff, Tina M

2016-04-28

The separation of oxygen from nitrogen using metal-organic frameworks (MOFs) is of great interest for potential pressure-swing adsorption processes for the generation of purified O2 on industrial scales. This study uses ab initio molecular dynamics (AIMD) simulations to examine for the first time the pure-gas and competitive gas adsorption of O2 and N2 in the M2(dobdc) (M = Cr, Mn, Fe) MOF series with coordinatively unsaturated metal centers. Effects of metal, temperature, and gas composition are explored. This unique application of AIMD allows us to study in detail the adsorption/desorption processes and to visualize the process of multiple guests competitively binding to coordinatively unsaturated metal sites of a MOF.

Structure-function relationships for the selective inhibition of human 3β-hydroxysteroid dehydrogenase type 1 by a novel androgen analog.

PubMed

Pham, Jenny H; Will, Catherine M; Mack, Vance L; Halbert, Matthew; Conner, Edward Alexander; Bucholtz, Kevin M; Thomas, James L

2017-11-01

3β-Hydroxysteroid dehydrogenase type 1 (3β-HSD1) is selectively expressed in human placenta, mammary glands and breast tumors in women. Human 3β-HSD2 is selectively expressed in adrenal glands and ovaries. Based on AutoDock 3 and 4 results, we have exploited key differences in the amino acid sequences of 3β-HSD1 (Ser194, Arg195) and 3β-HSD2 (Gly194, Pro195) by designing a selective inhibitor of 3β-HSD1. 2,16-Dicyano-4,5-epoxy-androstane-3,17-dione (16-cyano-17-keto-trilostane or DiCN-AND) was synthesized in a 4-step procedure from androstenedione. In purified 3β-HSD inhibition studies, DiCN-AND competitively inhibited 3β- HSD1 with K i =4.7μM and noncompetitively inhibited 3β-HSD2 with a 6.5-fold higher K i =30.7μM. We previously reported similar isoenzyme-specific inhibition profiles for trilostane. Based on our docking results, we created, expressed and purified the chimeric S194G-1 mutant of 3β-HSD1. Trilostane inhibited S194G-1 (K i =0.67μM) with a noncompetitive mode compared to its 6.7-fold higher affinity, competitive inhibition of 3β-HSD1 (K i =0.10μM). DiCN-AND inhibited S194G-1 with a 6.3-fold higher K i (29.5μM) than measured for 3β-HSD1 (K i =4.7μM) but with the same competitive mode for both enzyme species. Since DiCN-AND noncompetitively inhibits 3β-HSD2, which has the Gly194 and Pro195 of 3β-HSD2 in place of the Ser194 and Arg195 in 3β-HSD1, this suggests that Arg195 alone in 3β-HSD1 or S194G-1 is required to bind DiCN-AND in the substrate binding site (competitive inhibition). However, both Ser194 and Arg195 are required to bind trilostane in the 3β-HSD1 substrate site based on its noncompetitive inhibition of S194G-1 and 3β-HSD2. In support of this hypothesis, DiCN-AND inhibited our chimeric R195P-1 mutant noncompetitively with a K i =41.3μM (similar to the 3β-HSD2 inhibition profile). Since DiCN-AND competitively inhibited S194G-1 that still contains R195 but noncompetitively inhibited R195P-1 that still contains S194, our data provides strong evidence that the Arg195 being mutated to Pro195 (as present in 3β-HSD2) shifts the inhibition mode from competitive to noncompetitive in 3β-HSD1. This supports the key role of Arg195 in 3β-HSD1 for the high affinity, competitive binding of the trilostane analogs. Our new structure/function information for the design of targeted 3β-HSD1 inhibitors may lead to important new treatments for the prevention of spontaneous premature birth. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

PubMed

Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B; Bird, Kelly E; Ginther, Patrick S; Rivalta, Ivan; Ten, Nicholas S; Batista, Victor S; Loria, J Patrick

2017-01-10

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.

Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

PubMed

Nobrega, R Paul; Brown, Michael; Williams, Cody; Sumner, Chris; Estep, Patricia; Caffry, Isabelle; Yu, Yao; Lynaugh, Heather; Burnina, Irina; Lilov, Asparouh; Desroches, Jordan; Bukowski, John; Sun, Tingwan; Belk, Jonathan P; Johnson, Kirt; Xu, Yingda

2017-10-01

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

A novel anti-EGFR monoclonal antibody inhibiting tumor cell growth by recognizing different epitopes from cetuximab.

PubMed

Hong, Kwang-Won; Kim, Chang-Goo; Lee, Seung-Hyun; Chang, Ki-Hwan; Shin, Yong Won; Ryoo, Kyung-Hwan; Kim, Se-Ho; Kim, Yong-Sung

2010-01-01

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.

Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

PubMed

Tsuganezawa, Keiko; Shinohara, Yoshiyasu; Ogawa, Naoko; Tsuboi, Shun; Okada, Norihisa; Mori, Masumi; Yokoyama, Shigeyuki; Noda, Nobuo N; Inagaki, Fuyuhiko; Ohsumi, Yoshinori; Tanaka, Akiko

2013-10-01

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds

PubMed Central

Rahman, Mona N.; Vukomanovic, Dragic; Vlahakis, Jason Z.; Szarek, Walter A.; Nakatsu, Kanji; Jia, Zongchao

2013-01-01

The development of heme oxygenase (HO) inhibitors, especially those that are isozyme-selective, promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties and may play a role in several disease states, making it an enticing therapeutic target. Traditionally, the metalloporphyrins have been used as competitive HO inhibitors owing to their structural similarity with the substrate, heme. However, given heme's important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), non-selectivity is an unfortunate side-effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort to develop novel compounds as potent, selective inhibitors of HO. This resulted in the creation of non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated, which provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. PMID:23097500

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

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

Lee, J.H.; el-Fakahany, E.E.

1985-06-01

The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/supmore » 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.« less

Comprehensive insight into the binding of sunitinib, a multi-targeted anticancer drug to human serum albumin

NASA Astrophysics Data System (ADS)

Kabir, Md. Zahirul; Tee, Wei-Ven; Mohamad, Saharuddin B.; Alias, Zazali; Tayyab, Saad

2017-06-01

Binding studies between a multi-targeted anticancer drug, sunitinib (SU) and human serum albumin (HSA) were made using fluorescence, UV-vis absorption, circular dichroism (CD) and molecular docking analysis. Both fluorescence quenching data and UV-vis absorption results suggested formation of SU-HSA complex. Moderate binding affinity between SU and HSA was evident from the value of the binding constant (3.04 × 104 M-1), obtained at 298 K. Involvement of hydrophobic interactions and hydrogen bonds as the leading intermolecular forces in the formation of SU-HSA complex was predicted from the thermodynamic data of the binding reaction. These results were in good agreement with the molecular docking analysis. Microenvironmental perturbations around Tyr and Trp residues as well as secondary and tertiary structural changes in HSA upon SU binding were evident from the three-dimensional fluorescence and circular dichroism results. SU binding to HSA also improved the thermal stability of the protein. Competitive displacement results and molecular docking analysis revealed the binding locus of SU to HSA in subdomain IIA (Sudlow's site I). The influence of a few common ions on the binding constant of SU-HSA complex was also noticed.

Human La binds mRNAs through contacts to the poly(A) tail.

PubMed

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-05-04

In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

PubMed

Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

2018-01-24

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

Effects of nucleotides on [3H]bradykinin binding in guinea pig: further evidence for multiple B2 receptor subtypes.

PubMed

Seguin, L; Widdowson, P S

1993-02-01

We have suggested recently the existence of three subtypes of B2 bradykinin receptors in tissues of guinea pigs. We have classified these B2 bradykinin receptors into B2a, B2b, and B2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [3H]bradykinin to the B2 subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B2a and B2b subtypes, specific [3H]bradykinin binding was reduced with GDP (100 microM) and the nonmetabolized analogue of GTP, guanyl-5'-yl-imidodiphosphate (GppNHp; 100 microM). Competition studies with bradykinin and with [Hyp3]bradykinin, which shows approximately 20-fold greater selectivity for the B2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 microM). The competition experiments demonstrated that binding to the B2a subtype, which has higher affinity for [Hyp3]bradykinin and bradykinin than the B2b subtype, was lost in the presence of GppNHp, whereas binding to the B2b subtype was unaffected. In contrast, GppNHp (100 microM) and GDP (100 microM) failed to alter specific [3H]bradykinin binding to B2b and B2c subtypes in lung. [3H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 microM each). Based on this evidence, we suggest that the B2a bradykinin subtype is coupled to G proteins. The B2b and B2c subtypes are either not coupled to G proteins, or may be coupled to the Go-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of ferrihydrite- and Metsorb™-DGT techniques for measuring oxyanion species (As, Se, V, P): effective capacity, competition and diffusion coefficients.

PubMed

Price, Helen L; Teasdale, Peter R; Jolley, Dianne F

2013-11-25

This study investigated several knowledge gaps with respect to the diffusive gradients in thin films (DGT) technique for measurement of oxyanions (As(III), As(V), Se(IV), Se(VI), PO4(3-), and V(V)) using the ferrihydrite and Metsorb™ binding layers. Elution efficiencies for each binding layer were higher with 1:20 dilutions, as analytical interferences for ICP-MS were minimised. Diffusion coefficients measured by diffusion cell and by DGT time-series experiments were found to agree well and generally agreed with previously reported values, although a range of diffusion coefficients have been reported for inorganic As and Se species. The relative binding affinity for both ferrihydrite and Metsorb™ was PO4(3-) ≈ As(V)>V(V) ≈ As(III)>Se(IV) > Se(VI) and effective binding capacities were measured in single ion solutions, and spiked synthetic freshwater and seawater, advising practical decisions about DGT monitoring. Under the conditions tested the performance of both ferrihydrite and Metsorb™ binding layers was directly comparable for As(V), As(III) Se(IV), V(V) and PO4(3-) over a deployment spanning ≤ 2 days for both freshwater and seawater. In order to return quantitative data for several analytes we recommend that the DGT method using either ferrihydrite or Metsorb™ be deployed for a maximum of 2 days in marine waters likely to contain high levels of the most strongly adsorbing oxyanions contaminants. The high pH, the competitive ions present in seawater and the identity of co-adsorbing ions affect the capacity of each binding layer for the analytes of interest. In freshwaters, longer deployment times can be considered but the concentration and identity of co-adsorbing ions may impact on quantitative uptake of Se(IV). This study found ferrihydrite-DGT outperformed Metsorb-DGT while previous studies have found the opposite, with variation in binding materials masses used being a likely reason. Clearly, preparation of both binding layers should always be optimised to produce the highest capacity possible, especially for seawater deployments. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

PubMed Central

Huang, Tao; Long, Mian; Huo, Bo

2010-01-01

Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

Characterization of a novel non-peptide vasopressin V1 receptor antagonist (OPC-21268) in the rat.

PubMed

Burrell, L M; Phillips, P A; Stephenson, J; Risvanis, J; Hutchins, A M; Johnston, C I

1993-08-01

A non-peptide, orally effective, vasopressin (AVP) V1 receptor antagonist 1-(1-[4-(3-acetylaminopropoxy) benzoyl]-4-piperidyl)-3,4-dihydro-2(1H)-quinolinone (OPC-21268) has recently been described. This paper reports the in-vitro and in-vivo characterization of OPC-21268 binding to vasopressin receptors in rat liver and kidney. OPC-21268 caused a concentration-dependent displacement of the selective V1 receptor antagonist radioligand, 125I-labelled [d(CH2)5,sarcosine7]AVP to V1 receptors in both rat liver and kidney medulla membranes. The concentration of OPC-21268 that displaced 50% of specific AVP binding (IC50) was 40 +/- 3 nmol/l for liver V1 and 15 +/- 2 nmol/l for kidney V1 receptors (mean +/- S.E.M.; n = 3). OPC-21268 had little effect on the selective V2 antagonist radioligand [3H]desGly-NH2(9)]d(CH2)5,D-Ile2,Ile4] AVP binding to V2 receptors in renal medulla membranes (IC50 > 0.1 mmol/l). After oral administration to rats, OPC-21268 was an effective V1 antagonist in a time- and dose-dependent manner. Binding kinetic studies showed that OPC-21268 acted as a competitive antagonist at the liver V1 receptor in vitro and in vivo, in addition to its in-vitro competitive effects at the renal V1 receptor. OPC-21268 shows promise as an orally active V1 antagonist.

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

PubMed

Mou, Linkai; Cui, Tongwei; Liu, Weiguang; Zhang, Hong; Cai, Zhanxiu; Lu, Shaoyong; Gao, Guojun

2017-05-01

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity. © 2016 John Wiley & Sons A/S.

The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility.

PubMed

Rapp, Micah; Schein, Jessica; Hunt, Kevin A; Nalam, Vamsi; Mourad, George S; Schultes, Neil P

2016-03-01

The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor-based assay for monitoring the drug atenolol.

PubMed

Sapir, A; Shalev, A Hariton; Skalka, N; Bronshtein, A; Altstein, M

2013-03-01

Two approaches for monitoring atenolol (ATL) were applied: an immunochemical assay and a competitive-binding assay, based on the interaction between ATL and its target receptor, β1 adrenergic receptor (β1AR). Polyclonal antibodies (Abs) for ATL were generated, and a highly specific microplate immunochemical assay, that is, an enzyme-linked immunosorbent assay (ELISA), for its detection was developed. The ATL ELISA exhibited I50 and limit of detection (I20) values of 0.15 ± 0.048 and 0.032 ± 0.016 ng/ml, respectively, and the Abs did not cross-react with any of the tested beta-blocker drugs. Furthermore, a human β1AR (h-β1AR) was stably expressed in Spodoptera frugiperda cells (Sf9). The receptor was employed to develop a competitive-binding assay that monitored binding of ATL in the presence of isoproteranol by quantification of secondary messenger, cyclic adenosine monophosphate (cAMP), levels in the transfected cells. The assay showed that the recombinant h-β1AR was functional, could bind the agonistic ligand isoproterenol as well as the antagonist ATL, as indicated by a dose-dependent elevation of cAMP in the presence of isoproteranol, and decrease after ATL addition. The highly efficient and sensitive ELISA and the receptor assay represent two methods suitable for efficient and cost-effective large-scale, high-throughput monitoring of ATL in environmental, agricultural, and biological samples. Copyright © 2012 SETAC.

Estimation of relative free energies of binding using pre-computed ensembles based on the single-step free energy perturbation and the site-identification by Ligand competitive saturation approaches.

PubMed

Raman, E Prabhu; Lakkaraju, Sirish Kaushik; Denny, Rajiah Aldrin; MacKerell, Alexander D

2017-06-05

Accurate and rapid estimation of relative binding affinities of ligand-protein complexes is a requirement of computational methods for their effective use in rational ligand design. Of the approaches commonly used, free energy perturbation (FEP) methods are considered one of the most accurate, although they require significant computational resources. Accordingly, it is desirable to have alternative methods of similar accuracy but greater computational efficiency to facilitate ligand design. In the present study relative free energies of binding are estimated for one or two non-hydrogen atom changes in compounds targeting the proteins ACK1 and p38 MAP kinase using three methods. The methods include standard FEP, single-step free energy perturbation (SSFEP) and the site-identification by ligand competitive saturation (SILCS) ligand grid free energy (LGFE) approach. Results show the SSFEP and SILCS LGFE methods to be competitive with or better than the FEP results for the studied systems, with SILCS LGFE giving the best agreement with experimental results. This is supported by additional comparisons with published FEP data on p38 MAP kinase inhibitors. While both the SSFEP and SILCS LGFE approaches require a significant upfront computational investment, they offer a 1000-fold computational savings over FEP for calculating the relative affinities of ligand modifications once those pre-computations are complete. An illustrative example of the potential application of these methods in the context of screening large numbers of transformations is presented. Thus, the SSFEP and SILCS LGFE approaches represent viable alternatives for actively driving ligand design during drug discovery and development. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Fate of wastewater effluent hER-agonists and hER-antagonists during soil aquifer treatment.

PubMed

Otakuye, Conroy; Quanrud, David M; Ela, Wendell P; Wicke, Daniel; Lansey, Kevin E; Arnold, Robert G

2005-04-01

Estrogen activity was measured in wastewater effluent before and after polishing via soil-aquifer treatment (SAT) using both a (hER-beta) competitive binding assay and a transcriptional activation (yeast estrogen screen, YES) assay. From the competitive binding assay, the equivalent 17alpha-ethinylestradiol (EE2) concentration in secondary effluent was 4.7 nM but decreased to 0.22 nM following SAT. The YES assay indicated that the equivalent EE2 concentration in the same effluent sample was below the method-detection limit (<2.5 x 10(-3) nM) but increased to 0.68 nM in effluent polished via SAT processes. It was hypothesized thattest-dependent differences arose because the competitive binding assay responds positively to both estrogen mimics and anti-estrogens; the YES assay responds to estrogen mimics, but test response is inhibited by anti-estrogens. The hypothesis was supported when organics extracted from wastewater effluent inhibited the YES test response to EE2 (anti-estrogenic effect). A similar extract prepared from SAT-polished effluent augmented the EE2 curve (agonist response). When hydrophobic organics in secondary effluent were fractionated, assay results indicated that several physically distinct anti-estrogens were present in the sample. From this work, it is evident that transcription-activation bioassays alone should not be relied upon to measure estrogenic activity in complex environmental samples because the simultaneous presence of both agonists and antagonist compounds can yield false negatives. Multiple in vitro bioassays, sample fractionation or tests designed to measure anti-estrogenic activity can be used to overcome this problem. It is also clear that there are circumstances under which SAT does not completely remove estrogenic activity during municipal wastewater effluent polishing.

Crl binds to domain 2 of σ(S) and confers a competitive advantage on a natural rpoS mutant of Salmonella enterica serovar Typhi.

PubMed

Monteil, Véronique; Kolb, Annie; Mayer, Claudine; Hoos, Sylviane; England, Patrick; Norel, Françoise

2010-12-01

The RpoS sigma factor (σ(S)) is the master regulator of the bacterial response to a variety of stresses. Mutants in rpoS arise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one natural rpoS mutant of Salmonella enterica serovar Typhi (Ty19). We show that the rpoS allele of Ty19 (rpoS(Ty19)) led to the synthesis of a σ(S)(Ty19) protein carrying a single glycine-to-valine substitution at position 282 in σ(S) domain 4, which was much more dependent than the wild-type σ(S) protein on activation by Crl, a chaperone-like protein that increases the affinity of σ(S) for the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of σ(S) domain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of σ(S)(Ty19) to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the Eσ(S) holoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between σ(S) and E. The rpoS(Ty19) allele conferred a competitive fitness when the bacterial population was wild type for crl but was outcompeted in Δcrl populations. Thus, these results indicate that the competitive advantage of the rpoS(Ty19) mutant is dependent on Crl and suggest that crl plays a role in the appearance of rpoS mutants in bacterial populations.

