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Strong Inhibitors (strong + inhibitor)
Selected AbstractsExperimental validation of metabolic pathway modelingFEBS JOURNAL, Issue 13 2008An illustration with glycolytic segments from Entamoeba histolytica In the search for new drug targets in the human parasite Entamoeba histolytica, metabolic control analysis was applied to determine, experimentally, flux control distribution of amebal glycolysis. The first (hexokinase, hexose-6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase (PPi -PFK), aldolase and triose-phosphate isomerase) and final (3-phosphoglycerate mutase, enolase and pyruvate phosphate dikinase) glycolytic segments were reconstituted in vitro with recombinant enzymes under near-physiological conditions of pH, temperature and enzyme proportion. Flux control was determined by titrating flux with each enzyme component. In parallel, both glycolytic segments were also modeled by using the rate equations and kinetic parameters previously determined. Because the flux control distribution predicted by modeling and that determined by reconstitution were not similar, kinetic interactions among all the reconstituted components were experimentally revised to unravel the causes of the discrepancy. For the final segment, it was found that 3-phosphoglycerate was a weakly competitive inhibitor of enolase, whereas PPi was a moderate inhibitor of 3-phosphoglycerate mutase and enolase. For the first segment, PPi was both a strong inhibitor of aldolase and a nonessential mixed-type activator of amebal hexokinase; in addition, lower Vmax values for hexose-6-phosphate isomerase, PPi -PFK and aldolase were induced by PPi or ATP inhibition. It should be noted that PPi and other metabolites were absent from the 3-phosphoglycerate mutase and enolase or aldolase and hexokinase kinetics experiments, but present in reconstitution experiments. Only by incorporating these modifications in the rate equations, modeling predicted values of flux control distribution, flux rate and metabolite concentrations similar to those experimentally determined. The experimentally validated segment models allowed ,in silico experimentation' to be carried out, which is not easy to achieve in in vivo or in vitro systems. The results predicted a nonsignificant effect on flux rate and flux control distribution by adding parallel routes (pyruvate kinase for the final segment and ATP-dependent PFK for the first segment), because of the much lower activity of these enzymes in the ameba. Furthermore, modeling predicted full flux-control by 3-phosphoglycerate mutase and hexokinase, in the presence of low physiological substrate and product concentrations. It is concluded that the combination of in vitro pathway reconstitution with modeling and enzyme kinetics experimentation permits a more comprehensive understanding of the pathway behavior and control properties. [source] Comparative study of eight well-known polyphenolic antioxidantsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2003P. Cos ABSTRACT Eight antioxidants from five different polyphenolic classes (cinnamic acids, benzoic acids, flavonoids, proanthocyanidins and stilbenes), and the water-soluble vitamin E derivative trolox were examined for their antioxidant activity in-vitro. In addition, the compounds were tested for their cytotoxicity on growing fibroblasts and their inhibition of the classical pathway of the complement system. Procyanidin C1 was shown to be a good scavenger of both DPPH* and HO*, and a strong inhibitor of lipid peroxidation and the classical pathway of the complement system. Consequently, procyanidin C1 was classified as the most promising antioxidant in-vitro of all compounds tested. In contrast, genistein exhibited a very low antioxidant activity in both the lipid peroxidation and the DPPH* scavenging assay, a high cytotoxicity and a low complement-inhibiting activity. [source] New kinase regulation mechanism found in HipBA: a bacterial persistence switchACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Artem Evdokimov Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data. [source] Equally potent inhibitors of cholesterol synthesis in human hepatocytes have distinguishable effects on different cytochrome P450 enzymesBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2000L.H. Cohen Abstract Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC50 values: 0.2,8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug,drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6,-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S -mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1,-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O -dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 µM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC50 values around 200 µM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC50 value: 4 µM). Using the assay of testosterone 6,-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC50 values of about 200 µM and a little more by C and A (IC50 around 100 µM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug,drug interaction is considered. Copyright © 2000 John Wiley & Sons, Ltd. [source] Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenaseFEBS JOURNAL, Issue 24 2007Katy Montin 6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho- d -erythronate (4PE) and 4-phospho- d -erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho- d -gluconate (6PG), the inhibitors 5-phospho- d -ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho- d -gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (Kd = 5 µm) and its analogues (Kd =1.3 µm and Kd = 2.8 µm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the Kd value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state. [source] Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenesFEBS JOURNAL, Issue 24 2002Jihui Zhang A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100. The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organisms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can