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Enzyme Inhibition (enzyme + inhibition)
Selected AbstractsGrowth and pectate-lyase activity of the ruminal bacterium Lachnospira multiparus in the presence of short-chain organic acidsLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2005R.A. Paggi Abstract Aims:, Acetic, propionic, butyric and lactic acids are end products of feed fermentation by rumen microbes. The effects of these short chain acids on growth and pectate-lyase (PL) activity of Lachnospira multiparus were studied. Methods and Results:, The bacterial strain used was L. multiparus D32. Acids were tested between 50 and 300 mmol l,1. Growth and PL activity were measured by the increase in total protein content and by the increase in absorbance at 235 nm in the reaction medium respectively. With the exception of lactic acid, all acids decreased bacterial growth rates; generally, these effects were more pronounced at higher concentrations and with acids of longer chains. PL activity was inhibited by all the acids except by butyric acid at 50 and 100 mmol l,1. Enzyme inhibition increased with the concentrations of the acids and lactic acid was the most inhibitory. Conclusions:, High concentrations of short chain acids can differentially inhibit the growth rate and the PL activity of L. multiparus. Significance and Impact of the Study:, Products of fermentation generated by the ruminal microbiota could modify the degradation of pectic substances by this bacterium. [source] Comparing the relative toxicity of malathion and malaoxon in blue catfish Ictalurus furcatusENVIRONMENTAL TOXICOLOGY, Issue 4 2008Winfred G. Aker Abstract Malathion inhibits the critical body enzyme, acetylcholinesterase (AChE). This capability requires that malathion should first be converted to malaoxon to become an active anticholinesterase agent. Conversion can be caused by oxidation in mammals, insects, plants, and in sunlight. In this study, the effects of malathion and malaoxon on catfish Ictalurus furcatus were evaluated. After 96-h exposures, the LC50 (concentration that causes 50% mortality) and IC50 (concentration that causes 50% enzyme inhibition) for malaoxon were lower than corresponding values for malathion. The overall mean 96-h LC50 is 17.0 ppm for malathion and 3.1 ppm for malaoxon. IC50 values for malathion are 8.5 ppm for brain, 10.3 ppm for liver, and 16.6 ppm for muscle. Corresponding values for malaoxon are 2.3, 3.7, and 6.8 ppm, respectively. All the AChE activities in malathion- and malaoxon-exposed catfish brain showed significant inhibition. The oxidation product malaoxon demonstrated higher inhibition on AChE activity than did malathion. Moreover, malaoxon showed significant inhibition on butyrylcholinesterase (BChE) in the liver if the concentrations were increased to more than 1 ppm. Malathion showed no difference between treatment group and control group. Compared with malathion, malaoxon showed higher inhibition on monoamine activity than that of malathion. The results indicated that the oxidative product malaoxon is more toxic than the parent compound malathion. AChE, BChE, and monoamine activities are confirmed as bioindicators of malathion exposure in blue catfish, I. furcatus. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] "Inhibition" of the Enzyme Model TpPh,MeZn,OH by Diketo CompoundsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 24 2006Teame Tekeste Abstract In order to gain a structural understanding of zinc enzyme inhibition, the model complex TpPh,MeZn,OH was treated with various diketo compounds. ,-Keto carboxylic acids were attached to the zinc ion as anionic O,O-chelate ligands, of which benzoylformate was oxidatively decarboxylated in air to form the benzoate complex. Two functionalized ,-diketones did not use their functionality in forming the ,-diketonate complexes. Of the ,-diketones, 2,3-pentanedione formed the ,-keto enolate complex, while 1-phenyl-1,2-propanedione underwent oxidative C,C coupling resulting in a red dinuclear bis(,-keto enolato) complex. Of the diaryl-,-diketones, benzil did not react, but pyridil underwent hydrolytic cleavage to pyridine-2-carbaldehyde and picolinate, of which the latter was bound to the zinc ion as an N,O-chelate ligand.