Development of LSPR and SPR sensor for the detection of an anti-cancer drug for chemotherapy

NASA Astrophysics Data System (ADS)

Zhao, Sandy Shuo; Bolduc, Olivier R.; Colin, Damien Y.; Pelletier, Joelle N.; Masson, Jean-François

2012-03-01

The anti-cancer drug, methotrexate (MTX) as a strong inhibitor of human dihydrofolate reductase (hDHFR) has been studied in localized surface plasmon resonance (LSPR) and surface plasmon resonance (SPR) competitive binding assays with folic acid stabilized gold nanoparticles (FA AuNP). The latter with a diameter of 15 nm were prepared in a simple step with sequential characterization using UV-Vis, FTIR, and Raman. A LSPR competitive binding assay between different concentrations of MTX and FA AuNP for hDHFR in solution was designed to quantify MTX by using UV-Vis spectroscopy. Sensitivity of the assay was optimized with respect to both concentrations of the enzyme and FA. The detection and quantification of spiked MTX was demonstrated in phosphate buffer saline and in fetal bovine serum accompanied by solid-phase extraction treatment of the serum. In addition, this assay could also provide as a screening tool for potential inhibitors of hDHFR. In another perspective, MTX was measured in a competitive binding assay with FA AuNP for histidine-tagged hDHFR immobilized on a SPR sensitive surface. In this case, FA AuNP offer a secondary amplification of the analytical response which is indirectly proportional to the concentration of MTX. This alternative approach could contribute to the realization of direct detection of MTX in complex biological fluids. A comparison of characteristics and analytical parameters such as sensitivity, dynamic range and limit of detection between the LSPR and SPR sensing platforms will also be presented. Both assays offer potential in tackling real biological samples for the purpose of monitoring and validating anti-cancer drug levels in human serum during chemotherapy.

New opioid receptor antagonist: Naltrexone-14-O-sulfate synthesis and pharmacology.

PubMed

Zádor, Ferenc; Király, Kornél; Váradi, András; Balogh, Mihály; Fehér, Ágnes; Kocsis, Dóra; Erdei, Anna I; Lackó, Erzsébet; Zádori, Zoltán S; Hosztafi, Sándor; Noszál, Béla; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

2017-08-15

Opioid antagonists, naloxone and naltrexone have long been used in clinical practice and research. In addition to their low selectivity, they easily pass through the blood-brain barrier. Quaternization of the amine group in these molecules, (e.g. methylnaltrexone) results in negligible CNS penetration. In addition, zwitterionic compounds have been reported to have limited CNS access. The current study, for the first time gives report on the synthesis and the in vitro [competition binding, G-protein activation, isolated mouse vas deferens (MVD) and mouse colon assay] pharmacology of the zwitterionic compound, naltrexone-14-O-sulfate. Naltrexone, naloxone, and its 14-O-sulfate analogue were used as reference compounds. In competition binding assays, naltrexone-14-O-sulfate showed lower affinity for µ, δ or κ opioid receptor than the parent molecule, naltrexone. However, the μ/κ opioid receptor selectivity ratio significantly improved, indicating better selectivity. Similar tendency was observed for naloxone-14-O-sulfate when compared to naloxone. Naltrexone-14-O-sulfate failed to activate [ 35 S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile 5,6 deltorphin II and U69593), similarly to the reference compounds. Schild plot constructed in MVD revealed that naltrexone-14-O-sulfate acts as a competitive antagonist. In mouse colon, naltrexone-14-O-sulfate antagonized the inhibitory effect of morphine with lower affinity compared to naltrexone and higher affinity when compared to naloxone or naloxone-14-O-sulfate. In vivo (mouse tail-flick test), subcutaneously injected naltrexone-14-O-sulfate antagonized morphine's antinociception in a dose-dependent manner, indicating it's CNS penetration, which was unexpected from such zwitter ionic structure. Future studies are needed to evaluate it's pharmacokinetic profile. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of [3H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([3H]PSB-12150): A Useful Tool for Studying GPR17

PubMed Central

2014-01-01

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [3H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities. PMID:24900835

Development of [(3)H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([(3)H]PSB-12150): A Useful Tool for Studying GPR17.

PubMed

Köse, Meryem; Ritter, Kirsten; Thiemke, Katharina; Gillard, Michel; Kostenis, Evi; Müller, Christa E

2014-04-10

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [(3)H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.

Three-dimensional structure of human serum albumin

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; He, Xiao-Min; Twigg, Pamela D.; Casale, Elena

1991-01-01

The binding locations to human serum albumin (HSA) of several drug molecules were determined at low resolution using crystallographic methods. The principal binding sites are located within subdomains IIA and IIIA. Preliminary studies suggest that an approach to increasing the in vivo efficacy of drugs which are rendered less effective or ineffective by virtue of their interaction with HSA, would be the use of competitive displacement in drug therapies and/or the development of a general inhibitor to the site within subdomain IIIA. These findings also suggest that the facilitated transfer of various ligands across organ/circulatory interfaces such as liver, kidney, and brain may be associated with binding to the IIIA subdomain.

Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR†

PubMed Central

Golynskiy, Misha V.; Gunderson, William A.; Hendrich, Michael P.; Cohen, Seth M.

2007-01-01

Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in Bacillus subtilis. Prior biophysical studies have focused on the metal-mediated DNA binding of MntR [Lieser, S. A., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2003) Biochemistry 42, 12634-12642], as well as metal stabilization of the MntR structure [Golynskiy, M. V., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2005) Biochemistry 44, 3380-3389], but only limited data on the metal-binding affinities for MntR are available. Herein, the metal-binding affinities of MntR were determined by using electron paramagnetic resonance (EPR) spectroscopy, as well as competition experiments with the fluorimetric dyes Fura-2 and Mag-fura-2. MntR was not capable of competing with Fura-2 for the binding of transition metal ions. Therefore, the metal-binding affinities and stoichiometries of Mag-fura-2 for Mn2+, Co2+, Ni2+, Zn2+, and Cd2+ were determined and utilized in MntR/Mag-fura-2 competition experiments. The measured Kd values for MntR metal binding are comparable to those reported for DtxR metal binding [Kd from 10-7 to 10-4 M; D’Aquino, J. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 18408-18413], AntR [a homologue from Bacillus anthracis; Sen, K. I. et al. (2006) Biochemistry 45, 4295-4303], and generally follow the Irving-Williams series. Direct detection of the dinuclear Mn2+ site in MntR with EPR spectroscopy is presented, and the exchange interaction was determined, J = -0.2 cm-1. This value is lower in magnitude than most known dinuclear Mn2+ sites in proteins and synthetic complexes and is consistent with a dinuclear Mn2+ site with a longer Mn···Mn distance (4.4 Å) observed in some of the available crystal structures. MntR is found to have a surprisingly low binding affinity (∼160 μM) for its cognate metal ion Mn2+. Moreover, the results of DNA binding studies in the presence of limiting metal ion concentrations were found to be consistent with the measured metal-binding constants. The metal-binding affinities of MntR reported here help to elucidate the regulatory mechanism of this metal-dependent transcription factor. PMID:17176058

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

GPER-targeted, 99mTc-labeled, nonsteroidal ligands demonstrate selective tumor imaging and in vivo estrogen binding

PubMed Central

Nayak, Tapan K.; Ramesh, Chinnasamy; Hathaway, Helen J.; Norenberg, Jeffrey P.; Arterburn, Jeffrey B.; Prossnitz, Eric R.

2014-01-01

Our understanding of estrogen (E2) receptor biology has evolved in recent years with the discovery and characterization of a 7-transmembrane-spanning G protein-coupled estrogen receptor (GPER1/GPER/GPR30) and the development of GPER-selective functional chemical probes. GPER is highly expressed in certain breast, endometrial and ovarian cancers, establishing the importance of non-invasive methods to evaluate GPER expression in vivo. Herein, we developed 99mTc-labeled GPER ligands to demonstrate the in vivo status of GPER as an estrogen receptor and for GPER visualization in whole animals. A series of 99mTc(I)-labeled non-steroidal tetrahydro-3H-cyclopenta[c]quinolone derivatives was synthesized utilizing pyridin-2-yl hydrazine and picolylamine chelates. Radioligand receptor binding studies revealed binding affinities in the 10–30 nM range. Cell signaling assays previously demonstrated that derivatives retaining a ketone functionality displayed agonist properties whereas those lacking such a hydrogen bond acceptor were antagonists. In vivo biodistribution and imaging studies performed on mice bearing human endometrial and breast cancer cell xenografts yielded significant tumor uptake (0.4–1.1 %ID/g). Blocking studies revealed specific uptake in multiple organs (adrenals, uterus, mammary tissue) as well as tumor uptake with similar levels of competition by E2 and G-1, a GPER-selective agonist. In conclusion, we synthesized and evaluated a series of first generation 99mTc-labeled GPER-specific radioligands, demonstrating GPER as an estrogen-binding receptor for the first time in vivo using competitive binding principles, and establishing the utility of such ligands as tumor imaging agents. These results warrant further investigation into the role of GPER in estrogen-mediated carcinogenesis and as a target for diagnostic/therapeutic/ image-guided drug delivery. PMID:25030373

Insulin receptor in mouse neuroblastoma cell line N18TG2: binding properties and visualization with colloidal gold.

PubMed

Sartori, C; Stefanini, S; Bernardo, A; Augusti-Tocco, G

1992-08-01

Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335-347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system. We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20 degrees C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligand-receptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects. On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.

Multifunctional Nutrient-Binding Proteins Adapt Human Symbiotic Bacteria for Glycan Competition in the Gut by Separately Promoting Enhanced Sensing and Catalysis

PubMed Central

Cameron, Elizabeth A.; Kwiatkowski, Kurt J.; Lee, Byung-Hoo; Hamaker, Bruce R.; Koropatkin, Nicole M.

2014-01-01

ABSTRACT To compete for the dynamic stream of nutrients flowing into their ecosystem, colonic bacteria must respond rapidly to new resources and then catabolize them efficiently once they are detected. The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model for nutrient acquisition by symbiotic gut bacteria, which harbor thousands of related Sus-like systems. Structural investigation of the four Sus outer membrane proteins (SusD, -E, -F, and -G) revealed that they contain a total of eight starch-binding sites that we demonstrated, using genetic and biochemical approaches, to play distinct roles in starch metabolism in vitro and in vivo in gnotobiotic mice. SusD, whose homologs are abundant in the human microbiome, is critical for the initial sensing of available starch, allowing sus transcriptional activation at much lower concentrations than without this function. In contrast, seven additional binding sites across SusE, -F, and -G are dispensable for sus activation. However, they optimize the rate of growth on starch in a manner dependent on the expression of the bacterial polysaccharide capsule, suggesting that they have evolved to offset the diffusion barrier created by this structure. These findings demonstrate how proteins with similar biochemical behavior can serve orthogonal functions during different stages of cellular adaptation to nutrients. Finally, we demonstrated in gnotobiotic mice fed a starch-rich diet that the Sus binding sites confer a competitive advantage to B. thetaiotaomicron in vivo in a manner that is dependent on other colonizing microbes. This study reveals how numerically dominant families of carbohydrate-binding proteins in the human microbiome fulfill separate and sometimes cooperative roles to optimize gut commensal bacteria for nutrient acquisition. PMID:25205092

Milk Inhibits the Biological Activity of Ricin

PubMed Central

Rasooly, Reuven; He, Xiaohua; Friedman, Mendel

2012-01-01

Ricin is a highly toxic protein produced by the castor plant Ricinus communis. The toxin is relatively easy to isolate and can be used as a biological weapon. There is great interest in identifying effective inhibitors for ricin. In this study, we demonstrated by three independent assays that a component of reconstituted powdered milk has a high binding affinity to ricin. We discovered that milk can competitively bind to and reduce the amount of toxin available to asialofetuin type II, which is used as a model to study the binding of ricin to galactose cell-surface receptors. Milk also removes ricin bound to the microtiter plate. In parallel experiments, we demonstrated by activity assay and by immuno-PCR that milk can bind competitively to 1 ng/ml ricin, reducing the amount of toxin uptake by the cells, and thus inhibit the biological activity of ricin. The inhibitory effect of milk on ricin activity in Vero cells was at the same level as by anti-ricin antibodies. We also found that (a) milk did not inhibit ricin at concentrations of 10 or 100 ng/ml; (b) autoclaving 10 and 100 ng/ml ricin in DMEM at 121 °C for 30 min completely abolished activity; and (c) milk did not affect the activity of another ribosome inactivating protein, Shiga toxin type 2 (Stx2), produced by pathogenic Escherichia coli O157:H7. Unlike ricin, which is internalized into the cells via a galactose-binding site, Stx2 is internalized through the cell surface receptor glycolipid globotriasylceramides Gb3 and Gb4. These observations suggest that ricin toxicity may possibly be reduced at room temperature by a widely consumed natural liquid food. PMID:22733821

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.

PubMed

Baker, Christopher L; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M; Paigen, Kenneth

2015-09-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

PubMed Central

Baker, Christopher L.; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M.; Paigen, Kenneth

2015-01-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 +/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape. PMID:26368021

Detection of potential (anti)progestagenic endocrine disruptors using a recombinant human progesterone receptor binding and transactivation assay.

PubMed

Viswanath, Gunda; Halder, Sujata; Divya, Gunda; Majumder, Chandrajeet B; Roy, Partha

2008-11-25

The present work describes the identification of (anti)progestin endocrine disrupting chemicals (EDC) using a two step screening system. In the first step a competitive binding assay was developed using recombinant human progesterone receptor (hPR). The tested chemicals were of various classes like insecticides, their metabolites, industrial chemicals and waste water treatment plant (WWTP) effluents. All the tested chemicals demonstrated a high affinity binding for hPR. The average IC50 values of the test chemicals were within the range of 1-25microM. In the second step of screening, a mammalian cell-based hPR transactivation assay was developed where HEK 293 cells were co-transfected with hPR and luciferase reporter gene under the control of progesterone-response element. Stimulation of the cells with progesterone resulted in about 25-fold up regulation of luciferase activity, with EC50 value of 4nM. Potent anti-progesterone, RU486, significantly inhibited progesterone-induced transactivation and non-progestagenic steroids failed to transactivate hPR till 1microM concentrations. The chemicals showing high binding affinities in competitive binding assays were then tested in transactivation assay and all of them were found to be anti-progestative except WWTP effluents. Transactivation assays using extracted water samples from five different WWTP effluents showed that it was rich in progestative compounds. The levels of induction caused by these effluents were in the range of 15-25% of induction by progesterone and they represented about 6ng/l equivalent progesterone activities. In conclusion, we demonstrated that this two step assay provides an efficient screening tool for the detection of (anti)progestative EDC in various samples.

Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera.

PubMed

Ngwuta, Joan O; Chen, Man; Modjarrad, Kayvon; Joyce, M Gordon; Kanekiyo, Masaru; Kumar, Azad; Yassine, Hadi M; Moin, Syed M; Killikelly, April M; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S; Rundlet, Emily J; Sastry, Mallika; Stewart-Jones, Guillaume B E; Yang, Yongping; Zhang, Baoshan; Nason, Martha C; Capella, Cristina; Peeples, Mark E; Ledgerwood, Julie E; McLellan, Jason S; Kwong, Peter D; Graham, Barney S

2015-10-14

Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø-specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F-specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø. Copyright © 2015, American Association for the Advancement of Science.

Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Song, Daqian; Kan, Yuhe; Xu, Dong; Tian, Yuan; Zhou, Xin; Zhang, Hanqi

2005-11-01

The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 °C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the Föster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box.

PubMed

Jia, Chuandong; Zuo, Wei; Yang, Dong; Chen, Yanming; Cao, Liping; Custelcean, Radu; Hostaš, Jiří; Hobza, Pavel; Glaser, Robert; Wang, Yao-Yu; Yang, Xiao-Juan; Wu, Biao

2017-10-16

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. This similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives, as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, L-carnitine, and glycine betaine effectively.The choline-binding protein ChoX exhibits a synergistic dual-site binding mode that allows it to discriminate choline over structural analogues. Here, the authors design a biomimetic triple anion helicate receptor whose selectivity for choline arises from a similar binding mechanism.

Autoradiographic analysis of binding sites for sup 125 I-Bolton-Hunter-substance P in the human eye

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

Kieselbach, G.F.; Ragaut, R.; Knaus, H.G.

1990-07-01

Substance P is known to exert potent effects in peripheral tissues, and is thought to be important for ocular function. The mechanism of action of substance P in the human eye is not known. As a basis for biochemical characterization specific binding of {sup 125}I-Bolton-Hunter-substance P was demonstrated in the human eye using autoradiographic methods. Biochemical characterization on slide-mounted tissue preparations showed that binding was saturable with a KD of 0.27 +/- 0.1 nmol/l. Specific binding occurred at comparable autoradiographic densities to both human retina and choroid. Substance P and its carboxyterminal fragment, substance P(3-11), were shown to be highlymore » potent in binding competition experiments against {sup 125}I-Bolton-Hunter-substance P. Similar concentrations of substance P(1-9), neurokinin A and neurokinin B failed to significantly alter specific binding of {sup 125}I-Bolton-Hunter-substance P. The results indicate expression of high affinity substance P binding sites in human retina and choroid.« less

Kinetics of Binding of Caldesmon to Actin*

PubMed Central

Chalovich, Joseph M.; Chen, Yi-der; Dudek, Ronald; Luo, Hai

2005-01-01

The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using a Monte Carlo simulation. The derived association and dissociation rates were 107 m−1 s−1 and 18.2 s−1, respectively. Smooth muscle tropomyosin enhances the binding of caldesmon to actin, and this was found to be due to a reduction in the rate of dissociation to 6.3 s −1. There is no evidence from this study for a different mechanism of binding in the presence of tropomyosin. The fluorescence changes that occurred with the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or actin-tropomyosin were reversed by the addition of myosin subfragment 1 as predicted by a competitive binding mechanism. PMID:7730374

Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors

PubMed Central

Lipzig, Rosalinde van; Montagu, Marc Van; Cornelissen, Marc; Meulewaeter, Frank

2001-01-01

The satellite tobacco necrosis virus RNA is uncapped and requires a 3′ translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30. PMID:11222757

Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human

EPA Science Inventory

Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however...

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs.

PubMed

Alonso, A; Almendral, M J; Curto, Y; Criado, J J; Rodríguez, E; Manzano, J L

2006-08-15

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

Synergy of cAMP and calcium signaling pathways in CFTR regulation

PubMed Central

Bozoky, Zoltan; Ahmadi, Saumel; Milman, Tal; Kim, Tae Hun; Du, Kai; Di Paola, Michelle; Pasyk, Stan; Pekhletski, Roman; Keller, Jacob P.; Bear, Christine E.; Forman-Kay, Julie D.

2017-01-01

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport. PMID:28242698

A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-dependent Leukemia Cell Survival

PubMed Central

Cohen, Nicole A.; Stewart, Michelle L.; Gavathiotis, Evripidis; Tepper, Jared L.; Bruekner, Susanne R.; Koss, Brian; Opferman, Joseph T.; Walensky, Loren D.

2012-01-01

SUMMARY Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an anti-apoptotic surface groove that neutralizes the pro-apoptotic BH3 α-helix of death proteins. Anti-apoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Whereas targeting the BCL-2 anti-apoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lockhold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence. PMID:22999885

Performance improvement of the one-dot lateral flow immunoassay for aflatoxin B1 by using a smartphone-based reading system.

PubMed

Lee, Sangdae; Kim, Giyoung; Moon, Jihea

2013-04-18

This study was conducted to develop a simple, rapid, and accurate lateral flow immunoassay (LFIA) detection method for point-of-care diagnosis. The one-dot LFIA for aflatoxin B1 (AFB1) was based on the modified competitive binding format using competition between AFB1 and colloidal gold-AFB1-BSA conjugate for antibody binding sites in the test zone. A Smartphone-based reading system consisting of a Samsung Galaxy S2 Smartphone, a LFIA reader, and a Smartphone application for the image acquisition and data analysis. The detection limit of one-dot LFIA for AFB1 is 5 μg/kg. This method provided semi-quantitative analysis of AFB1 samples in the range of 5 to 1,000 μg/kg. Using combination of the one-dot LFIA and the Smartphone-based reading system, it is possible to conduct a more fast and accurate point-of-care diagnosis.