convert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7,8 and 48,56 °C, respectively. The Km of NaoA for nitroethane is ,,26.8 mm. NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA. [source] Genetic features of circular bacteriocins produced by Gram-positive bacteriaFEMS MICROBIOLOGY REVIEWS, Issue 1 2008Mercedes Maqueda Abstract This review highlights the main genetic features of circular bacteriocins, which require the co-ordinated expression of several genetic determinants. In general terms, it has been demonstrated that the expression of such structural genes must be combined with the activity of proteins involved in maturation (cleavage/circularization) and secretion outside the cell via different transporter systems, as well as multifaceted immunity mechanisms essential to ensuring the bacteria's self-protection against such strong inhibitors. Several circular antibacterial peptides produced by Gram-positive bacteria have been described to date, including enterocin AS-48, from Enterococcus faecalis S-48 (the first one characterized), gassericin A, from Lactobacillus gasseri LA39, and a similar one, reutericin 6, from Lactobacillus reuteri LA6, butyrivibriocin AR10, from the ruminal anaerobe Butyrivibrio fibrisolvens AR10, uberolysin, from Streptococcus uberis, circularin A, from Clostridium beijerinckii ATCC 25752, and subtilosin A, from Bacillus subtilis. We summarize here the progress made in the understanding of their principal genetic features over the last few years, during which the functional roles of circular proteins with wide biological activity have become clearer. [source] Synthesis of Monosaccharide-Derived Spirocyclic Cyclopropylamines and Their Evaluation as Glycosidase InhibitorsHELVETICA CHIMICA ACTA, Issue 9 2003Christian Blüchel The glucose-, mannose-, and galactose-derived spirocyclic cyclopropylammonium chlorides 1a,1d, 2a,2d and 3a,3d were prepared as potential glycosidase inhibitors. Cyclopropanation of the diazirine 5 with ethyl acrylate led in 71% yield to a 4,:,5,:,1,:,20 mixture of the ethyl cyclopropanecarboxylates 7a,7d, while the Cu-catalysed cycloaddition of ethyl diazoacetate to the exo -glycal 6 afforded 7a,7d (6,:,2,:,5,:,3) in 93,98% yield (Scheme,1). Saponification, Curtius degradation, and subsequent addition of BnOH or t- BuOH led in 60,80% overall yield to the Z- or Boc-carbamates 11a,11d and 12a,12d, respectively. Hydrogenolysis of 11a,11d afforded 1a,1d, while 12a,12d was debenzylated to 13a,13d prior to acidic cleavage of the N -Boc group. The manno - and galacto -isomers 2a,2d and 3a,3d, respectively, were similarly obtained in comparable yields (Schemes,2 and 4). Also prepared were the differentially protected manno- configured esters 24a,24d; they are intermediates for the synthesis of analogous N -acetylglucosamine-derived cyclopropanes (Scheme,3). The cyclopropylammonium chlorides 1a,1d, 2a,2d and 3a,3d are very weak inhibitors of several glycosidases (Tables,1 and 2). Traces of Pd compounds, however, generated upon catalytic debenzylation, proved to be strong inhibitors. PdCl is, indeed, a reversible, micromolar inhibitor for the ,- glucosidases from C. saccharolyticum and sweet almonds (non-competitive), the , -galactosidases from bovine liver and from E. coli (both non-competitive), the , -galactosidase from Aspergillus niger (competitive), and an irreversible inhibitor of the , -glucosidase from yeast and the , -galactosidase from coffee beans. The cyclopropylamines derived from 1a,1d or 3a,3d significantly enhance the inhibition of the ,- glucosidase from C. saccharolyticum by PdCl, lowering the Ki value from 40,,M (PdCl) to 0.5,,M for a 1,:,1 mixture of PdCl and 1d. A similar effect is shown by cyclopropylamine, but not by several other amines. [source] Weak inhibitors protect cholinesterases from strong inhibitors (paraoxon): in vitro effect of tiaprideJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2005G. A. Petroianu Abstract Weak and reversible inhibitors of cholinesterases, when administered before potent organophosphorus inhibitors (pretreatment), have the ability, to a certain extent, to protect enzymes from inhibition. Such a protective effect was demonstrated in vitro for metoclopramide and ranitidine. The putative mode of protective action of these substances is, when administered in excess, competition for the active site of the enzyme with the more potent organophosphate. The present paper presents results using another benzamide with weak cholinesterase inhibitory properties: tiapride (TIA). The purpose of the study was to quantify in vitro the extent that TIA conferred protection, using paraoxon (POX) as an inhibitor, and to compare the results with existing data obtained using TIA as a protective agent against dichlorvos (DDVP). POX is a highly toxic non-neuropathic organophosphate. While the use of parathion (the inactive prodrug which is metabolically converted to POX) has been restricted in most countries, the organophosphate is still responsible for a large number of accidental or suicidal exposures. DDVP is a moderately toxic, non-neuropathic organophosphate. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood and butyrylcholinesterase (BChE) activities in human plasma were measured photometrically in the presence of different POX and TIA concentrations and the IC50 was calculated. Determinations were repeated in the presence of increasing TIA concentrations. The IC50 of POX increases with the TIA concentration in a linear manner. The protective effect of tiapride on cholinesterase could be of practical relevance in the pretreatment of organophosphate poisoning. It is concluded that in vivo testing of TIA as an organophosphate protective agent is warranted. Copyright © 2005 John Wiley & Sons, Ltd. [source] The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosomeMOLECULAR MICROBIOLOGY, Issue 5 2001Susan M. Poulsen The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058,9, U2506 and U2584,5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer. [source] | ||||||||||||||||