Performance Improvement of the One-Dot Lateral Flow Immunoassay for Aflatoxin B1 by Using a Smartphone-Based Reading System

PubMed Central

Lee, Sangdae; Kim, Giyoung; Moon, Jihea

2013-01-01

This study was conducted to develop a simple, rapid, and accurate lateral flow immunoassay (LFIA) detection method for point-of-care diagnosis. The one-dot LFIA for aflatoxin B1 (AFB1) was based on the modified competitive binding format using competition between AFB1 and colloidal gold-AFB1-BSA conjugate for antibody binding sites in the test zone. A Smartphone-based reading system consisting of a Samsung Galaxy S2 Smartphone, a LFIA reader, and a Smartphone application for the image acquisition and data analysis. The detection limit of one-dot LFIA for AFB1 is 5 μg/kg. This method provided semi-quantitative analysis of AFB1 samples in the range of 5 to 1,000 μg/kg. Using combination of the one-dot LFIA and the Smartphone-based reading system, it is possible to conduct a more fast and accurate point-of-care diagnosis. PMID:23598499

Analysis of oxidative stress biomarkers using a simultaneous competitive/non-competitive micromosaic immunoassay.

PubMed

Murphy, Brian M; Dandy, David S; Henry, Charles S

2009-04-27

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 microL sample and 45 min for completion. Logistic curve fits and LOD (limits of detection) statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

High-throughput screening and mechanism-based evaluation of estrogenic botanical extracts

PubMed Central

Overk, Cassia R.; Yao, Ping; Chen, Shaonong; Deng, Shixing; Imai, Ayano; Main, Matthew; Schinkovitz, Andreas; Farnsworth, Norman R.; Pauli, Guido F.; Bolton, Judy L.

2009-01-01

Symptoms associated with menopause can greatly affect the quality of life for women. Botanical dietary supplements have been viewed by the public as safe and effective despite a lack of evidence indicating a urgent necessity to standardize these supplements chemically and biologically. Seventeen plants were evaluated for estrogenic biological activity using standard assays: competitive estrogen receptor (ER) binding assay for both alpha and beta subtypes, transient transfection of the estrogen response element luciferase plasmid into MCF-7 cells expressing either ER alpha or ER beta, and the Ishikawa alkaline phosphatase induction assay for both estrogenic and antiestrogenic activities. Based on the combination of data pooled from these assays, the following was determined: a) a high rate of false positive activity for the competitive binding assays, b) some extracts had estrogenic activity despite a lack of ability to bind the ER, c) one extract exhibited selective estrogen receptor modulator (SERM) activity, and d) several extracts show additive/synergistic activity. Taken together, these data indicate a need to reprioritize the order in which the bioassays are performed for maximal efficiency of programs involving bioassay-guided fractionation. In addition, possible explanations for the conflicts in the literature over the estrogenicity of Cimicifuga racemosa (black cohosh) are suggested. PMID:18473738

A novel principle for partial agonism of liver X receptor ligands. Competitive recruitment of activators and repressors.

PubMed

Albers, Michael; Blume, Beatrix; Schlueter, Thomas; Wright, Matthew B; Kober, Ingo; Kremoser, Claus; Deuschle, Ulrich; Koegl, Manfred

2006-02-24

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs.

Spectroscopic and electrochemical studies of the interaction between oleuropein, the major bio-phenol in olives, and salmon sperm DNA

NASA Astrophysics Data System (ADS)

Mohamadi, Maryam; Afzali, Daryoush; Esmaeili-Mahani, Saeed; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

2015-09-01

Interaction of oleuropein, the major bio-phenol in olive leaf and fruit, with salmon sperm double-stranded DNA was investigated by employing electronic absorption titrations, fluorescence quenching spectroscopy, competitive fluorescence spectroscopy, thermal denaturation and voltammetric studies. Titration of oleuropein with the DNA caused a hypochromism accompanied with a red shift indicating an intercalative mode of interaction. Binding constant of 1.4 × 104 M-1 was obtained for this interaction. From the curves of fluorescence titration of oleuropein with the DNA, binding constant and binding sites were calculated to be 8.61 × 103 M-1 and 1.05, respectively. Competitive studies with ethidium bromide (a well-known DNA intercalator) showed that the bio-phenol could take the place of ethidium bromide in the DNA intercalation sites. The interaction of oleuropein with DNA was also studied electrochemically. In the presence of the DNA, the anodic and cathodic peak currents of oleuropein decreased accompanied with increases in peak-to-peak potential separation and formal potential, indicating the intercalation of oleuropein into the DNA double helix. Moreover, melting temperature of the DNA was found to increase in the presence of oleuropein, indicating the stabilization of the DNA double helix due to an intercalative interaction.

Kinetic mechanism of the dimeric ATP sulfurylase from plants

PubMed Central

Ravilious, Geoffrey E.; Herrmann, Jonathan; Goo Lee, Soon; Westfall, Corey S.; Jez, Joseph M.

2013-01-01

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively. PMID:23789618

Sugar and pH dual-responsive mesoporous silica nanocontainers based on competitive binding mechanisms

NASA Astrophysics Data System (ADS)

Yilmaz, M. Deniz; Xue, Min; Ambrogio, Michael W.; Buyukcakir, Onur; Wu, Yilei; Frasconi, Marco; Chen, Xinqi; Nassar, Majed S.; Stoddart, J. Fraser; Zink, Jeffrey I.

2014-12-01

A sugar and pH dual-responsive controlled release system, which is highly specific towards molecular stimuli, has been developed based on the binding between catechol and boronic acid on a platform of mesoporous silica nanoparticles (MSNs). By grafting phenylboronic acid stalks onto the silica surface, catechol-containing β-cyclodextrins can be attached to the orifices of the MSNs' nanopores through formation of boronate esters which block access to the nanopores. These esters are stable enough to prevent cargo molecules from escaping. The boronate esters disassociate in the presence of sugars, enabling the molecule-specific controlled-release feature of this hybrid system. The rate of release has been found to be tunable by varying both the structures and the concentrations of sugars, as a result of the competitive binding nature associated with the mechanism of its operation. Acidification also induces the release of cargo molecules. Further investigations show that the presence of both a low pH and sugar molecules provides cooperative effects which together control the rate of release.A sugar and pH dual-responsive controlled release system, which is highly specific towards molecular stimuli, has been developed based on the binding between catechol and boronic acid on a platform of mesoporous silica nanoparticles (MSNs). By grafting phenylboronic acid stalks onto the silica surface, catechol-containing β-cyclodextrins can be attached to the orifices of the MSNs' nanopores through formation of boronate esters which block access to the nanopores. These esters are stable enough to prevent cargo molecules from escaping. The boronate esters disassociate in the presence of sugars, enabling the molecule-specific controlled-release feature of this hybrid system. The rate of release has been found to be tunable by varying both the structures and the concentrations of sugars, as a result of the competitive binding nature associated with the mechanism of its operation. Acidification also induces the release of cargo molecules. Further investigations show that the presence of both a low pH and sugar molecules provides cooperative effects which together control the rate of release. Electronic supplementary information (ESI) available: Synthetic schemes, electron microscopy images and nitrogen adsorption/desorption isotherms of the nanoparticles, FT-IR spectra, isothermal titration calorimetry, X-ray photoelectron spectra and time-of-flight secondary ion mass spectra. DLS results for nanoparticle stability. See DOI: 10.1039/c4nr04796f

Funcationalized nanomaterials and their biological applications

NASA Astrophysics Data System (ADS)

Vedantam, Pallavi

Bionanomaterials have been used in drug delivery, cancer therapy and biodiagnosis of pathogens based on their size and surface functionalization. In this present work, different kinds of nanoparticles (NPs), their cellular interactions, cytotoxicity profiles, and finally role of gold nanoparticles (GNPs) in biodetection of E. coli was investigated. Firstly, cytotoxicity profiles of commercial, laser ablation, and green synthetic NPs were studied. Induction of apoptosis in cancer cells was found to be size dependent. The plain 80 nm GNPs and AgNPs enhanced toxic effects in cancer cells when compared to 20 nm ones. Apoptotic profiles of ALB- or FBS-coated NPs were significantly low in cancer cells when compared to plain NPs. The FBS-coated NPs were relatively bigger. Whereas, green synthetic NPs prepared from floral extracts of Tacoma stans and Tagetus erecta caused significant increase in cell membrane damage in cancer cells when compared to commercial GNPs. The phytochemicals in the extract were found to have a synergistic effect on pathogens like E. coli, Staphylococcus aureus and Enterococcus faecalis when used with antibiotics like tetracycline, ampicillin and vancomycin. This effect can be capitalized in developing NPs as effective drug carriers. Next, use of commercial GNPs as diagnostic tools to detect competitive binding in DU-145 in the presence of UTI-causing E. coli ORN178 was studied. GNPs functionalized with D-mannose (Mn) showed competitive binding between Mn-GNPs and E. coli ORN178 when presented together with DU-145 cells. Cytotoxicity assays of plain and Mn-GNPs showed significant decrease in viability of DU-145 cells. The plain/Mn 20 nm GNPs were taken up more by the cell when compared to the 200 nm ones. The protein-coated GNPs were found to be stable in culture medium. This competitive binding can be further developed to prevent/detect recurrent UTI in DU-45 cells. Lastly, primary and fine sugar specificity of fimbrial lectins of E.coli ORN178 and E. coli 13762 to D-mannose and Neualphac(alpha2-3)-Gal-(beta1-4)Glc-Paa functionalized GNPs showed that E. coli ORN178 binds specifically only to Mn-GNPs and E. coli 13762 to the latter. Hence, adhesin-specific adhesion shows great potential for designing NPs to specifically bind to microorganisms as biodiagnostic tools.

Development of binding assays in microfabricated picoliter vials: an assay for biotin.

PubMed

Grosvenor, A L; Feltus, A; Conover, R C; Daunert, S; Anderson, K W

2000-06-01

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers.

PubMed

Pang, T; Su, X; Wakabayashi, S; Shigekawa, M

2001-05-18

The Na+/H+ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na+/H+ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.

Structural, biochemical, and biophysical characterization of idelalisib binding to phosphoinositide 3-kinase δ

DOE PAGES

Somoza, John R.; Koditek, David; Villaseñor, Armando G.; ...

2015-01-28

Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Here, our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. Lastly, a crystal structure of idelalisib bound to the p110δ subunit of PI3Kδmore » furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.« less

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

PubMed

Schuller, Marion; Höfner, Georg; Wanner, Klaus T

2017-10-09

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

PubMed Central

Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

2009-01-01

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication.

PubMed

Abdul Ahmad, Siti Aisyah; Palanisamy, Uma D; Tejo, Bimo A; Chew, Miaw Fang; Tham, Hong Wai; Syed Hassan, Sharifah

2017-11-21

The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated. Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC 50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay. Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC 50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity. Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Equol is more active than soy isoflavone itself to compete for binding to thromboxane A(2) receptor in human platelets.

PubMed

Muñoz, Yenny; Garrido, Argelia; Valladares, Luis

2009-03-01

Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A(2) (TxA(2)) receptor. Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA(2) receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA(2) was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA(2) mimic) and the calcium ionophore A23187. The data showed that aglycone isoflavones and equol bind to TxA(2) receptor in the micromol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [(3)H]-SQ29585 binding was: equol>genistein>daidzein>glycitein>genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA(2) binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA(2) analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP. The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A(2) receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.

Competition Between Co(NH3)63+ and Inner Sphere Mg2+ Ions in the HDV Ribozyme

PubMed Central

Gong, Bo; Chen, Jui-Hui; Bevilacqua, Philip C.; Golden, Barbara L.; Carey, Paul R.

2009-01-01

Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg2+ are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH3)63+, an analog of Mg(H2O)62+. Here, the binding of Mg2+ and Co(NH3)63+ to the HDV ribozyme are studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg2+ alone is introduced to the ribozyme, inner sphere coordination of Mg(H2O)x2+ (x≤5) to non-bridging PO2− oxygen, and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg2+ induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH3)63+ alone is introduced, deprotonation of C75 is again observed, as are distinctive changes in base vibrational ring modes and phosphodiester backbone conformation. In contrast to Mg2+ binding, Co(NH3)63+ binding does not perturb PO2− group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH3)63+ ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO2− groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH3)63+ ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg2+-PO2− binding sites: sites that Co(NH3)63+ can displace, and others it cannot. PMID:19888753

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

PubMed

Doern, Adam; Cao, Xianjun; Sereno, Arlene; Reyes, Christopher L; Altshuler, Angelina; Huang, Flora; Hession, Cathy; Flavier, Albert; Favis, Michael; Tran, Hon; Ailor, Eric; Levesque, Melissa; Murphy, Tracey; Berquist, Lisa; Tamraz, Susan; Snipas, Tracey; Garber, Ellen; Shestowsky, William S; Rennard, Rachel; Graff, Christilyn P; Wu, Xiufeng; Snyder, William; Cole, Lindsay; Gregson, David; Shields, Michael; Ho, Steffan N; Reff, Mitchell E; Glaser, Scott M; Dong, Jianying; Demarest, Stephen J; Hariharan, Kandasamy

2009-04-10

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

Inhibition of tyrosinase by 4H-chromene analogs: Synthesis, kinetic studies, and computational analysis.

PubMed

Brasil, Edikarlos M; Canavieira, Luciana M; Cardoso, Érica T C; Silva, Edilene O; Lameira, Jerônimo; Nascimento, José L M; Eifler-Lima, Vera L; Macchi, Barbarella M; Sriram, Dharmarajan; Bernhardt, Paul V; Silva, José Rogério Araújo; Williams, Craig M; Alves, Cláudio N

2017-11-01

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a K i value of 4 μm, comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site. © 2017 John Wiley & Sons A/S.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules.

PubMed

Newton, Ana S; Deiana, Luca; Puleo, David E; Cisneros, José A; Cutrona, Kara J; Schlessinger, Joseph; Jorgensen, William L

2017-06-08

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as K d results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules

PubMed Central

2017-01-01

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses. PMID:28626520

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.

PubMed

Pai, Sudipta; Das, Mili; Banerjee, Rahul; Dasgupta, Dipak

2011-08-01

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.

Crystal structure of the Escherichia coli regulator of sigma70, Rsd, in complex with sigma70 domain 4.

PubMed

Patikoglou, Georgia A; Westblade, Lars F; Campbell, Elizabeth A; Lamour, Valérie; Lane, William J; Darst, Seth A

2007-09-21

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.

Crystal structure of the Escherichia coli regulator of σ70, Rsd, in complex with σ70 domain 4

PubMed Central

Patikoglou, Georgia A.; Westblade, Lars F.; Campbell, Elizabeth A.; Lamour, Valérie; Lane, William J.; Darst, Seth A.

2007-01-01

Summary The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) σ70 factor. Rsd plays a role in alternative σ factor-dependent transcription by biasing the competition between σ70 and alternative σ factors for the available core RNAP. Here, we determined the 2.6 Å-resolution X-ray crystal structure of Rsd bound to σ70 domain 4 (σ704), the primary determinant for Rsd binding within σ70. The structure reveals that Rsd binding interferes with the two primary functions of σ704, core RNAP binding and promoter –35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the σ704-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between σ704 binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation. PMID:17681541

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

Patikoglou,G.; Westblade, L.; Campbell, E.

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) {sigma}{sup 70} factor. Rsd plays a role in alternative {sigma} factor-dependent transcription by biasing the competition between {sigma}{sup 70} and alternative {sigma} factors for the available core RNAP. Here, we determined the 2.6 {angstrom}-resolution X-ray crystal structure of Rsd bound to {sigma}{sup 70} domain 4 ({sigma}{sup 70}{sub 4}), the primary determinant for Rsd binding within {sigma}{sup 70}. The structure reveals that Rsd binding interferes with the two primary functions of {sigma}{sup 70}{sub 4}, core RNAP binding and promoter -35 element binding. Interestingly, the most highly conservedmore » Rsd residues form a network of interactions through the middle of the Rsd structure that connect the {sigma}{sup 70}{sub 4}-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between {sigma}{sup 70}{sub 4} binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.« less

Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

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

Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.

The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using ({sup 125}I)melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37{degree}C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of ({sup 125}I)melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8more » fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of ({sup 125}I)melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the ({sup 125}I)melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.« less

78 FR 78370 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-26

... via the antibody binding fragment of the CAR. By engineering a T-cell to express a CAR that is...) developed in tissue that has been propagated by serial transplantation (rather than cell culture), (b... marrow transplantation, stem cell therapy, tissue regeneration) Competitive Advantages: Labeling markers...

Zn(II) stimulation of Fe(II)-activated repression in the iron-dependent repressor from Mycobacterium tuberculosis.

PubMed

Stapleton, Brian; Walker, Lawrence R; Logan, Timothy M

2013-03-19

Thermodynamic measurements of Fe(II) binding and activation of repressor function in the iron-dependent repressor from Mycobacterium tuberculosis (IdeR) are reported. IdeR, a member of the diphtheria toxin repressor family of proteins, regulates iron homeostasis and contributes to the virulence response in M. tuberculosis. Although iron is the physiological ligand, this is the first detailed analysis of iron binding and activation in this protein. The results showed that IdeR binds 2 equiv of Fe(II) with dissociation constants that differ by a factor of 25. The high- and low-affinity iron binding sites were assigned to physical binding sites I and II, respectively, using metal binding site mutants. IdeR was also found to contain a high-affinity Zn(II) binding site that was assigned to physical metal binding site II through the use of binding site mutants and metal competition assays. Fe(II) binding was modestly weaker in the presence of Zn(II), but the coupled metal binding-DNA binding affinity was significantly stronger, requiring 30-fold less Fe(II) to activate DNA binding compared to Fe(II) alone. Together, these results suggest that IdeR is a mixed-metal repressor, where Zn(II) acts as a structural metal and Fe(II) acts to trigger the physiologically relevant promoter binding. This new model for IdeR activation provides a better understanding of IdeR and the biology of iron homeostasis in M. tuberculosis.

Competitive binding effects on surface-enhanced Raman scattering of peptide molecules

NASA Astrophysics Data System (ADS)

Seballos, Leo; Richards, Nicole; Stevens, Daniel J.; Patel, Mira; Kapitzky, Laura; Lokey, Scott; Millhauser, Glenn; Zhang, Jin Z.

2007-10-01

Surface enhanced Raman scattering (SERS) has been conducted on tryptophan (W), proline (P) and tyrosine (Y) containing peptides that include W-P-Y, Y-P-W, W-P-P-P-Y, Y-P-P-P-W, W-P-P-P-P-P-Y, and Y-P-P-P-P-P-W to gain insight into molecular binding behavior on a metal substrate to eventually apply in protein SERS detection. The peptides are shown to bind through the molecule's carboxylic end, but the strong affinity of the tryptophan residue to the substrate surface, in conjunction with its large polarizability, dominates each molecule's SERS signal with the strong presence of its ring modes in all samples. These results are important for understanding SERS of protein molecules.

Boron-based phosphodiesterase inhibitors show novel binding of boron to PDE4 bimetal center.

PubMed

Freund, Yvonne R; Akama, Tsutomu; Alley, M R K; Antunes, Joana; Dong, Chen; Jarnagin, Kurt; Kimura, Richard; Nieman, James A; Maples, Kirk R; Plattner, Jacob J; Rock, Fernando; Sharma, Rashmi; Singh, Rajeshwar; Sanders, Virginia; Zhou, Yasheen

2012-09-21

We have used boron-based molecules to create novel, competitive, reversible inhibitors of phosphodiesterase 4 (PDE4). The co-crystal structure reveals a binding configuration which is unique compared to classical catechol PDE4 inhibitors, with boron binding to the activated water in the bimetal center. These phenoxybenzoxaboroles can be optimized to generate submicromolar potency enzyme inhibitors, which inhibit TNF-α, IL-2, IFN-γ, IL-5 and IL-10 activities in vitro and show safety and efficacy for topical treatment of human psoriasis. They provide a valuable new route for creating novel potent anti-PDE4 inhibitors. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Synthesis and biological evaluation of cyclopropyl analogues of 2-amino-5-phosphonopentanoic acid

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

Dappen, M.S.; Pellicciari, R.; Natalini, B.

1991-01-01

A series of cyclopropyl analogues related to 2-amino-5-phosphonopentanoic acid (AP5) were synthesized and their biological activity was assessed as competitive antagonists for the N-methyl-D-aspartate (NMDA) receptor. In vitro receptor binding using (3H)-L-glutamate as the radioligand provided affinity data, while modulation of (3H)MK-801 binding was used as a functional assay. The analogues were also evaluated in (3H)kainate binding to assess selectivity over non-NMDA glutamate receptors. Of the compounds tested, 4,5-methano-AP5 analogue 26 was the most potent selective NMDA antagonist; however, potency was lower than that for (((+/-)-2-carboxypiperidin-4-yl)methyl)phosphonic acid (CGS 19755, 5).

pH-insensitive electrostatic interaction of carmoisine with two serum proteins: a possible caution on its uses in food and pharmaceutical industry.

PubMed

Datta, Shubhashis; Mahapatra, Niharendu; Halder, Mintu

2013-07-05

Here we have investigated the binding of carmoisine, a water-soluble azo food colorant, with serum proteins (HSA and BSA) by fluorescence and UV-VIS spectroscopy, circular dichroism and molecular docking studies. Results indicate that fluorescence quenching of protein has been due to site-specific binding of the dye with biomacromolecules. Site marker competitive binding and molecular docking explorations show that interaction occurs in the sub-domain ІІA of HSA and the sub-domains ІІA and ІB in the case of BSA. Conformational investigation indicates that dye binding modifies the secondary structure of proteins and this also alters the microenvironment of the tryptophan(s). The interaction is found to be pH-insensitive which can have relevance to the toxicological profiles of the dye, and ionic strength dependence of binding can be exploited in protein purification mediated by such food colorants. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of guanyl nucleotides on CCKB receptor binding in brain tissue and continuous cell lines: a comparative study.

PubMed

Kaufmann, R; Schöneberg, T; Henklein, P; Meyer, R; Martin, H; Ott, T

1995-07-01

The effects of non-hydrolyzable guanyl nucleotide analogue GTP-gamma S on CCKB receptor binding in human and guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells, rat glioma C6 cells and human small cell lung cancer NCI-H69 cells were investigated by using [3H]CCK-8S saturation and competition binding studies. GTP-gamma S caused inhibition of specific [3H]CCK-8S binding in a concentration dependent manner with a plateau at 10-25 microM. 25 microM GTP-gamma S resulted in a small but significant increase in Kd and IC50 values with amount very similar in all CCKB receptor models tested. However, the maximal number of specific [3H]CCK-8S binding sites (Bmax) was unaffected. Results suggest that CCKB receptors are G-protein coupled in a similar way to human and guinea-pig cortex, Jurkat cells, GH3 cells, C6 cells and NCI-H69 cells.

Quantitatively and Kinetically Identifying Binding Motifs of Amelogenin Proteins to Mineral Crystals Through Biochemical and Spectroscopic Assays

PubMed Central

Zhu, Li; Hwang, Peter; Witkowska, H. Ewa; Liu, Haichuan; Li, Wu

2014-01-01

Tooth enamel is the hardest tissue in vertebrate animals. Consisting of millions of carbonated hydroxyapatite crystals, this highly mineralized tissue develops from a protein matrix in which amelogenin is the predominant component. The enamel matrix proteins are eventually and completely degraded and removed by proteinases to form mineral-enriched tooth enamel. Identification of the apatite-binding motifs in amelogenin is critical for understanding the amelogenin–crystal interactions and amelogenin–proteinases interactions during tooth enamel biomineralization. A stepwise strategy is introduced to kinetically and quantitatively identify the crystal-binding motifs in amelogenin, including a peptide screening assay, a competitive adsorption assay, and a kinetic-binding assay using amelogenin and gene-engineered amelogenin mutants. A modified enzyme-linked immunosorbent assay on crystal surfaces is also applied to compare binding amounts of amelogenin and its mutants on different planes of apatite crystals. We describe the detailed protocols for these assays and provide the considerations for these experiments in this chapter. PMID:24188774

Binding characteristics and protective capacity of cyanidin-3-glucoside and its aglycon to calf thymus DNA.

PubMed

Zhang, Chao; Guo, Xiaofei; Cai, Wenqian; Ma, Yue; Zhao, Xiaoyan

2015-04-01

The binding characteristics and protective capacity of cyanidin (Cy) and cyanidin-3-glucoside (C3G) to calf thymus DNA were explored for the first time. The Cy and C3G gave a bathochromic shift to the ultraviolet-visible spectra of the DNA, indicating the formation of the DNA-Cy and DNA-C3G complexes. The complexes were formed by an intercalative binding mode based on the results of the fluorescence spectra and competitive binding analysis. Meanwhile, the Cy and C3G protected the DNA from the damage induced by the hydroxyl radical. The binding capacity and protective capacity of the C3G were stronger than that of the Cy. Furthermore, the formation of the DNA-anthocyanin complexes was spontaneous when the hydrogen bond and hydrophobic force played a key role. Hence, the Cy and C3G could protect the DNA automatically from the damage induced by the hydroxyl radical. © 2015 Institute of Food Technologists®

Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

NASA Astrophysics Data System (ADS)

Dacosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

2015-08-01

α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

Developing tools for risk assessment in protected species: relative potencies inferred from competitive binding of halogenated aromatic hydrocarbons to aryl hydrocarbon receptors from beluga (Delphinapterus leucas) and mouse

PubMed Central

Jensen, Brenda A.; Reddy, Christopher M.; Nelson, Robert K.; Hahn, Mark E.

2011-01-01

Persistent organic pollutants such as halogenated aromatic hydrocarbons (HAHs) biomagnify in food webs and accumulate to high concentrations in top predators like odontocete cetaceans (toothed whales). The most toxic HAHs are the 2,3,7,8-substituted halogenated dibenzo-p-dioxins and furans, and non-ortho-substituted polychlorinated biphenyls (PCBs), which exert their effects via the aryl hydrocarbon receptor (AHR). Understanding the impact of HAHs in wildlife is limited by the lack of taxon-specific information about the relative potencies of toxicologically important congeners. To assess whether Toxic Equivalency Factors (TEFs) determined in rodents are predictive of HAH relative potencies in a cetacean, we used beluga and mouse AHRs expressed in vitro from cloned cDNAs to measure the relative AHR-binding affinities of ten HAHs from five different structural classes. The rank order of mean IC50s for competitive binding to beluga AHR was: TCDD

Developing tools for risk assessment in protected species: Relative potencies inferred from competitive binding of halogenated aromatic hydrocarbons to aryl hydrocarbon receptors from beluga (Delphinapterus leucas) and mouse.

PubMed

Jensen, Brenda A; Reddy, Christopher M; Nelson, Robert K; Hahn, Mark E

2010-11-01

Persistent organic pollutants such as halogenated aromatic hydrocarbons (HAHs) biomagnify in food webs and accumulate to high concentrations in top predators like odontocete cetaceans (toothed whales). The most toxic HAHs are the 2,3,7,8-substituted halogenated dibenzo-p-dioxins and furans, and non-ortho-substituted polychlorinated biphenyls (PCBs), which exert their effects via the aryl hydrocarbon receptor (AHR). Understanding the impact of HAHs in wildlife is limited by the lack of taxon-specific information about the relative potencies of toxicologically important congeners. To assess whether Toxic Equivalency Factors (TEFs) determined in rodents are predictive of HAH relative potencies in a cetacean, we used beluga and mouse AHRs expressed in vitro from cloned cDNAs to measure the relative AHR-binding affinities of ten HAHs from five different structural classes. The rank order of mean IC(50)s for competitive binding to beluga AHR was: TCDD

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

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

Liang, B.T.

1989-06-01

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or themore » maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).« less

Insulation and wiring specificity of BceR-like response regulators and their target promoters in Bacillus subtilis.

PubMed

Fang, Chong; Nagy-Staroń, Anna; Grafe, Martin; Heermann, Ralf; Jung, Kirsten; Gebhard, Susanne; Mascher, Thorsten

2017-04-01

BceRS and PsdRS are paralogous two-component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, P bceA or P psdA , resulting in a strong up-regulation of target gene expression and ultimately antibiotic resistance. Despite high sequence similarity between the RRs BceR and PsdR and their known binding sites, no cross-regulation has been observed between them. We therefore investigated the specificity determinants of P bceA and P psdA that ensure the insulation of these two paralogous pathways at the RR-promoter interface. In vivo and in vitro analyses demonstrate that the regulatory regions within these two promoters contain three important elements: in addition to the known (main) binding site, we identified a linker region and a secondary binding site that are crucial for functionality. Initial binding to the high-affinity, low-specificity main binding site is a prerequisite for the subsequent highly specific binding of a second RR dimer to the low-affinity secondary binding site. In addition to this hierarchical cooperative binding, discrimination requires a competition of the two RRs for their respective binding site mediated by only slight differences in binding affinities. © 2016 John Wiley & Sons Ltd.

Down-regulation of tryptamine binding sites following chronic molindone administration. A comparison with responses of dopamine and 5-hydroxytryptamine receptors.

PubMed

Nguyen, T V; Juorio, A V

1989-10-01

The present study assessed changes of tryptamine, dopamine D2, 5-HT1 and 5-HT2 binding sites in rat brain following chronic treatment with low (5 mg/kg/day) and high (40 mg/kg/day) doses of molindone, a clinically effective psychotropic drug. The high-dose molindone treatment produced a decrease in the number of tryptamine binding sites while both high and low doses caused an increase in the number of dopamine D2 binding sites in the striatum. No significant changes were observed in either 5-HT1 or 5-HT2 binding sites in the cerebral cortex. Competition binding experiments showed that molindone was a potent inhibitor at dopamine D2 but less effective at tryptamine, 5-HT1 and 5-HT2 binding sites. The inhibition activity of molindone towards type A monoamine oxidase produced a significant increase in endogenous tryptamine accumulation rate which was much higher than that of dopamine and 5-HT. These findings suggest that the reduction in the number of tryptamine binding sites produced by chronic molindone administration is related to monoamine oxidase inhibition and that the increase in the number of dopamine D2 binding sites is correlated to receptor blocking activity of the drug.

Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS

DOE PAGES

Kirby, Thomas W.; Gassman, Natalie R.; Smith, Cassandra E.; ...

2015-08-25

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs ofmore » the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior gives a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.« less

Human La binds mRNAs through contacts to the poly(A) tail

PubMed Central

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-01-01

Abstract In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3’OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3’OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail. PMID:29447394

MATLAB-Based Teaching Modules in Biochemical Engineering

ERIC Educational Resources Information Center

Lee, Kilho; Comolli, Noelle K.; Kelly, William J.; Huang, Zuyi

2015-01-01

Mathematical models play an important role in biochemical engineering. For example, the models developed in the field of systems biology have been used to identify drug targets to treat pathogens such as Pseudomonas aeruginosa in biofilms. In addition, competitive binding models for chromatography processes have been developed to predict expanded…

Inhibition of ferric ion to oxalate oxidase shed light on the substrate binding site.

PubMed

Pang, Yu; Lan, Wanjun; Huang, Xuelei; Zuo, Guanke; Liu, Hui; Zhang, Jingyan

2015-10-01

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe(3+) ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe(3+) is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe(3+) ions, two residue clusters of OxOx were assigned as potential Fe(3+) binding sites, the mechanism of the inhibition of Fe(3+) was delineated. Importantly, the residues that interact with Fe(3+) ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe(3+) highlights insight into substrate binding site in OxOx.

Human serum albumin binding of certain antimalarials

NASA Astrophysics Data System (ADS)

Marković, Olivera S.; Cvijetić, Ilija N.; Zlatović, Mario V.; Opsenica, Igor M.; Konstantinović, Jelena M.; Terzić Jovanović, Nataša V.; Šolaja, Bogdan A.; Verbić, Tatjana Ž.

2018-03-01

Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37 °C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

Sequence-specific binding of counterions to B-DNA

PubMed Central

Denisov, Vladimir P.; Halle, Bertil

2000-01-01

Recent studies by x-ray crystallography, NMR, and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove of B form DNA. Such groove-bound ions potentially could play an important role in AT-tract bending and groove narrowing, thereby modulating DNA function in vivo. To address this issue, we report here 23Na magnetic relaxation dispersion measurements on oligonucleotides, including difference experiments with the groove-binding drug netropsin. The exquisite sensitivity of this method to ions in long-lived and intimate association with DNA allows us to detect sequence-specific sodium ion binding in the minor groove AT tract of three B-DNA dodecamers. The sodium ion occupancy is only a few percent, however, and therefore is not likely to contribute importantly to the ensemble of B-DNA structures. We also report results of ion competition experiments, indicating that potassium, rubidium, and cesium ions bind to the minor groove with similarly weak affinity as sodium ions, whereas ammonium ion binding is somewhat stronger. The present findings are discussed in the light of previous NMR and diffraction studies of sequence-specific counterion binding to DNA. PMID:10639130

In situ imaging of single carbohydrate-binding modules on cellulose microfibrils.

PubMed

Dagel, Daryl J; Liu, Yu-San; Zhong, Lanlan; Luo, Yonghua; Himmel, Michael E; Xu, Qi; Zeng, Yining; Ding, Shi-You; Smith, Steve

2011-02-03

The low efficiency of enzymes used in the bioprocessing of biomass for biofuels is one of the primary bottlenecks that must be overcome to make lignocellulosic biofuels cost-competitive. One of the rate-limiting factors is the accessibility of the cellulase enzymes to insoluble cellulolytic substrates, facilitated by surface absorption of the carbohydrate-binding modules (CBMs), a component of most cellulase systems. Despite their importance, reports of direct observation of CBM function and activity using microscopic methods are still uncommon. Here, we examine the site-specific binding of individual CBMs to crystalline cellulose in an aqueous environment, using the single molecule fluorescence method known as Defocused Orientation and Position Imaging (DOPI). Systematic orientations were observed that are consistent with the CBMs binding to the two opposite hydrophobic faces of the cellulose microfibril, with a well-defined orientation relative to the fiber axis. The approach provides in situ physical evidence indicating the CBMs bind with a well-defined orientation on those planes, thus supporting a binding mechanism driven by chemical and structural recognition of the cellulose surface.

Detection of Z DNA binding proteins in tissue culture cells.

PubMed Central

Leith, I R; Hay, R T; Russell, W C

1988-01-01

A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA. Images PMID:3419919

Binding of mercury(II) to aquatic humic substances: Influence of pH and source of humic substances

USGS Publications Warehouse

Haitzer, M.; Aiken, G.R.; Ryan, J.N.

2003-01-01

Conditional distribution coefficients (KDOM???) for Hg(II) binding to seven dissolved organic matter (DOM) isolates were measured at environmentally relevant ratios of Hg(II) to DOM. The results show that KDOM??? values for different types of samples (humic acids, fulvic acids, hydrophobic acids) isolated from diverse aquatic environments were all within 1 order of magnitude (1022.5??1.0-1023.5??1.0 L kg-1), suggesting similar Hg(II) binding environments, presumably involving thiol groups, for the different isolates. KDOM??? values decreased at low pHs (4) compared to values at pH 7, indicating proton competition for the strong Hg(II) binding sites. Chemical modeling of Hg(II)-DOM binding at different pH values was consistent with bidentate binding of Hg(II) by one thiol group (pKa = 10.3) and one other group (pKa = 6.3) in the DOM, which is in agreement with recent results on the structure of Hg(II)-DOM bonds obtained by extended X-ray absorption fine structure spectroscopy (EXAFS).

Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein

PubMed Central

Min, Hongping; Niu, Miaomiao; Zhang, Weilin; Yan, Jia; Li, Jiachang; Tan, Xiying; Li, Bo; Su, Mengxiang; Di, Bin; Yan, Fang

2017-01-01

Development of multidrug resistance (MDR) is a continuous clinical challenge partially due to the overexpression of P-glycoprotein (P-gp) for chronic myelogenous leukemia (CML) patients. Herein, we evaluated the inhibitory potency of emodin, a natural anthraquinone derivative isolated from Rheum palmatum L, on P-gp in P-gp positive K562/ADM cells. Competition experiments combined with molecular docking analysis were utilized to investigate the binding modes between emodin and binding sites of P-gp. Emodin reversed adriamycin resistance in K562/ADM cells accompanied with the decrease of P-gp protein expression, further increasing the uptake of rhodamine123 in both K562/ADM and Caco-2 cells, indicating the inhibition of P-gp efflux function. Moreover, when incubated with emodin under different conditions where P-gp was inhibited, K562/ADM cells displayed increasing intracellular uptake of emodin, suggesting that emodin may be the potential substrate of P-gp. Importantly, rhodamine 123 could increase the Kintrinsic (Ki) value of emodin linearly, whereas, verapamil could not, implying that emodin competitively bound to the R site of P-gp and noncompetition existed between emodin and verapamil at the M site, in a good accordance with the results of molecular docking that emodin bound to the R site of P-gp with higher affinity. Based on our results, we suggest that emodin might be used to modulate P-gp function and expression. PMID:29121121

Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein.

PubMed

Min, Hongping; Niu, Miaomiao; Zhang, Weilin; Yan, Jia; Li, Jiachang; Tan, Xiying; Li, Bo; Su, Mengxiang; Di, Bin; Yan, Fang

2017-01-01

Development of multidrug resistance (MDR) is a continuous clinical challenge partially due to the overexpression of P-glycoprotein (P-gp) for chronic myelogenous leukemia (CML) patients. Herein, we evaluated the inhibitory potency of emodin, a natural anthraquinone derivative isolated from Rheum palmatum L, on P-gp in P-gp positive K562/ADM cells. Competition experiments combined with molecular docking analysis were utilized to investigate the binding modes between emodin and binding sites of P-gp. Emodin reversed adriamycin resistance in K562/ADM cells accompanied with the decrease of P-gp protein expression, further increasing the uptake of rhodamine123 in both K562/ADM and Caco-2 cells, indicating the inhibition of P-gp efflux function. Moreover, when incubated with emodin under different conditions where P-gp was inhibited, K562/ADM cells displayed increasing intracellular uptake of emodin, suggesting that emodin may be the potential substrate of P-gp. Importantly, rhodamine 123 could increase the Kintrinsic (Ki) value of emodin linearly, whereas, verapamil could not, implying that emodin competitively bound to the R site of P-gp and noncompetition existed between emodin and verapamil at the M site, in a good accordance with the results of molecular docking that emodin bound to the R site of P-gp with higher affinity. Based on our results, we suggest that emodin might be used to modulate P-gp function and expression.

UNDERSTANDING POLYSPECIFICITY WITHIN THE SUBSTRATE-BINDING CAVITY OF THE HUMAN MULTIDRUG RESISTANCE P-GLYCOPROTEIN

PubMed Central

Martinez, Lorena; Arnaud, Ophélie; Henin, Emilie; Tao, Houchao; Chaptal, Vincent; Doshi, Rupak; Andrieu, Thibaud; Dussurgey, Sébastien; Tod, Michel; Di Pietro, Attilio; Zhang, Qinghai; Chang, Geoffrey; Falson, Pierre

2015-01-01

Human P-glycoprotein (P-gp) controls drugs bioavailability by pumping out of the cells many structurally-unrelated drugs. The x-ray structure of the mouse P-gp ortholog was solved with two SSS- and one RRR-enantiomers of the selenohexapeptide inhibitor QZ59, found within the putative drug-binding pocket of the membrane domain outer leaflet. This offered the first opportunity to localize the well-known H- and R- drug-substrate sites in light of QZ59 inhibition mechanisms that were characterized here in cellulo and modelled towards Hoechst 33342 and daunorubicin transport. We found that QZ59-SSS competes efficiently with both substrates, displaying KI,app values of 0.15 and 0.3 μM, respectively 13 and 2 times lower than corresponding Km,app. In contrast, QZ59-RRR non-competitively inhibited daunorubicin transport with moderate efficacy (KI,app = 1.9 μM) and displayed a mixed-type inhibition towards Hoechst 33342 transport, resulting from a mainly non-competitive (Ki2,app = 1.6 μM) and a poor but significant competitive tendency (Ki1,app = 5 μM). These results suppose a positional overlap of QZ59 – drug-transport sites, total for the SSS enantiomer and partial for the RRR one. Crystal structures analysis suggests that the H site overlaps both QZ59-SSS locations while the R-site overlaps the most embedded one. PMID:24219411

Steroid hormones are novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

PubMed

Kaneko, Masahiro; Hakuno, Fumihiko; Kamei, Hiroyasu; Yamanaka, Daisuke; Chida, Kazuhiro; Minami, Shiro; Coe, Imogen R; Takahashi, Shin-Ichiro

2014-01-10

Nucleoside transport is important for nucleic acid synthesis in cells that cannot synthesize nucleosides de novo, and for entry of many cytotoxic nucleoside analog drugs used in chemotherapy. This study demonstrates that various steroid hormones induce inhibition of nucleoside transport in mammalian cells. We analyzed the inhibitory effects of estradiol (E2) on nucleoside transport using SH-SY5Y human neuroblastoma cells. We observed inhibitory effects after acute treatment with E2, which lasted in the presence of E2. However, when E2 was removed, the effect immediately disappeared, suggesting that E2 effects are not mediated through the canonical regulatory pathway of steroid hormones, such as transcriptional regulation. We also discovered that E2 could competitively inhibit thymidine uptake and binding of the labeled nucleoside transporter inhibitor, S-[4-nitrobenzyl]-6-thioinosine (NBTI), indicating that E2 binds to endogenous nucleoside transporters, leading to inhibition of nucleoside transport. We then tested the effects of various steroids on nucleoside uptake in NBTI-sensitive cells, SH-SY5Y and NBTI-insensitive cells H9c2 rat cardiomyoblasts. We found E2 and progesterone clearly inhibited both NBTI-sensitive and insensitive uptake at micromolar concentrations. Taken together, we concluded that steroid hormones function as novel nucleoside transport inhibitors by competition with nucleosides for their transporters. Copyright © 2013 Elsevier Inc. All rights reserved.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques.

PubMed

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R; Abyar, Fatemeh

2018-06-05

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H 2 O] (1) and [Zn (L)] (2) [L=N-2-hydroxyacetophenon-N'-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1 H and 13 C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities. Copyright © 2018 Elsevier B.V. All rights reserved.

Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

PubMed Central

Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

2014-01-01

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

PubMed

Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

2014-01-01

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

Lessons learned in induced fit docking and metadynamics in the Drug Design Data Resource Grand Challenge 2

NASA Astrophysics Data System (ADS)

Baumgartner, Matthew P.; Evans, David A.

2018-01-01

Two of the major ongoing challenges in computational drug discovery are predicting the binding pose and affinity of a compound to a protein. The Drug Design Data Resource Grand Challenge 2 was developed to address these problems and to drive development of new methods. The challenge provided the 2D structures of compounds for which the organizers help blinded data in the form of 35 X-ray crystal structures and 102 binding affinity measurements and challenged participants to predict the binding pose and affinity of the compounds. We tested a number of pose prediction methods as part of the challenge; we found that docking methods that incorporate protein flexibility (Induced Fit Docking) outperformed methods that treated the protein as rigid. We also found that using binding pose metadynamics, a molecular dynamics based method, to score docked poses provided the best predictions of our methods with an average RMSD of 2.01 Å. We tested both structure-based (e.g. docking) and ligand-based methods (e.g. QSAR) in the affinity prediction portion of the competition. We found that our structure-based methods based on docking with Smina (Spearman ρ = 0.614), performed slightly better than our ligand-based methods (ρ = 0.543), and had equivalent performance with the other top methods in the competition. Despite the overall good performance of our methods in comparison to other participants in the challenge, there exists significant room for improvement especially in cases such as these where protein flexibility plays such a large role.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R.; Abyar, Fatemeh

2018-06-01

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H2O] (1) and [Zn (L)] (2) [L = N-2-hydroxyacetophenon-N‧-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1H and 13C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities.

Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.

PubMed

Feltus, A; Grosvenor, A L; Conover, R C; Anderson, K W; Daunert, S

2001-04-01

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

Photocaged Competitor Guests: A General Approach Toward Light-Activated Cargo Release From Cucurbiturils.

PubMed

Romero, Miguel A; Basílio, Nuno; Moro, Artur J; Domingues, Mara; González-Delgado, José A; Arteaga, Jesús F; Pischel, Uwe

2017-09-21

A general approach toward the light-induced guest release from cucurbit[7]uril by means of a photoactivatable competitor was devised. An o-nitrobenzyl-caged competitor is photolyzed to generate a competitive guest that can displace cargo from the host macrocycle solely based on considerations of chemical equilibrium. With this method the release of terpene guests from inclusion complexes with cucurbit[7]uril was demonstrated. The binding of the herein investigated terpenes, all being lead fragrant components in essential oils, has been characterized for the first time. They feature binding constants of up to 10 8  L mol -1 and a high differential binding selectivity (spanning four orders of magnitude for the binding constants for the particular set of terpenes). By fine-tuning the photoactivatable competitor guest, selective and also sequential release of the terpenes was achieved. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC.

PubMed

Su, Xingye; Kong, Liang; Li, Xin; Chen, Xueguo; Guo, Ming; Zou, Hanfa

2005-05-27

Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box

DOE PAGES

Jia, Chuandong; Zuo, Wei; Yang, Dong; ...

2017-10-16

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. Here, this similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives,more » as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, l-carnitine, and glycine betaine effectively.« less

Molecular Simulation of Receptor Occupancy and Tumor Penetration of an Antibody and Smaller Scaffolds: Application to Molecular Imaging.

PubMed

Orcutt, Kelly D; Adams, Gregory P; Wu, Anna M; Silva, Matthew D; Harwell, Catey; Hoppin, Jack; Matsumura, Manabu; Kotsuma, Masakatsu; Greenberg, Jonathan; Scott, Andrew M; Beckman, Robert A

2017-10-01

Competitive radiolabeled antibody imaging can determine the unlabeled intact antibody dose that fully blocks target binding but may be confounded by heterogeneous tumor penetration. We evaluated the hypothesis that smaller radiolabeled constructs can be used to more accurately evaluate tumor expressed receptors. The Krogh cylinder distributed model, including bivalent binding and variable intervessel distances, simulated distribution of smaller constructs in the presence of increasing doses of labeled antibody forms. Smaller constructs <25 kDa accessed binding sites more uniformly at large distances from blood vessels compared with larger constructs and intact antibody. These observations were consistent for different affinity and internalization characteristics of constructs. As predicted, a higher dose of unlabeled intact antibody was required to block binding to these distant receptor sites. Small radiolabeled constructs provide more accurate information on total receptor expression in tumors and reveal the need for higher antibody doses for target receptor blockade.

Interaction of berberine with human platelet. alpha. sub 2 adrenoceptors

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

Hui, Ka Kit; Yu, Jun Liang; Chan, Wai Fong A.

1991-01-01

Berberine was found to inhibit competitively the specific binding of ({sup 3}H)-yohimbine. The displacement curve was parallel to those of clonidine, epinephrine, norepinephrine, with the rank order of potency (IC{sub 50}) being clonidine {gt} epinephrine {gt} norepinephrine (14.5 {mu}M) = berberine. Increasing concentrations of berberine from 0.1 {mu}M to 10 {mu}M inhibited ({sup 3}H)-yohimbine binding, shifting the saturation binding curve to the right without decreasing the maximum binding capacity. In platelet cyclic AMP accumulation experiments, berberine at concentrations of 0.1 {mu}M to 0.1 mM inhibited the cAMP accumulation induced by 10 {mu}M prostaglandin E{sub 1} in a dose dependent manner,more » acting as an {alpha}{sub 2} adrenoceptor agonist. In the presence of L-epinephrine, berberine blocked the inhibitory effect of L-epinephrine behaving as an {alpha}{sub 2} adrenoceptor antagonist.« less

Method for screening inhibitors of the toxicity of Bacillus anthracis

DOEpatents

Cirino, Nick M.; Jackson, Paul J.; Lehnert, Bruce E.

2001-01-01

The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

PubMed Central

Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

2015-01-01

Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity. PMID:25600932

Non-steroidal anti-inflammatory drug naproxen destabilizes Aβ amyloid fibrils: A molecular dynamics investigation

PubMed Central

Takeda, Takako; Kumar, Rashmi; Raman, E. Prabhu; Klimov, Dmitri K.

2010-01-01

Using implicit solvent model and replica exchange molecular dynamics we examine the propensity of non-steroidal anti-inflammatory drug, naproxen, to interfere with Aβ fibril growth. We also compare the anti-aggregation propensity of naproxen with that of ibuprofen. Naproxen anti-aggregation effect is influenced by two factors. Similar to ibuprofen, naproxen destabilizes binding of incoming Aβ peptides to the fibril due to direct competition between the ligands and the peptides for the same binding location on the fibril surface (the edge). However, in contrast to ibuprofen naproxen binding also alters the conformational ensemble of Aβ monomers by promoting β-structure. The second factor weakens naproxen anti-aggregation effect. These findings appear to explain the experimental observations, according to which naproxen binds to Aβ fibril with higher affinity than ibuprofen, yet produces weaker anti-aggregation action. PMID:20979356

Ibogaine, a noncompetitive inhibitor of serotonin transport, acts by stabilizing the cytoplasm-facing state of the transporter.

PubMed

Jacobs, Miriam T; Zhang, Yuan-Wei; Campbell, Scott D; Rudnick, Gary

2007-10-05

Ibogaine, a hallucinogenic alkaloid with purported anti-addiction properties, inhibited serotonin transporter (SERT) noncompetitively by decreasing V(max) with little change in the K(m) for serotonin (5-HT). Ibogaine also inhibited binding to SERT of the cocaine analog 2beta-2-carbomethoxy-3-(4-[(125)I]iodophenyl)tropane. However, inhibition of binding was competitive, increasing the apparent K(D) without much change in B(max). Ibogaine increased the reactivity of cysteine residues positioned in the proposed cytoplasmic permeation pathway of SERT but not at nearby positions out of that pathway. In contrast, cysteines placed at positions in the extracellular permeation pathway reacted at slower rates in the presence of ibogaine. These results are consistent with the proposal that ibogaine binds to and stabilizes the state of SERT from which 5-HT dissociates to the cytoplasm, in contrast with cocaine, which stabilizes the state that binds extracellular 5-HT.

Fungal toxins bind to the URF13 protein in maize mitochondria and Escherichia coli.

PubMed Central

Braun, C J; Siedow, J N; Levings, C S

1990-01-01

Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein. Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13. Binding analysis in E. coli reveals cooperative toxin binding. A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URF13 important in toxin binding. A third toxin-insensitive URF13 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13. PMID:2136632

Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization

PubMed Central

Wong, Jason Alexander Ji-Xhin; Bhat, Rakesh; Hockman, Darren; Logan, Michael; Chen, Chao; Levin, Aviad; Frey, Sharon E.; Belshe, Robert B.; Tyrrell, D. Lorne

2014-01-01

ABSTRACT Although effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization. IMPORTANCE An effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4+ and CD8+ T cell responses. PMID:25275133

PA1 Protein, a New Competitive Decelerator Acting at More than One Step to Impede Glucocorticoid Receptor-mediated Transactivation*

PubMed Central

Zhang, Zhenhuan; Sun, Yunguang; Cho, Young-Wook; Chow, Carson C.; Simons, S. Stoney

2013-01-01

Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (Amax), the potency of agonists (EC50), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses Amax, increases the EC50, and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC50, and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene. PMID:23161582

PA1 protein, a new competitive decelerator acting at more than one step to impede glucocorticoid receptor-mediated transactivation.

PubMed

Zhang, Zhenhuan; Sun, Yunguang; Cho, Young-Wook; Chow, Carson C; Simons, S Stoney

2013-01-04

Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (A(max)), the potency of agonists (EC(50)), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses A(max), increases the EC(50), and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC(50), and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene.

Monitoring methotrexate in clinical samples from cancer patients during chemotherapy with a LSPR-based competitive sensor.

PubMed

Zhao, Sandy Shuo; Bichelberger, Mathilde A; Colin, Damien Y; Robitaille, Robert; Pelletier, Joelle N; Masson, Jean-François

2012-10-21

A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.

Type II Kinase Inhibitors Show an Unexpected Inhibition Mode against Parkinson’s Disease-Linked LRRK2 Mutant G2019S

PubMed Central

Liu, Min; Bender, Samantha A.; Cuny, Gregory D; Sherman, Woody; Glicksman, Marcie; Ray, Soumya S.

2014-01-01

A number of well-known type II inhibitors (ATP non-competitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson’s disease-linked mutation G2019S. We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between wild type (WT) and the G2019S mutant of LRRK2. The type II kinase inhibitors were found to work by an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected non-competitive mechanism against WT. Since the G2019S mutation lies in the DXG-motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics. The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form. The conformational bias toward the active form of LRRK2 mutants has two primary consequences: 1) the mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being “locked” into the activated state and 2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. Our results suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors due to desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant. PMID:23379419

Ab initio molecular dynamics determination of competitive O 2 vs. N 2 adsorption at open metal sites of M 2 (dobdc)

DOE PAGES

Parkes, Marie V.; Greathouse, Jeffery A.; Hart, David B.; ...

2016-04-04

The separation of oxygen from nitrogen using metal–organic frameworks (MOFs) is of great interest for potential pressure-swing adsorption processes for the generation of purified O 2 on industrial scales. This study uses ab initio molecular dynamics (AIMD) simulations to examine for the first time the pure-gas and competitive gas adsorption of O 2 and N 2 in the M 2(dobdc) (M = Cr, Mn, Fe) MOF series with coordinatively unsaturated metal centers. Effects of metal, temperature, and gas composition are explored. Lastly, this unique application of AIMD allows us to study in detail the adsorption/desorption processes and to visualize themore » process of multiple guests competitively binding to coordinatively unsaturated metal sites of a MOF.« less

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

NASA Astrophysics Data System (ADS)

Zhang, Z.; Birkedal, V.; Gothelf, K. V.

2016-05-01

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection.

Photochemical and DFT studies on DNA-binding ability and antibacterial activity of lanthanum(III)-phenanthroline complex

NASA Astrophysics Data System (ADS)

Niroomand, Sona; Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Jahani, Shohreh; Moodi, Asieh

2017-02-01

The binding of the lanthanum(III) complex containing 1,10-phenanthroline (phen), [La(phen)3Cl3·OH2], to DNA is investigated by absorption and emission methods. This complex shows absorption decreasing in a charge transfer band, and fluorescence decrement when it binds to DNA. Electronic absorption spectroscopy (UV-Vis), fluorescence spectra, iodide quenching experiments, salt effect and viscosity measurements, ethidium bromide (EB) competition test, circular dichroism (CD) spectra as well as variable temperature experiments indicate that the La(III) complex binds to fish salmon (FS) DNA, presumably via groove binding mode. The binding constants (Kb) of the La(III) complex with DNA is (2.55 ± 0.02) × 106 M-1. Furthermore, the binding site size, n, the Stern-Volmer constant KSV and thermodynamic parameters; enthalpy change (ΔH0) and entropy change (ΔS0) and Gibb's free energy (ΔG0), are calculated according to relevant fluorescent data and the Van't Hoff equation. The La(III) complex has been screened for its antibacterial activities by the disc diffusion method. Also, in order to supplement the experimental findings, DFT computation and NBO analysis are carried out.

Action of tremorgenic mycotoxins on GABA/sub A/ receptor

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

Gant, D.B.; Cole, R.J.; Valdes, J.J.

1987-11-09

The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on ..gamma..-aminobutyric acid (GABA/sub A/) receptor binding and function in rat brain and on binding of a voltage-operated Cl/sup -/ channel in Torpedo electric organ. None of the mycotoxins had significant effect on (/sup 3/H)muscimol or (/sup 3/H)flunitrazepam binding to the GAMA/sup A/ receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced /sup 36/Cl/sup -/ influx and (/sup 35/S)t-butylbicyclophosphorothionate ((/sup 35/S)TBPS) binding in rate brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of (/sup 35/S)TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABA/submore » A/ receptor function by binding close to the receptor's Cl/sup -/ channel. On the voltage-operated Cl/sup -/ channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of (/sup 35/S)TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABA/sub A/ receptor function. 21 references, 4 figures, 2 tables.« less

Exploring the site-selective binding of jatrorrhizine to human serum albumin: spectroscopic and molecular modeling approaches.

PubMed

Mi, Ran; Hu, Yan-Jun; Fan, Xiao-Yang; Ouyang, Yu; Bai, Ai-Min

2014-01-03

This paper exploring the site-selective binding of jatrorrhizine to human serum albumin (HSA) under physiological conditions (pH=7.4). The investigation was carried out using fluorescence spectroscopy, UV-vis spectroscopy, and molecular modeling. The results of fluorescence quenching and UV-vis absorption spectra experiments indicated the formation of the complex of HSA-jatrorrhizine. Binding parameters calculating from Stern-Volmer method and Scatchard method were calculated at 298, 304 and 310 K, with the corresponding thermodynamic parameters ΔG, ΔH and ΔS as well. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that jatrorrhizine bind to HSA with the binding affinities of the order 10(4) L mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for jatrorrhizine-HSA association. Site marker competitive displacement experiments and molecular modeling calculation demonstrating that jatrorrhizine is mainly located within the hydrophobic pocket of the subdomain IIIA of HSA. Furthermore, the synchronous fluorescence spectra suggested that the association between jatrorrhizine and HSA changed molecular conformation of HSA. Copyright © 2013. Published by Elsevier B.V.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

NASA Astrophysics Data System (ADS)

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-03-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

PubMed Central

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-01-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses. PMID:26928635

Spectroscopic and molecular modeling approaches to investigate the interaction of bisphenol A, bisphenol F and their diglycidyl ethers with PPARα.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Ruan, Ping; Ren, Dayong; Dai, Weichang; Yu, Hansong; Li, Tiezhu

2017-08-01

A fluorescence polarization (FP) assay for the simultaneous determination of bisphenol A (BPA), bisphenol F (BPF), bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) was developed. The method was based on the competition between bisphenols (BPs) and fluorescein-labeled dexamethasone derivative (Dex-fl) for mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD). A recombinant soluble protein derivative mPPARα-LBD* was prepared, then in vitro binding of 4 BPs to mPPARα-LBD* was investigated. Fluorescence polarization assay showed that these compounds exhibited different binding potencies with mPPARα-LBD*. Additionally, molecular dynamics simulations were performed to further understand the mechanism of BPs binding affinity for mPPARα-LBD*. Docking results elucidated that the driving forces for the binding of BPs to mPPARα-LBD* were predominantly dependent on hydrophobic and hydrogen-bonding interactions. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.7258). The proposed method has potential for multi-residue detection of BPA, BPF, BADGE, and BFDGE. Copyright © 2017 Elsevier Ltd. All rights reserved.

Calculating binding free energies of host-guest systems using the AMOEBA polarizable force field.

PubMed

Bell, David R; Qi, Rui; Jing, Zhifeng; Xiang, Jin Yu; Mejias, Christopher; Schnieders, Michael J; Ponder, Jay W; Ren, Pengyu

2016-11-09

Molecular recognition is of paramount interest in many applications. Here we investigate a series of host-guest systems previously used in the SAMPL4 blind challenge by using molecular simulations and the AMOEBA polarizable force field. The free energy results computed by Bennett's acceptance ratio (BAR) method using the AMOEBA polarizable force field ranked favorably among the entries submitted to the SAMPL4 host-guest competition [Muddana, et al., J. Comput.-Aided Mol. Des., 2014, 28, 305-317]. In this work we conduct an in-depth analysis of the AMOEBA force field host-guest binding thermodynamics by using both BAR and the orthogonal space random walk (OSRW) methods. The binding entropy-enthalpy contributions are analyzed for each host-guest system. For systems of inordinate binding entropy-enthalpy values, we further examine the hydrogen bonding patterns and configurational entropy contribution. The binding mechanism of this series of host-guest systems varies from ligand to ligand, driven by enthalpy and/or entropy changes. Convergence of BAR and OSRW binding free energy methods is discussed. Ultimately, this work illustrates the value of molecular modelling and advanced force fields for the exploration and interpretation of binding thermodynamics.

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Effects of acute nicotine on hemodynamics and binding of [11C]raclopride to dopamine D2,3 receptors in pig brain.

PubMed

Cumming, Paul; Rosa-Neto, Pedro; Watanabe, Hideaki; Smith, Donald; Bender, Dirk; Clarke, Paul B S; Gjedde, Albert

2003-07-01

Positive reinforcing properties of nicotine and the psychostimulants have been attributed to elevated dopamine release in the basal ganglia. It is well known that the specific binding of [(11)C]raclopride to dopamine D(2,3) receptors in living striatum is reduced by cocaine and amphetamines, revealing increased competition between endogenous dopamine and [(11)C]raclopride for dopamine D(2,3) receptors. However, the sensitivity of [(11)C]raclopride binding to nicotine-induced dopamine release is less well documented. In order to provide the basis for mapping effects of nicotine, we first optimized reference tissue methods for quantifying [(11)C]raclopride binding sites in striatum of living pigs (n = 16). In the same animals, the rate of cerebral blood flow (CBF) was mapped using [(15)O]water. Neither a low dose of nicotine (50 mu kg(-1), iv) nor a high dose of nicotine (500 microg kg(-1), iv) altered CBF in the pig brain, an important condition for calculating the binding of radioligands when using a reference tissue to estimate the free ligand concentration. The methods of Logan and of Lammertsma were compared using the cerebellum or the occipital cortex as reference tissues for calculating the binding potential (pB) of [(11)C]raclolpride in brain. Irrespective of the method used, the mean undrugged baseline pB in striatum (ca. 2.0) was significantly asymmetric, with highest binding in the left caudate and right putamen. Test-retest estimates of pB were stable. Subtraction of Logan pB maps revealed that the low dose of nicotine reduced the pB of [(11)C]raclopride by 10% in a cluster of voxels in the left anteroventral striatum, but this effect did not persist after correction for multiple comparisons. The high dose of nicotine (n = 9) acutely reduced pB by 10% bilaterally in the ventral striatum; 3 h after the high nicotine dose, the reductions had shifted dorsally and caudally into the caudate and putamen. Evidently, nicotine challenge enhances the competition between endogenous dopamine for [(11)C]raclopride binding sites with a complex temporal and spacial pattern in pig brain, initially presenting in the left ventral striatum.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

PubMed

Bellio, Pierangelo; Di Pietro, Letizia; Mancini, Alisia; Piovano, Marisa; Nicoletti, Marcello; Brisdelli, Fabrizia; Tondi, Donatella; Cendron, Laura; Franceschini, Nicola; Amicosante, Gianfranco; Perilli, Mariagrazia; Celenza, Giuseppe

2017-06-15

RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC 50 ) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity. Copyright © 2017 Elsevier GmbH. All rights reserved.

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies.

PubMed

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Sun, Guohui; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2.

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein.

PubMed

Muñoz-Martínez, Francisco; Reyes, Carolina P; Pérez-Lomas, Antonio L; Jiménez, Ignacio A; Gamarro, Francisco; Castanys, Santiago

2006-01-01

Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies

PubMed Central

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2. PMID:29301250

Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes.

PubMed

Treuter, E; Johansson, L; Thomsen, J S; Wärnmark, A; Leers, J; Pelto-Huikko, M; Sjöberg, M; Wright, A P; Spyrou, G; Gustafsson, J A

1999-03-05

Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.

Investigating the effect of hardness cations on coagulation: The aspect of neutralisation through Al(III)-dissolved organic matter (DOM) binding.

PubMed

Zhou, Yuxuan; Yan, Mingquan; Liu, Ruiping; Wang, Dongsheng; Qu, Jiuhui

2017-05-15

Hardness cations are ubiquitous and abundant in source water, while the effect of hardness on the performance of coagulation for dissolved organic matter (DOM) removal in water treatment remains unclear due to the limitation of methods that can characterise the subtle interactions between DOM, coagulant and hardness cations. This work quantified the competition between coagulant Al 3+ and hardness cations to bind onto DOM using absorbance spectroscopy acquired at different Al 3+ concentrations in the absence and presence of Ca 2+ or Mg 2+ . The results indicate that, in the presence of either Mg 2+ or Ca 2+ , an increasing depression of the binding of Al 3+ -DOM could be observed in the differential spectra of DOM with the increasing of Mg 2+ or Ca 2+ at a level of 10, 100 and 1000 μM, with the observation being more significant at higher pH from 6.5 to 8.5. The results of zeta potentials of DOM indicate that the competition of hardness cations results in the negative DOM being less efficiently neutralised by Al 3+ . This study demonstrates that the removal of DOM by coagulation would significantly deteriorate with the presence of hardness cations, which would compete with coagulant Al 3+ to neutralise the unsaturated sites in DOM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Competitive and noncompetitive phage immunoassays for the determination of benzothiostrobin.

PubMed

Hua, Xiude; Zhou, Liangliang; Feng, Lu; Ding, Yuan; Shi, Haiyan; Wang, Limin; Gee, Shirley J; Hammock, Bruce D; Wang, Minghua

2015-08-26

Twenty-three phage-displayed peptides that specifically bind to an anti-benzothiostrobin monoclonal antibody (mAb) in the absence or presence of benzothiostrobin were isolated from a cyclic 8-residue peptide phage library. Competitive and noncompetitive phage enzyme linked immunosorbent assays (ELISAs) for benzothiostrobin were developed by using a clone C3-3 specific to the benzothiostrobin-free mAb and a clone N6-18 specific to the benzothiostrobin immunocomplex, respectively. Under the optimal conditions, the half maximal inhibition concentration (IC50) of the competitive phage ELISA and the concentration of analyte producing 50% saturation of the signal (SC50) of the noncompetitive phage ELISA for benzothiostrobin were 0.94 and 2.27 ng mL(-1), respectively. The noncompetitive phage ELISA showed higher selectivity compared to the competitive. Recoveries of the competitive and the noncompetitive phage ELISAs for benzothiostrobin in cucumber, tomato, pear and rice samples were 67.6-119.6% and 70.4-125.0%, respectively. The amounts of benzothiostrobin in the containing incurred residues samples detected by the two types of phage ELISAs were significantly correlated with that detected by high-performance liquid chromatography (HPLC). Copyright © 2015 Elsevier B.V. All rights reserved.

Decavanadate, a P2X receptor antagonist, and its use to study ligand interactions with P2X7 receptors.

PubMed

Michel, Anton D; Xing, Mengle; Thompson, Kyla M; Jones, Clare A; Humphrey, Patrick P A

2006-03-18

In this study we have studied decavanadate effects at P2X receptors. Decavanadate competitively blocked 2'- and 3'-O-(4benzoylbenzoyl) ATP (BzATP) stimulated ethidium accumulation in HEK293 cells expressing human recombinant P2X7 receptors (pK(B) 7.5). The effects of decavanadate were rapid (minutes) in both onset and offset and contrasted with the much slower kinetics of pyridoxal 5-phosphate (P5P), Coomassie brilliant blue (CBB) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62). Decavanadate competitively blocked the slowly reversible, or irreversible, blockade of the P2X7 receptor produced by P5P and oxidised ATP suggesting competition for a common binding site. However, the interaction between decavanadate and KN62 was non-competitive. Decavanadate also blocked P2X2 and P2X4 receptors but with slightly lower potency. These data demonstrate that decavanadate is the first reversible and competitive antagonist of the P2X7 receptor and is a useful tool for studying the mechanism of interaction of ligands with the P2X7 receptor.

Interactions of Monoamine Oxidases with the Antiepileptic Drug Zonisamide: Specificity of Inhibition and Structure of the Human Monoamine oxidase B Complex

PubMed Central

Binda, Claudia; Aldeco, Milagros; Mattevi, Andrea; Edmondson, Dale E.

2010-01-01

The binding of zonisamide to purified, recombinant monoamine oxidases (MAOs) has been investigated. It is a competitive inhibitor of human MAO B (Ki = 3.1 ± 0.3 μM), of rat MAO B (Ki = 2.9 ± 0.5 μM), and of zebrafish MAO (Ki = 30.8 ± 5.3 μM). No inhibition is observed with purified human or rat MAO A. The 1.8 Å structure of the MAO B complex demonstrates that it binds within the substrate cavity. PMID:21175212

Zinc(II) complexation by some biologically relevant pH buffers.

PubMed

Wyrzykowski, D; Tesmar, A; Jacewicz, D; Pranczk, J; Chmurzyński, L

2014-12-01

The isothermal titration calorimetry (ITC) technique supported by potentiometric titration data was used to study the interaction of zinc ions with pH buffer substances, namely 2-(N-morpholino)ethanesulfonic acid (Mes), piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), and dimethylarsenic acid (Caco). The displacement ITC titration method with nitrilotriacetic acid as a strong, competitive ligand was applied to determine conditional-independent thermodynamic parameters for the binding of Zn(II) to Mes, Pipes, and Caco. Furthermore, the relationship between the proposed coordination mode of the buffers and the binding enthalpy has been discussed. Copyright © 2014 John Wiley & Sons, Ltd.

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

Habara, Y.; Williams, J.A.; Hootman, S.R.

Chloroquine inhibited carbachol-induced amylase release in a dose-dependent fashion in rat pancreatic acini; cholecystokinin- and bombesin-induced secretory responses were almost unchanged by the antimalarial drug. The inhibition of carbachol-induced amylase release by chloroquine was competitive in nature with a K/sub i/ of 11.7 ..mu..M. Chloroquine also inhibited (/sup 3/H)N-methylscopolamine binding to acinar muscarinic receptors. The IC/sub 50/ for chloroquine inhibition of (/sup 3/H)N-methylscopolamine binding was lower than that for carbachol or the other antimalarial drugs, quinine and quinidine. These results demonstrate that chloroquine is a muscarinic receptor antagonist in the exocrine pancreas.

Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid β Fibrils Predicted by Computational Methods.

PubMed

Kawai, Ryoko; Araki, Mitsugu; Yoshimura, Masashi; Kamiya, Narutoshi; Ono, Masahiro; Saji, Hideo; Okuno, Yasushi

2018-05-16

Development of new diagnostic imaging probes for Alzheimer's disease, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes, has been strongly desired. In this study, we investigated the most accessible amyloid β (Aβ) binding site of [ 123 I]IMPY, a Thioflavin-T-derived SPECT probe, using experimental and computational methods. First, we performed a competitive inhibition assay with Orange-G, which recognizes the KLVFFA region in Aβ fibrils, suggesting that IMPY and Orange-G bind to different sites in Aβ fibrils. Next, we precisely predicted the IMPY binding site on a multiple-protofilament Aβ fibril model using computational approaches, consisting of molecular dynamics and docking simulations. We generated possible IMPY-binding structures using docking simulations to identify candidates for probe-binding sites. The binding free energy of IMPY with the Aβ fibril was calculated by a free energy simulation method, MP-CAFEE. These computational results suggest that IMPY preferentially binds to an interfacial pocket located between two protofilaments and is stabilized mainly through hydrophobic interactions. Finally, our computational approach was validated by comparing it with the experimental results. The present study demonstrates the possibility of computational approaches to screen new PET/SPECT probes for Aβ imaging.

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

PubMed

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

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

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

Assessment of the binding performance of histamine-imprinted microspheres by frontal analysis capillary electrophoresis.

PubMed

Romano, Edwin F; Quirino, Joselito P; Holdsworth, John L; So, Regina C; Holdsworth, Clovia I

2017-05-01

Frontal analysis capillary electrophoresis was used to evaluate the binding performance of molecularly imprinted microspheres (MIM) toward its template histamine and analogs at pH 7, and compared to the high performance liquid chromatographic method. In both methods, batch binding was employed and the binding parameters were calculated from the measured concentration of unbound amine analytes and afforded comparable histamine equilibrium dissociation constants (K d ∼ 0.4 mM). FACE was easily carried out at shorter binding equilibration time (i.e. 30 min) and without the need to separate the microspheres, circumventing laborious and, in the case of the system under study, inefficient sample filtration. It also allowed for competitive binding studies by virtue of its ability to distinctly separate intact microspheres and all tested amines which could not be resolved in HPLC. K d 's for nonimprinted (control) microspheres (NIM) from FACE and HPLC were also comparable (∼ 0.6 mM) but at higher histamine concentrations, HPLC gave lower histamine binding. This discrepancy was attributed to inefficient filtration of the batch binding samples prior to HPLC analysis resulting in an over-estimation of the concentration of free histamine brought about by the presence of unfiltered histamine-bound microspheres. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

PubMed

Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

2015-02-01

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered. Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

Differential binding of /sup 3/H-imipramine and /sup 3/H-mianserin in rat cerebral cortex

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

Dumbrille-Ross, A.; Tang, S.W.; Coscina, D.V.

1981-11-16

Drug competition profiles, effect of raphe lesion, and sodium dependency of the binding of two antidepressant drugs /sup 3/H-imipramine and /sup 3/H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common ''antidepressant receptor.'' Of the neurotransmitters tested, only serotonin displaced binding of both /sup 3/H-imipramine and /sup 3/H-mianserin. /sup 3/H-Mianserin binding was potently displaced by serotonin S/sub 2/ antagonists and exhibited a profile similar to that of /sup 3/H-spiperone binding. In the presence of the serotonin S/sub 2/ antagonist spiperone, antihistamines (H/sub 1/) potently displaced /sup 3/H-mianserin binding. /sup 3/H-Imipramine binding was displacedmore » potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing /sup 3/H-imipramine binding was not similar to their order in displacing /sup 3/H-spiperone or -3H-serotonin binding. Prior midbrain raphe lesions greatly decreased the binding of /sup 3/H-imipramine but did not alter binding of /sup 3/H-mianserin. Binding of /sup 3/H-imipramine but not /sup 3/H-mianserin was sodium dependent. These results show that /sup 3/H-imipramine and /sup 3/H-mianserin bind to different receptors. /sup 3/H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. /sup 3/H-Mianserin binds to postsynaptic receptors, possibly both serotonin S/sub 2/ and histamine H/sub 1/ receptors, the binding of which is sodium independent.« less

MELATONIN-INDUCED SUPPRESSION OF PC12 CELL GROWTH IS MEDIATED BY ITS GI COUPLED TRANSMEMBRANE RECEPTORS. (R826248)

EPA Science Inventory

The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the mela...

Targeting Prostate Cancer with Bifunctional Modulators of the Androgen Receptor

DTIC Science & Technology

2013-10-01

linker lengths were prepared and assessed to determine the impact of linker length on the activity of the molecules. HEK293T cells were transfected as...M. Determination of...15 derivatives were determined experimentally by radiolabeled competition binding assays using an extract 16 from Hi5 insect cells expressing an N

The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 [L-(+)-lactate dehydrogenase

PubMed Central

Genet, R; Lederer, F

1990-01-01

Although nitroethane does not bind to the active site of flavocytochrome b2, its anion, ethane nitronate, behaves as a competitive inhibitor, with a Ki of 2.2 mM. No electron transfer can be detected between the nitronate and the enzyme, in contrast with the observations of other workers on D-amino acid oxidase. Propionate is a competitive inhibitor, with a Ki of 28 mM. The significance of these results with respect to the proposed carbanion mechanism and the putative existence of a covalent enzyme-substrate intermediate is discussed. PMID:2178603

Demonstration of four immunoassay formats using the array biosensor

NASA Technical Reports Server (NTRS)

Sapsford, Kim E.; Charles, Paul T.; Patterson, Charles H Jr; Ligler, Frances S.

2002-01-01

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

The IP vs. VDE competition as a key factor determining the stability of the MgBX 5 (X = F, Cl) compounds

NASA Astrophysics Data System (ADS)

Sikorska, Celina; Skurski, Piotr

2010-11-01

The explanation of the MgBX 5 (X = F, Cl) systems existence and stability is provided on the basis of theoretical considerations supported by ab initio calculations. The equilibrium structures and most probable fragmentation processes of 26 (including isomers) MgBX 5 molecules and six OBX 5 compounds (for comparison) were examined and discussed. The competition between the ionization potential (IP) of the MgX subunit and the electron binding energy (VDE) of the BX 4 fragment was found to be the key factor determining the ability to form a stable compound of MgBX 5 type.

A multi-spectroscopic and molecular docking approach to investigate the interaction of antiviral drug oseltamivir with ct-DNA.

PubMed

Moghadam, Neda Hosseinpour; Salehzadeh, Sadegh; Shahabadi, Nahid; Golbedaghi, Reza

2017-07-03

The possible interaction between the antiviral drug oseltamivir and calf thymus DNA at physiological pH was studied by spectrophotometry, competitive spectrofluorimetry, differential pulse voltammogram (DPV), circular dichroism spectroscopy (CD), viscosity measurements, salt effect, and computational studies. Intercalation of oseltamivir between the base pairs of DNA was shown by a sharp increase in specific viscosity of DNA and a decrease of the peak current and a positive shift in differential pulse voltammogram. Competitive fluorescence experiments were performed using neutral red (NR) as a probe for the intercalation binding mode. The studies showed that oseltamivir is able to release the NR.

Novel propanamides as fatty acid amide hydrolase inhibitors.

PubMed

Deplano, Alessandro; Morgillo, Carmine Marco; Demurtas, Monica; Björklund, Emmelie; Cipriano, Mariateresa; Svensson, Mona; Hashemian, Sanaz; Smaldone, Giovanni; Pedone, Emilia; Luque, F Javier; Cabiddu, Maria G; Novellino, Ettore; Fowler, Christopher J; Catalanotti, Bruno; Onnis, Valentina

2017-08-18

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Modern drug design: the implication of using artificial neuronal networks and multiple molecular dynamic simulations

NASA Astrophysics Data System (ADS)

Yakovenko, Oleksandr; Jones, Steven J. M.

2018-01-01

We report the implementation of molecular modeling approaches developed as a part of the 2016 Grand Challenge 2, the blinded competition of computer aided drug design technologies held by the D3R Drug Design Data Resource (https://drugdesigndata.org/). The challenge was focused on the ligands of the farnesoid X receptor (FXR), a highly flexible nuclear receptor of the cholesterol derivative chenodeoxycholic acid. FXR is considered an important therapeutic target for metabolic, inflammatory, bowel and obesity related diseases (Expert Opin Drug Metab Toxicol 4:523-532, 2015), but in the context of this competition it is also interesting due to the significant ligand-induced conformational changes displayed by the protein. To deal with these conformational changes we employed multiple simulations of molecular dynamics (MD). Our MD-based protocols were top-ranked in estimating the free energy of binding of the ligands and FXR protein. Our approach was ranked second in the prediction of the binding poses where we also combined MD with molecular docking and artificial neural networks. Our approach showed mediocre results for high-throughput scoring of interactions.

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

PubMed

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

A portable measuring system for a competitive binding glucose biosensor

NASA Astrophysics Data System (ADS)

Colvin, Lydia E.; Means, A. Kristen; Grunlan, Melissa A.; Coté, Gerard L.

2018-02-01

Central to minimizing the long- and short-term complications associated with diabetes is careful monitoring and maintenance of blood glucose at normal levels. Towards replacing conventionally used finger-prick glucose testing, indwelling continuous glucose monitors (CGMs) based on amperometric electrodes have been introduced to the market. Envisioned to lead to a CGM with an increased lifetime, we report herein a fluorescently-labeled competitive binding assay contained within a hydrogel membrane whose glucose response is measured via a novel portable system. The optical system design included a laser source, bifurcated fiber, laser filter and simple fiber coupled spectrometer to obtain the change in FRET pair ratio of the assay. Glucose response of the assay in free solution was measured using this system across the physiologic range (0-200 mg/dL). The FRET pair ratio signal was seen to increase with glucose and the standard error of calibration was 22.42 mg/dL with a MARD value of 14.85%. When the assay was contained within the hydrogel membrane's central cavity and similarly analyzed, the standard error increased but the assay maintained its reversibility.

Inactivation of muscle adenylate kinase by site-specific destruction of tyrosine 95 using potassium ferrate.

PubMed

Crivellone, M D; Hermodson, M; Axelrod, B

1985-03-10

Potassium ferrate, an analog of orthophosphate and a potent oxidizing agent, was found to irreversibly inactivate porcine muscle adenylate kinase. Inhibition was prevented by competitive inhibitors or substrates, indicating that the action of ferrate was site-specific. Inactivation was accompanied by the loss of Cys-25 and Tyr-95. P1,P5-di(adenosine 5')-pentaphosphate (10(-7) M), a powerful competitive inhibitor, gave 50% protection to the enzyme from ferrate inactivation. No loss of tyrosine or cysteine residues was observed under conditions of total protection. The degree of inactivation was proportional to the amount of Tyr-95 destroyed. However, Cys-25 was totally oxidized when only 55% inactivation had occurred. Partially inactivated enzyme exhibited a Km for ATP and AMP similar to that of the untreated enzyme. It appears that Cys-25 may be proximate to a phosphate-binding site but is not directly involved in the catalytic reaction. The results suggest that Tyr-95 is located in the vicinity of a phosphate-binding region of adenylate kinase and is essential for enzyme activity.

Characterization of the binding of 2-mercaptobenzimidazole to bovine serum albumin.

PubMed

Teng, Yue; Zou, Luyi; Huang, Ming; Zong, Wansong

2015-04-01

2-Mercaptobenzimidazole (MBI) is widely utilized as a corrosion inhibitor, copper-plating brightener and rubber accelerator. The residue of MBI in the environment is potentially harmful to human health. In this article, the interaction of MBI with bovine serum albumin (BSA) was explored using spectroscopic and molecular docking methods under physiological conditions. The positively charged MBI can spontaneously bind with the negatively charged BSA through electrostatic forces with one binding site. The site marker competition experiments and the molecular docking study revealed that MBI bound into site II (subdomain IIIA) of BSA, which further led to some secondary structure and microenvironmental changes of BSA. This work provides useful information on understanding the toxicological actions of MBI at the molecular level. Copyright © 2015 John Wiley & Sons, Ltd.

Anion Binding to Hydrophobic Concavity is Central to the Salting-in Effects of Hofmeister Chaotropes

PubMed Central

Gibb, Corinne L. D.; Gibb, Bruce C.

2011-01-01

For over 120 years it has been appreciated that certain salts (kosmotropes) cause the precipitation of proteins, whilst others (chaotropes) increase their solubility. The cause of this, “Hofmeister effect” is still unclear; especially with the original concept that kosmotropic anions “make” water structure and chaotropes “break” it being countered by recent studies suggesting otherwise. Here, we present the first direct evidence that chaotropic anions have an affinity for hydrophobic concavity, and that it is competition between a convex hydrophobe and the anion for a binding site that leads to the apparent weakening of the hydrophobic effect by chaotropes. In combination, these results suggest that chaotropes primarily induce protein solubilization by direct binding to concavity in the molten globule state of a protein. PMID:21524086

Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin.

PubMed Central

Little, S F; Leppla, S H; Cora, E

1988-01-01

Thirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin have been characterized for affinity, antibody subtype, competitive binding to antigenic regions, and ability to neutralize lethal and edema toxin activities. At least 23 antigenic regions were detected on protective antigen by a blocking, enzyme-linked immunosorbent assay. Two clones, 3B6 and 14B7, competed for a single antigenic region and neutralized the activity of both the lethal toxin in vivo (Fisher 344 rat) and the edema toxin in vitro (CHO cells). These two antibodies blocked the binding of 125I-labeled protective antigen to FRL-103 cells. Our results support the proposal that binding of protective antigen to cell receptors is required for expression of toxicity. Images PMID:3384478

Malathion-induced inhibition of human plasma cholinesterase studied by the fluorescence spectroscopy method

NASA Astrophysics Data System (ADS)

Pavelkić, V. M.; Krinulović, K. S.; Savić, J. Z.; Ilić, M. A.

2008-05-01

The in vitro effect of technical grade malathion was assessed via the kinetic parameters of human plasma butyrylcholinesterase (BChE) using N-methylindoxyl acetate as a substrate for BChE. An inhibitor kinetics study demonstrated the existence of a biphasic inhibition curve, indicating high-and low-affinity binding sites of malathion. The IC 50 values as calculated from the experimental inhibition curves were 1.33 × 10-9 and 1.48 × 10-5 M for the high-and low-affinity binding sites, respectively; Hill’s analysis gave 1.29 × 10-9 and 1.38 × 10-6 M. The Cornish-Bowden plots and their secondary plots indicated that the nature of inhibition was of mixed type with the predominant competitive character of both affinity binding sites.

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.

PubMed

Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K

2006-11-01

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.

Spectroscopic investigation of the effect of salt on binding of tartrazine with two homologous serum albumins: quantification by use of the Debye-Hückel limiting law and observation of enthalpy-entropy compensation.

PubMed

Bolel, Priyanka; Datta, Shubhashis; Mahapatra, Niharendu; Halder, Mintu

2012-08-30

Formation of ion pair between charged molecule and protein can lead to interesting biochemical phenomena. We report the evolution of thermodynamics of the binding of tartrazine, a negatively charged azo colorant, and serum albumins with salt. The dye binds predominantly electrostatically in low buffer strengths; however, on increasing salt concentration, affinity decreases considerably. The calculated thermodynamic parameters in high salt indicate manifestation of nonelectrostatic interactions, namely, van der Waals force and hydrogen bonding. Site-marker competitive binding studies and docking simulations indicate that the dye binds with HSA in the warfarin site and with BSA at the interface of warfarin and ibuprofen binding sites. The docked poses indicate nearby amino acid positive side chains, which are possibly responsible for electrostatic interaction. Using the Debye-Hückel interionic attraction theory for binding equilibria, it is shown that, for electrostatic binding the calculated free energy change increases linearly with square root of ionic strength. Also UV-vis, fluorescence, CD data indicate a decrease of interaction with salt concentration. This study quantitatively relates how ionic strength modulates the strength of the protein-ligand electrostatic interaction. The binding enthalpy and entropy have been found to compensate one another. The enthalpy-entropy compensation (EEC), general property of weak intermolecular interactions, has been discussed.

Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.

PubMed Central

Dubois, B W; Cherian, S F; Evers, A S

1993-01-01

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein. PMID:8341659

Comparison of Fe2O3 and Fe2CoO4 core-shell plasmonic nanoparticles for aptamer mediated SERS assays

NASA Astrophysics Data System (ADS)

Marks, Haley; Mabbott, Samuel; Huang, Po-Jung; Jackson, George W.; Kameoka, Jun; Graham, Duncan; Coté, Gerard L.

2016-03-01

Conjugation of oligonucleotides or aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound bundles of nanoparticles which cause a measurable change in the colloid's optical properties. Here, we present further optimization of a "SERS off" competitive binding assay utilizing plasmonic and magnetic nanoparticles for the detection of the toxin bisphenol A (BPA). The assay involves 1) a `target' silver nanoparticle functionalized with a Raman reporter dye and PEGylated BPA-binding DNA aptamers, and 2) a version of the toxin BPA, bisphenol A diglycidyl ether (BADGE), PEGylated and immobilized onto a silver coated magnetic 'probe' nanoparticle. When mixed, these target and probe nanoparticles cluster into magnetic dimers and trimers and an enhancement in their SERS spectra is observed. Upon introduction of free BPA in its native form, target AgNPs are competitively freed; reversing the nanoparticle assembly and causing the SERS signal to "turn-off" and decrease in response to the competitive binding event. The assay particles were housed inside two types of optofluidic chips containing magnetically active nickel pads, in either a straight or spotted pattern, and both Fe2O3 and Fe2CoO4 were compared as magnetic cores for the silver coated probe nanoparticle. We found that the Ag@ Fe2O3 particles were, on average, more uniform in size and more stable than Ag@ Fe2CoO4, while the addition of cobalt significantly improved the collection time of particles within the magnetic chips. Using 3D Raman mapping, we found that the straight channel design with the Ag@ Fe2O3 particles provided the most uniform nanoparticle organization, while the spotted channel design with Ag@ Fe2CoO4 demonstrated a larger SERS enhancement, and thus a lower limit of detection.

LncRNA MT1JP functions as a ceRNA in regulating FBXW7 through competitively binding to miR-92a-3p in gastric cancer.

PubMed

Zhang, Gang; Li, Shuwei; Lu, Jiafei; Ge, Yuqiu; Wang, Qiaoyan; Ma, Gaoxiang; Zhao, Qinghong; Wu, Dongdong; Gong, Weida; Du, Mulong; Chu, Haiyan; Wang, Meilin; Zhang, Aihua; Zhang, Zhengdong

2018-05-02

Emerging evidence has shown that dysregulation function of long non-coding RNAs (lncRNAs) implicated in gastric cancer (GC). However, the role of the differentially expressed lncRNAs in GC has not fully explained. LncRNA expression profiles were determined by lncRNA microarray in five pairs of normal and GC tissues, further validated in another 75 paired tissues by quantitative real-time PCR (qRT-PCR). Overexpression of lncRNA MT1JP was conducted to assess the effect of MT1JP in vitro and in vivo. The biological functions were demonstrated by luciferase reporter assay, western blotting and rescue experiments. LncRNA MT1JP was significantly lower in GC tissues than adjacent normal tissues, and higher MT1JP was remarkably related to lymph node metastasis and advance stage. Besides, GC patients with higher MT1JP expression had a well survival. Functionally, overexpression of lncRNA MT1JP inhibited cell proliferation, migration, invasion and promoted cell apoptosis in vitro, and inhibited tumor growth and metastasis in vivo. Functional analysis showed that lncRNA MT1JP regulated FBXW7 expression by competitively binding to miR-92a-3p. MiR-92a-3p and down-regulated FBXW7 reversed cell phenotypes caused by lncRNA MT1JP by rescue analysis. MT1JP, a down-regulated lncRNA in GC, was associated with malignant tumor phenotypes and survival of GC. MT1JP regulated the progression of GC by functioning as a competing endogenous RNA (ceRNA) to competitively bind to miR-92a-3p and regulate FBXW7 expression. Our study provided new insight into the post-transcriptional regulation mechanism of lncRNA MT1JP, and suggested that MT1JP may act as a potential therapeutic target and prognosis biomarker for GC.

HTLV-I Tax transrepresses the human c-Myb promoter independently of its interaction with CBP or p300.

PubMed

Nicot, C; Mahieux, R; Opavsky, R; Cereseto, A; Wolff, L; Brady, J N; Franchini, G

2000-04-20

The c-Myb proto-oncogene is preferentially expressed in hematopoietic lineages, and highly expressed in several leukemia types. The Human T-cell Leukemia Virus Type I (HTLV-I) is the etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL). A previous report suggested that Tax, the viral transactivator, is able to suppress the transactivation potential of c-Myb protein by competing for recruitment of CBP. We tested whether such a competition could affect transcription from the c-Myb promoter in Tax expressing T-cells. Using several c-Myb promoter reporter constructs carrying mutations in various regions, we demonstrate that Tax suppression of c-Myb transactivation results in transrepression of the c-Myb promoter through the Myb responsive elements in Jurkat T-cells. The ability of Tax mutants M22, M47 and V89A to interact with the full-length CBP and p300 proteins in vitro, and their ability to repress the c-Myb promoter, was then evaluated. Although both M47 and M22 bind to CBP and p300 to a similar extent, only M47 was able to repress the c-Myb promoter, suggesting that competition for CBP/p300 binding was not the mechanism underlying Tax's effect. This concept was further supported by the fact that the Tax mutant V89A transrepresses the c-Myb promoter efficiently in spite of an impaired binding to CBP and p300. Therefore, Tax-mediated repression of the c-Myb promoter appears to be independent from a direct competition between c-Myb and Tax for recruitment of CBP/p300. Interestingly, a decreased transcription from the endogenous c-Myb promoter was observed in several HTLV-I transformed T-cell lines. Finally, the ability of Tax to directly repress the endogenous c-Myb promoter was demonstrated in a Jurkat cell line stably transfected with a tax gene driven by a cadmium-inducible promoter.

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

PubMed

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

2012-03-23

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

GPER-targeted, 99mTc-labeled, nonsteroidal ligands demonstrate selective tumor imaging and in vivo estrogen binding.

PubMed

Nayak, Tapan K; Ramesh, Chinnasamy; Hathaway, Helen J; Norenberg, Jeffrey P; Arterburn, Jeffrey B; Prossnitz, Eric R

2014-11-01

Our understanding of estrogen (17β-estradiol, E2) receptor biology has evolved in recent years with the discovery and characterization of a 7-transmembrane-spanning G protein-coupled estrogen receptor (GPER/GPR30) and the development of GPER-selective functional chemical probes. GPER is highly expressed in certain breast, endometrial, and ovarian cancers, establishing the importance of noninvasive methods to evaluate GPER expression in vivo. Here, we developed (99m)Tc-labeled GPER ligands to demonstrate the in vivo status of GPER as an estrogen receptor (ER) and for GPER visualization in whole animals. A series of (99m)Tc(I)-labeled nonsteroidal tetrahydro-3H-cyclopenta[c]quinolone derivatives was synthesized utilizing pyridin-2-yl hydrazine and picolylamine chelates. Radioligand receptor binding studies revealed binding affinities in the 10 to 30 nmol/L range. Cell signaling assays previously demonstrated that derivatives retaining a ketone functionality displayed agonist properties, whereas those lacking such a hydrogen bond acceptor were antagonists. In vivo biodistribution and imaging studies performed on mice bearing human endometrial and breast cancer cell xenografts yielded significant tumor uptake (0.4-1.1%ID/g). Blocking studies revealed specific uptake in multiple organs (adrenals, uterus, and mammary tissue), as well as tumor uptake with similar levels of competition by E2 and G-1, a GPER-selective agonist. In conclusion, we synthesized and evaluated a series of first-generation (99m)Tc-labeled GPER-specific radioligands, demonstrating GPER as an estrogen-binding receptor for the first time in vivo using competitive binding principles, and establishing the utility of such ligands as tumor imaging agents. These results warrant further investigation into the role of GPER in estrogen-mediated carcinogenesis and as a target for diagnostic/therapeutic/image-guided drug delivery. These studies provide a molecular basis to evaluate GPER expression and function as an ER through in vivo imaging. ©2014 American Association for Cancer Research.

Dual interaction of agmatine with the rat α2D-adrenoceptor: competitive antagonism and allosteric activation

PubMed Central

Molderings, G J; Menzel, S; Kathmann, M; Schlicker, E; Göthert, M

2000-01-01

In segments of rat vena cava preincubated with [3H]-noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [3H]-noradrenaline release, the EP3 prostaglandin receptor-mediated and the α2D-adrenoceptor-mediated inhibition of evoked [3H]-noradrenaline release was investigated. Agmatine (0.1–10 μM) by itself was without effect on evoked [3H]-noradrenaline release. In the presence of 10 μM agmatine, the prostaglandin E2(PGE2)-induced EP3-receptor-mediated inhibition of [3H]-noradrenaline release was not modified, whereas the α2D-adrenoceptor-mediated inhibition of [3H]-noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine-induced inhibition of [3H]-noradrenaline release. Agmatine concentration-dependently inhibited the binding of [3H]-clonidine and [3H]-rauwolscine to rat brain cortex membranes (Ki values 6 μM and 12 μM, respectively). In addition, 30 and 100 μM agmatine increased the rate of association and decreased the rate of dissociation of [3H]-clonidine resulting in an increased affinity of the radioligand for the α2D-adrenoceptors. [14C]-agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [14C]-agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine. In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the α2D-adrenoceptor and enhances the effects of α2-adrenoceptor agonists probably by binding to an allosteric binding site of the α2D-adrenoceptor which seems to be labelled by [14C]-agmatine. PMID:10928978

Complex Transcriptional Control of the Antibiotic Regulator afsS in Streptomyces: PhoP and AfsR Are Overlapping, Competitive Activators▿

PubMed Central

Santos-Beneit, Fernando; Rodríguez-García, Antonio; Martín, Juan F.

2011-01-01

The afsS gene of several Streptomyces species encodes a small sigma factor-like protein that acts as an activator of several pathway-specific regulatory genes (e.g., actII-ORF4 and redD in Streptomyces coelicolor). The two pleiotropic regulators AfsR and PhoP bind to overlapping sequences in the −35 region of the afsS promoter and control its expression. Using mutated afsS promoters containing specific point mutations in the AfsR and PhoP binding sequences, we proved that the overlapping recognition sequences for AfsR and PhoP are displaced by 1 nucleotide. Different nucleotide positions are important for binding of AfsR or PhoP, as shown by electrophoretic mobility shift assays and by reporter studies using the luxAB gene coupled to the different promoters. Mutant promoter M5 (with a nucleotide change at position 5 of the consensus box) binds AfsR but not PhoP with high affinity (named “superAfsR”). Expression of the afsS gene from this promoter led to overproduction of actinorhodin. Mutant promoter M16 binds PhoP with extremely high affinity (“superPhoP”). Studies with ΔafsR and ΔphoP mutants (lacking AfsR and PhoP, respectively) showed that both global regulators are competitive transcriptional activators of afsS. AfsR has greater influence on expression of afsS than PhoP, as shown by reverse transcriptase PCR (RT-PCR) and promoter reporter (luciferase) studies. These two high-level regulators appear to integrate different nutritional signals (particularly phosphate limitation sensed by PhoR), S-adenosylmethionine, and other still unknown environmental signals (leading to AfsR phosphorylation) for the AfsS-mediated control of biosynthesis of secondary metabolites. PMID:21378195

Targeting Phosphatidylserine with a 64Cu-Labeled Peptide for Molecular Imaging of Apoptosis.

PubMed

Perreault, Amanda; Richter, Susan; Bergman, Cody; Wuest, Melinda; Wuest, Frank

2016-10-03

Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 ( 64 Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with 64 Cu to prepare radiotracer 64 Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6, and nat Cu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer 64 Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide 64 Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC 50 values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6, and nat Cu-NOTA-PSBP-6 were 600 μM, 30 μM, and 23 μM, respectively. A competitive radiometric cell binding assay confirmed binding of 64 Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca 2+ -independent manner. PET imaging studies demonstrated significantly higher uptake of 64 Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV 5min 0.95 ± 0.04) compared to control tumors (SUV 5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-Ava-PSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer 64 Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional "fine-tuning" of 64 Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding potency and in vivo stability to improve tumor uptake and retention.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

PubMed

Perdih, Andrej; Hrast, Martina; Pureber, Kaja; Barreteau, Hélène; Grdadolnik, Simona Golič; Kocjan, Darko; Gobec, Stanislav; Solmajer, Tom; Wolber, Gerhard

2015-06-01

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Inhibitor recognition specificity of MERS-CoV papain-like protease may differ from that of SARS-CoV.

PubMed

Lee, Hyun; Lei, Hao; Santarsiero, Bernard D; Gatuz, Joseph L; Cao, Shuyi; Rice, Amy J; Patel, Kavankumar; Szypulinski, Michael Z; Ojeda, Isabel; Ghosh, Arun K; Johnson, Michael E

2015-06-19

The Middle East Respiratory Syndrome coronavirus (MERS-CoV) papain-like protease (PLpro) blocking loop 2 (BL2) structure differs significantly from that of SARS-CoV PLpro, where it has been proven to play a crucial role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead inhibitors were tested against MERS-CoV PLpro, none of which were effective against MERS-CoV PLpro. Structure and sequence alignments revealed that two residues, Y269 and Q270, responsible for inhibitor binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV PLpro, making critical binding interactions difficult to form for similar types of inhibitors. High-throughput screening (HTS) of 25 000 compounds against both PLpro enzymes identified a small fragment-like noncovalent dual inhibitor. Mode of inhibition studies by enzyme kinetics and competition surface plasmon resonance (SPR) analyses suggested that this compound acts as a competitive inhibitor with an IC50 of 6 μM against MERS-CoV PLpro, indicating that it binds to the active site, whereas it acts as an allosteric inhibitor against SARS-CoV PLpro with an IC50 of 11 μM. These results raised the possibility that inhibitor recognition specificity of MERS-CoV PLpro may differ from that of SARS-CoV PLpro. In addition, inhibitory activity of this compound was selective for SARS-CoV and MERS-CoV PLpro enzymes over two human homologues, the ubiquitin C-terminal hydrolases 1 and 3 (hUCH-L1 and hUCH-L3).

Interaction mechanisms between organic UV filters and bovine serum albumin as determined by comprehensive spectroscopy exploration and molecular docking.

PubMed

Ao, Junjie; Gao, Li; Yuan, Tao; Jiang, Gaofeng

2015-01-01

Organic UV filters are a group of emerging PPCP (pharmaceuticals and personal care products) contaminants. Current information is insufficient to understand the in vivo processes and health risks of organic UV filters in humans. The interaction mechanism of UV filters with serum albumin provides critical information for the health risk assessment of these active ingredients in sunscreen products. This study investigates the interaction mechanisms of five commonly used UV filters (2-hydroxy-4-methoxybenzophenone, BP-3; 2-ethylhexyl 4-methoxycinnamate, EHMC; 4-methylbenzylidene camphor, 4-MBC; methoxydibenzoylmethane, BDM; homosalate, HMS) with bovine serum albumin (BSA) by spectroscopic measurements of fluorescence, circular dichroism (CD), competitive binding experiments and molecular docking. Our results indicated that the fluorescence of BSA was quenched by these UV filters through a static quenching mechanism. The values of the binding constant (Ka) ranged from (0.78±0.02)×10(3) to (1.29±0.01)×10(5) L mol(-1). Further exploration by synchronous fluorescence and CD showed that the conformation of BSA was demonstrably changed in the presence of these organic UV filters. It was confirmed that the UV filters can disrupt the α-helical stability of BSA. Moreover, the results of molecular docking revealed that the UV filter molecule is located in site II (sub-domain IIIA) of BSA, which was further confirmed by the results of competitive binding experiments. In addition, binding occurred mainly through hydrogen bonding and hydrophobic interaction. This study raises critical concerns regarding the transportation, distribution and toxicity effects of organic UV filters in human body. Copyright © 2014 Elsevier Ltd. All rights reserved.

Usefulness of ELISA Methods for Assessing LPS Interactions with Proteins and Peptides

PubMed Central

Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A.; Romarís, Fernanda; Paniagua, Esperanza; Ubeira, Florencio M.

2016-01-01

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, can trigger severe inflammatory responses during bacterial infections, possibly leading to septic shock. One approach to combatting endotoxic shock is to neutralize the most conserved part and major mediator of LPS activity (lipid A) with LPS-binding proteins or peptides. Although several available assays evaluate the biological activity of these molecules on LPS (e.g. inhibition of LPS-induced TNF-α production in macrophages), the development of simple and cost-effective methods that would enable preliminary screening of large numbers of potential candidate molecules is of great interest. Moreover, it would be also desirable that such methods could provide information about the possible biological relevance of the interactions between proteins and LPS, which may enhance or neutralize LPS-induced inflammatory responses. In this study, we designed and evaluated different types of ELISA that could be used to study possible interactions between LPS and any protein or peptide. We also analysed the usefulness and limitations of the different ELISAs. Specifically, we tested the capacity of several proteins and peptides to bind FITC-labeled LPSs from Escherichia coli serotypes O111:B4 and O55:B5 in an indirect ELISA and in two competitive ELISAs including casein hydrolysate (hCAS) and biotinylated polymyxin B (captured by deglycosylated avidin; PMX) as LPS-binding agents in the solid phase. We also examined the influence of pH, detergents and different blocking agents on LPS binding. Our results showed that the competitive hCAS-ELISA performed under mildly acidic conditions can be used as a general method for studying LPS interactions, while the more restrictive PMX-ELISA may help to identify proteins/peptides that are likely to have neutralizing properties in vitro or in vivo. PMID:27249227

Square-wave voltammetry assays for glycoproteins on nanoporous gold

PubMed Central

Pandey, Binod; Bhattarai, Jay K.; Pornsuriyasak, Papapida; Fujikawa, Kohki; Catania, Rosa; Demchenko, Alexei V.; Stine, Keith J.

2014-01-01

Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A – ALP (or soybean agglutinin – ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A–ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL−1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay. PMID:24611035

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, M C; Boyfield, I; Brown, A M; Stemp, G; Middlemiss, D N

1999-01-01

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (−)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells. PMID:10455259

Gonadotropin-Inhibitory Hormone, the Piscine Ortholog of LPXRFa, Participates in 17β-Estradiol Feedback in Female Goldfish Reproduction.

PubMed

Qi, Xin; Zhou, Wenyi; Wang, Qingqing; Guo, Liang; Lu, Danqi; Lin, Haoran

2017-04-01

Gonadotropin-inhibitory hormone (GnIH) plays a critical role in regulating gonadotropin-releasing hormone, gonadotropin hormone, and steroidogenesis in teleosts. In the present study, we sought to determine whether 17β-estradiol (E2) acts directly on GnIH neurons to regulate reproduction in goldfish, a seasonal breeder, and we investigated the role of estrogen receptors (ERs) in mediating this process. We found that GnIH neurons coexpress three types of ERs. Ovariectomy and letrozole implantation into female goldfish at the vitellogenic stage elicited a substantial decrease in the expression of GnIH messenger RNA (mRNA), and E2 supplementation abolished this effect. In primary cultured hypothalamus cells, E2 increased GnIH mRNA levels; surprisingly, selective ERα and ERβ agonists showed opposite effects in regulating GnIH mRNA levels. Using genome walking, we isolated a 2329-bp section of the GnIH promoter sequence, and 7 half-estrogen response elements (EREs) were found in the promoter region. Luciferase assays and electrophoretic mobility shift assay results show that the half-ERE element at -2203 is the key site for competitive binding between ERα and ERβ. Ovariectomy and letrozole implantation into female goldfish in the maturating stage did not change the GnIH mRNA expression levels. Taken together, these findings suggest that E2 binds to multiple types of ERs, which competitively bind to the same half-ERE binding site of the GnIH promoter to achieve both positive and negative feedback in response to estrogen to regulate goldfish reproduction at different stages of ovarian development. Copyright © 2017 Endocrine Society.

Protective effects and mechanisms of curcumin on podophyllotoxin toxicity in vitro and in vivo

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

Li, Juan; Dai, Cai-Xia; Sun, Hua

2012-12-01

Podophyllotoxin (POD) is a naturally occurring lignan with pronounced antineoplastic and antiviral properties. POD binds to tubulin and prevents the formation of mitotic spindle. Although cases of overdose or accidental ingestion are quite often, no specific therapy is currently available to treat the POD intoxication. In the current investigation, the protective effects and mechanisms of curcumin (CUR) on podophyllotoxin toxicity were evaluated in vitro and in vivo. The results showed that CUR could protect POD-induced cytotoxicity by recovering the G2/M arrest and decrease the changes of membrane potential and microtubule structure in Vero cells. A significant decrease of mortality ratesmore » was observed in Swiss mice treated by intragastrical administration of POD + CUR as compared with POD alone. The POD + CUR group also exhibited decreases in plasma transaminases, alkaline phosphatase, lactate dehydrogenase, plasma urea, creatinine and malondialdehyde level but elevated superoxide dismutase and glutathione levels as compared to the POD group. Histological examination of the liver and kidney demonstrated less morphological changes in the treatment of POD + CUR as compared with POD alone. The mechanism of the protective effects might be due to the competitive binding of CUR with POD in the same colchicines binding site as revealed by the tubulin polymerization assay and the molecular docking analysis, and the antioxidant activity against the oxidative stress induced by POD. In summary, both in vitro and in vivo data indicated the promising role of CUR as a protective agent against the POD poisoning. Highlights: ► A potential antidote to treat the podophyllotoxin (POD) intoxication is found. ► Curcumin showed promising effects against POD poisoning in vitro and in vivo. ► The mechanisms lie in the antioxidant activity and competitive binding with tubulin.« less

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization

NASA Astrophysics Data System (ADS)

Perdih, Andrej; Hrast, Martina; Pureber, Kaja; Barreteau, Hélène; Grdadolnik, Simona Golič; Kocjan, Darko; Gobec, Stanislav; Solmajer, Tom; Wolber, Gerhard

2015-06-01

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/ d-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/ d-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8- 11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Escape from Lethal Bacterial Competition through Coupled Activation of Antibiotic Resistance and a Mobilized Subpopulation

PubMed Central

Stubbendieck, Reed M.; Straight, Paul D.

2015-01-01

Bacteria have diverse mechanisms for competition that include biosynthesis of extracellular enzymes and antibiotic metabolites, as well as changes in community physiology, such as biofilm formation or motility. Considered collectively, networks of competitive functions for any organism determine success or failure in competition. How bacteria integrate different mechanisms to optimize competitive fitness is not well studied. Here we study a model competitive interaction between two soil bacteria: Bacillus subtilis and Streptomyces sp. Mg1 (S. Mg1). On an agar surface, colonies of B. subtilis suffer cellular lysis and progressive degradation caused by S. Mg1 cultured at a distance. We identify the lytic and degradative activity (LDA) as linearmycins, which are produced by S. Mg1 and are sufficient to cause lysis of B. subtilis. We obtained B. subtilis mutants spontaneously resistant to LDA (LDAR) that have visibly distinctive morphology and spread across the agar surface. Every LDAR mutant identified had a missense mutation in yfiJK, which encodes a previously uncharacterized two-component signaling system. We confirmed that gain-of-function alleles in yfiJK cause a combination of LDAR, changes in colony morphology, and motility. Downstream of yfiJK are the yfiLMN genes, which encode an ATP-binding cassette transporter. We show that yfiLMN genes are necessary for LDA resistance. The developmental phenotypes of LDAR mutants are genetically separable from LDA resistance, suggesting that the two competitive functions are distinct, but regulated by a single two-component system. Our findings suggest that a subpopulation of B. subtilis activate an array of defensive responses to counter lytic stress imposed by competition. Coordinated regulation of development and antibiotic resistance is a streamlined mechanism to promote competitive fitness of bacteria. PMID:26647299

Kinetics and Mechanism Study of Competitive Inhibition of Jack-Bean Urease by Baicalin

PubMed Central

Tan, Lirong; Su, Jiyan; Wu, Dianwei; Yu, Xiaodan; Su, Zuqing; Wu, Xiaoli; Kong, Songzhi; Lai, Xiaoping; Lin, Ji; Su, Ziren

2013-01-01

Baicalin (BA) is the principal component of Radix Scutellariae responsible for its pharmacological activity. In this study, kinetics and mechanism of inhibition by BA against jack-bean urease were investigated for its therapeutic potential. It was revealed that the IC50 of BA against jack-bean urease was 2.74 ± 0.51 mM, which was proved to be a competitive and concentration-dependent inhibition with slow-binding progress curves. The rapid formation of initial BA-urease complex with an inhibition constant of K i = 3.89 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant of K i* = 1.47 × 10−4 mM. High effectiveness of thiol protectors against BA inhibition indicated that the strategic role of the active-site sulfhydryl group of the urease was involved in the blocking process. Moreover, the inhibition of BA was proved to be reversible due to the fact that urease could be reactivated by dithiothreitol but not reactant dilution. Molecular docking assay suggested that BA made contacts with the important activating sulfhydryl group Cys-592 residues and restricted the mobility of the active-site flap. Taken together, it could be deduced that BA was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for treatments on urease-related diseases. PMID:24198731

Complex coacervation in charge complementary biopolymers: Electrostatic versus surface patch binding.

PubMed

Pathak, Jyotsana; Priyadarshini, Eepsita; Rawat, Kamla; Bohidar, H B

2017-12-01

In this review, a number of systems are described to demonstrate the effect of polyelectrolyte chain stiffness (persistence length) on the coacervation phenomena, after we briefly review the field. We consider two specific types of complexation/coacervation: in the first type, DNA is used as a fixed substrate binding to flexible polyions such as gelatin A, bovine serum albumin and chitosan (large persistence length polyelectrolyte binding to low persistence length biopolymer), and in the second case, different substrates such as gelatin A, bovine serum albumin, and chitosan were made to bind to a polyion gelatin B (low persistence length substrate binding to comparable persistence length polyion). Polyelectrolyte chain flexibility was found to have remarkable effect on the polyelectrolyte-protein complex coacervation. The competitive interplay of electrostatic versus surface patch binding (SPB) leading to associative interaction followed by complex coacervation between these biopolymers is elucidated. We modelled the SPB interaction in terms of linear combination of attractive and repulsive Coulombic forces with respect to the solution ionic strength. The aforesaid interactions were established via a universal phase diagram, considering the persistence length of polyion as the sole independent variable. Copyright © 2017 Elsevier B.V. All rights reserved.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder*

PubMed Central

Ortiz de Orué Lucana, Darío; Fedosov, Sergey N.; Wedderhoff, Ina; Che, Edith N.; Torda, Andrew E.

2014-01-01

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (Kd = 34 μm) to aquo-cobalamin (H2OCbl+) but not to other cobalamins. Competition experiments with the H2OCbl+-coordinating ligand CN− and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H2OCbl+ and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H2OCbl+. Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins. PMID:25342754

The Binding of Four Licorice Flavonoids to Bovine Serum Albumin by Multi-Spectroscopic and Molecular Docking Methods: Structure-Affinity Relationship

NASA Astrophysics Data System (ADS)

Hou, J.; Liang, Q.; Shao, S.

2017-03-01

Flavanones are the main compound of licorice, and the C'-4 position substitution is a significant structural feature for their biological activity. The ability of three selected flavanones (liquiritigenin, liquiritin, and liquiritin apioside) bearing different substituents (hydroxyl groups, glucose, and glucose-apiose sugar moiety) at the C'-4 position and a chalcone ( isoliquiritigenin, an isomer of liquiritigenin) to bind bovine serum albumin (BSA) was studied by multispectroscopic and molecular docking methods under physiological conditions. The binding mechanism of fl avonoids to BSA can be explained by the formation of a flavonoids-BSA complex, and the binding affinity is the strongest for isoliquiritigenin, followed by liquiritin apioside, liquiritin, and liquiritigenin. The thermodynamic analysis and the molecular docking indicated that the interaction between flavonoids and BSA was dominated by the hydrophobic force and hydrogen bonds. The competitive experiments as well as the molecular docking results suggested the most possible binding site of licorice flavonoids on BSA at subdomain IIA. These results revealed that the basic skeleton structure and the substituents at the C'-4 position of flavanones significantly affect the structure-affinity relationships of the licorice flavonoid binding to BSA.

High-affinity 3H-substance P binding to longitudinal muscle membranes of the guinea pig small intestine.

PubMed

Buck, S H; Maurin, Y; Burks, T F; Yamamura, H I

1984-01-30

The binding of 3H-substance P (3H-SP) to longitudinal muscle membranes of the guinea pig small intestine has been characterized. The binding of 3H-SP exhibited a high affinity (Kd = 0.5nM). It was saturable (Bmax = 2 fmoles/mg tissue), reversible, and temperature-dependent. Kinetic studies and competition of 3H-SP binding by unlabeled SP yielded Kd and Ki values, respectively, which were in good agreement with the Kd calculated from saturation studies. The binding of 3H-SP appeared to be dependent on the presence of divalent cations in the incubation buffer. It was displaced by SP and various analogs and fragments in the rank order of SP greater than SP-(2-11) = SP-(3-11) greater than Nle11- SP = physalaemin greater than SP-(4-11) greater than SP-(5-11) greater than eledoisin much greater than SP-(7-11). Our results indicate that 3H-SP binds in longitudinal muscle of the guinea pig small intestine to a biologically relevant receptor which in many respects resembles the SP receptor characterized in the brain and the salivary gland of the rat.

Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

PubMed

Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

PubMed

Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

2003-09-01

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria.

DNA binding properties and biological evaluation of dihydropyrimidinones derivatives as potential antitumor agents.

PubMed

Wang, Gongke; Li, Xiangrong; Gou, Yaping; Chen, Yuhan; Yan, Changling; Lu, Yan

2013-10-01

The binding properties of two medicinally important dihydropyrimidinones derivatives 5-(Ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (EMPD) and 5-(Ethoxycarbonyl)-6-methyl-4-(4-chlorophenyl)-3,4-dihydropyrimidin-2(1H)-one (EMCD) with calf-thymus DNA (ctDNA) were investigated by spectroscopy, viscosity, isothermal titration calorimetry (ITC) and molecular modeling techniques. Simultaneously, their biological activities were evaluated with MTT assay method. The binding constants determined with spectroscopic titration and ITC were found to be in the same order of 10(4)M(-1). According to the results of viscosity studies, fluorescence competitive binding experiment and ITC investigations, intercalative binding was evaluated as the dominant binding modes between the two compounds and ctDNA. Furthermore, the results of molecular modeling corroborated those obtained from spectroscopic, viscosimetric and ITC investigations. Evaluation of the antitumor activities of the two derivatives against different tumor cell lines proved that they exhibited significant tumor cell inhibition rate, accordingly blocking DNA transcription and replication. The present results favor the development of potential drugs related with dihydropyrimidinones derivatives in the treatment of some diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

[Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus].

PubMed

Hang, Xiao-Tong; Li, Jian-Dong; Zhang, Quan-Fu; Li, Chuan; Zhang, Shuo; Liang, Mi-Fang; Li, De-Xin

2010-02-01

Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.