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] The thioredoxin-independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylationFEBS JOURNAL, Issue 1 2007Mirko Zaffagnini In animal cells, many proteins have been shown to undergo glutathionylation under conditions of oxidative stress. By contrast, very little is known about this post-translational modification in plants. In the present work, we showed, using mass spectrometry, that the recombinant chloroplast A4 -glyceraldehyde-3-phosphate dehydrogenase (A4 -GAPDH) from Arabidopsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H2O2. The formation of a mixed disulfide between glutathione and A4 -GAPDH resulted in the inhibition of enzyme activity. A4 -GAPDH was also inhibited by oxidants such as H2O2. However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation. On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e. the AnBn -GAPDH isozyme in either A2B2 or A8B8 conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly. GAPDH catalysis is based on Cys149 forming a covalent intermediate with the substrate 1,3-bisphosphoglycerate. In the presence of 1,3-bisphosphoglycerate, A4 -GAPDH was fully protected from either oxidation or glutathionylation. Site-directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active-site Cys149, did not affect enzyme inhibition by glutathionylation or oxidation. Catalytic Cys149 is thus suggested to be the target of both glutathionylation and thiol oxidation. Glutathionylation could be an important mechanism of regulation and protection of chloroplast A4 -GAPDH from irreversible oxidation under stress. [source] Preventive medicine beyond 65GERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 2 2006Lionel S. Lim Preventive health care in adults aged 65 and older is essential to ensure that quality of life is maintained with longevity. The first half of this article will focus on the two major causes of mortality in the US adult population: cancer and cardiovascular disease. We will address current screening and chemoprevention issues pertaining to breast, cervical, colorectal, prostate and skin cancer. For cardiovascular disease prevention, we will discuss the importance of screening for and treating hypertension, hyperlipidemia, diabetes mellitus, and the use of aspirin chemoprophylaxis and angiotensin-converting enzyme inhibition. In the latter half, we will discuss other aspects of preventive health care including fall prevention, motor vehicle safety, immunizations and screening issues. Health screening can help detect conditions like osteoporosis, subclinical thyroid disease, hearing impairment, nutritional status, and oral and dental problems. Finally, we will also address psychosocial health issues that affect older people including dementia, depression, elder abuse, lifestyle habits and advanced directives. Our recommendations are based on the latest available evidence and include the US Preventive Services Task Force and other leading health professional organizations. [source] Insulin Secretagogues from Moringa oleifera with Cyclooxygenase Enzyme and Lipid Peroxidation Inhibitory ActivitiesHELVETICA CHIMICA ACTA, Issue 2 2004Jayaraj Bioassay-directed isolation and purification of the methanol extract of Moringa oleifera fruits yielded bioactive N -benzyl thiocarbamates, N -benzyl carbamates, benzyl nitriles, and a benzyl ester. Among these, methyl 2-[4-(, - L -rhamnopyranosyl)phenyl]acetate (2), N -[4-(, - L -rhamnopyranosyl)benzyl]-1- O - , - D -glucopyranosylthiocarboxamide (3), 1- O -phenyl- , - L -rhamnopyranoside (5), and 4-[(, - D -glucopyranosyl)-(1,3)-(, - L -rhamnopyranosyl)]phenylacetonitrile (6) are novel, and their structures were determined by spectroscopic methods. The known compounds isolated and characterized from the MeOH extract were niazirin (=4-(, - L -rhamnopyranosyl)phenylacetonitrile; 1), niazicin A (=methyl N -{4-[(4,- O -acetyl- , - L -rhamnopyranosyl)benzyl]}thiocarbamate; 4), methyl N -{4-[(, - L -rhamnopyranosyl)benzyl]}carbamate (7), and methyl N -{4-[(4,- O -acetyl- , - L -rhamnopyranosyl)benzyl]}carbamate (8). The combined yield of these compounds from dried M. oleifera fruits was 1.63%. In rodent pancreatic , -cells (INS-1), compounds 4, 5, 6, 7, and 8 at 100,ppm significantly stimulated insulin release. Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme inhibition assays revealed that 5 and 6 were most active at 83,ppm. Compound 6, however, demonstrated greater specificity for inhibition of COX-2 enzyme (46%) than COX-1 enzyme. Lipid peroxidation assays revealed that 4 and 6 at 50,ppm inhibited peroxidation reactions by 80 and 95%, respectively, while 3 and 8 inhibited lipid peroxidation by 35%. These compounds did not inhibit the cell growth when tested with human breast (MCF-7), central nervous system (CNS, SF-268), lung (NCI-H460), or colon (HCT-116) cancer cell lines. Moreover, these compounds were not cytotoxic at the concentrations tested. [source] HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti-hepatitis C virus activity In Vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus,HEPATOLOGY, Issue 3 2009Norman M. Kneteman Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC50) values of 0.01 to 0.14 ,M for genotype 1, with half maximal effective concentration (EC50s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ± 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV-796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA-dependent RNA polymerase inhibitor HCV796 demonstrated potent anti-HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose-related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti-HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment. (HEPATOLOGY 2009.) [source] Kinetics of inhibition of peroxidase activity of myeloperoxidase by quercetinINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 7 2008Tatjana Momi The inhibition of myeloperoxidase (MPO), isolated from human neutrophils, by quercetin was investigated by following peroxidase activity of the enzyme using o -dianisidine as the substrate. The inhibition parameters (IC50) were obtained by graphical analysis of the inhibition curves. A reaction mechanism, which involved the enzyme inhibition by quercetin and H2O2 in excess, was proposed. The rate and equilibrium constants for the proposed reaction path were calculated from experimental data. Kinetic analysis in noninhibiting H2O2 concentration range in the absence and the presence of quercetin revealed that the reaction mechanism underwent Michaelis,Menten kinetics. K and V values indicated that quercetin was a mixed inhibitor of MPO activity. The initial reaction rates were recalculated using the obtained results. Calculated curves fitted the experimental results within the range of experimental error. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 384,394, 2008 [source] What is the impact of PRIME on real-life diabetic nephropathy?INTERNATIONAL JOURNAL OF CLINICAL PRACTICE, Issue 3 2004L. M. Ruilope Summary Type 2 diabetes is increasing globally and is a major cause of conditions such as cardiovascular disease, retinopathy and nephropathy. The Diabetes Control and Complications Trial and the UK Prospective Diabetes Study demonstrated that the progression of renal disease could be slowed by tight glycaemic control and treating any associated hypertension with angiotensin-converting enzyme inhibition. Recent clinical trials have supported the use of angiotensin II receptor antagonists in the treatment of diabetic nephropathy, resulting in the approval of new therapeutic indications in the United States and Europe. The objective of this review is to demonstrate how results from the Program for Irbesartan Mortality and morbidity Evaluation studies apply to clinical practice, and to show how the benefits of irbesartan therapy can be realised at any stage of renal disease in patients with diabetes. [source] [Fe-Fe]-hydrogenase reactivated by residue mutations as bridging carbonyl rearranges: A QM/MM studyINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 14 2010Stefan Motiu Abstract In this work, we found aqueous enzyme phase reaction pathways for the reactivation of the exogenously inhibited [Fe-Fe]-hydrogenases by O2, or OH,, which metabolizes to H2O (Dogaru et al., Int J Quantum Chem 2008, 108; Motiu et al., Int J Quantum Chem 2007, 107, 1248). We used the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study the reactivation pathways of the exogenously inhibited enzyme matrix. The ONIOM calculations performed on the enzyme agree with experimental results (Liu et al., J Am Chem Soc 2002, 124, 5175), that is, wild-type [Fe-Fe]-hydrogenase H-cluster is inhibited by oxygen metabolites. An enzyme spherical region with a radius of 8 Å (from the distal iron, Fed) has been screened for residues that prevent H2O from leaving the catalytic site and reactivate the [Fe-Fe]-hydrogenase H-cluster. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in the Gibbs' energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs' energy decrease, further residue substitutions have been carried out. Following each substitution, geometry optimization and frequency calculations have been performed to assess the change in the Gibbs' energy for the elimination of H2O. Favorable thermodynamic results have been obtained for both single residue removal (,G,Glu374 = ,1.6 kcal/mol), single substitution (,GGlu374His = ,3.1 kcal/mol), and combined residue substitutions (,GArg111Glu;Thr145Val;Glu374His;Tyr375Phe = ,7.5 kcal/mol). Because the wild-type enzyme has only an endergonic step to overcome, that is, for H2O removal, by eliminating several residues, one at a time, the endergonic step was made to proceed spontaneously. Thus, the most promising residue deletions which enhance H2O elimination are ,Arg111, ,Thr145, ,Ser177, ,Glu240, ,Glu374, and ,Tyr375. The thermodynamics and electronic structure analyses show that the bridging carbonyl (COb) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, COb shifts towards Fed to compensate for the electron density donated to oxygen upon the elimination of H2O. However, this is not possible in the wild-type enzyme because the protein matrix hinders the displacement of COb towards Fed, which leads to enzyme inhibition. Nevertheless, enzyme reactivation can be achieved by means of appropriate amino acid substitutions. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010 [source] X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibitionIUBMB LIFE, Issue 6 2010Koustav Maity Abstract Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2, substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2, position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2, and 4, unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2, and 4,. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2, and 4, positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. © 2010 IUBMB IUBMB Life, 467,476, 2010 [source] Perspectives of data analysis of enzyme inhibition and activation, Part 1: Use of the three-dimensional Km, V,I coordinate system for data analysis of enzyme inhibition and activationJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2009Vladimir I. Krupyanko Abstract The possibility of construction of the three-dimensional (unfolded and folded) Km, V,I rectangular coordinate systems convenient for vector representation of inhibited and activated enzymatic reactions as well as of a two-dimensional Km, V, scalar rectangular coordinate system convenient for diagrammatic representation of enzymatic reactions is considered. The perspectives of using the properties of the three-dimensional L vectors and their scalar L projections for data analysis of enzyme inhibition and activation are analyzed. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:97,100, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20273 [source] Perspectives of data analysis of enzyme inhibition and activation, Part 2: Parametrical classification of types of enzymatic reactionsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2009Vladimir I. Krupyanko Abstract A parametrical classification of types of enzymatic reactions that counts 15 types,7 inhibited reactions, 7 activated ones, and 1 type of initial (uninhibited and nonactivated) reaction,is considered. The system permits taking into account the number of parameters of enzymatic reactions and symmetricity of a course of their change. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:101,107, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20272 [source] Evaluation of the inhibition effect of thiolated poly(acrylates) on vaginal membrane bound aminopeptidase N and release of the model drug LH-RHJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2002Claudia Valenta The purpose of this study was to evaluate the inhibitory effect of thiolated carbopol 974P (carbcys) on the enzymatic activity of vaginal aminopeptidase N in-vitro. Mediated by a carbodiimide, L-cysteine was covalently linked to carbopol 974P. Depending on the weight ratio of polymer to cysteine during the coupling reaction, resulting conjugates displayed 31.3,54.4 ,mol thiol groups per g polymer. The inhibitory effect of carb-cys conjugates was evaluated towards isolated aminopeptidase N and aminopeptidase-N-like activity of excised vaginal mucosa covered with native mucus, respectively. Enzymatic activity was assayed spectrophotometrically using L-leucine- p -nitroanilide (L-leu-pNA) as a synthetic substrate. Carb-cys thereby showed a significantly higher inhibitory effect than unmodified polymer towards both isolated enzyme and vaginal mucosa. Moreover, enzyme inhibition was strongly dependent on the amount of thiol groups being immobilised. The more thiol groups available the higher was the inhibitory effect. Due to its additional high cohesive properties and the possibility of a sustained drug release, which could be shown for the model drug LH-RH, carb-cys appears interesting for the development of vaginal peptide drug-delivery systems. [source] Angiotensin converting enzyme inhibition of fish protein hydrolysates prepared from alkaline-aided channel catfish protein isolateJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 12 2007Ann E Theodore Abstract Peptides derived from aquatic animals have been shown to have inhibitory activity against angiotensin converting enzyme (ACE), which is a key enzyme behind elevated blood pressure. In this study a catfish protein isolate was prepared and hydrolyzed to 5%, 15% and 30% degrees of hydrolysis (% DH) and soluble peptides separated from the total hydrolysate. The hydrolysate and its soluble peptide fraction were studied separately. Increased hydrolysis produced smaller peptides, with the smallest peptides remaining in the soluble fraction. Both hydrolysates and its soluble fraction had high ACE inhibition activities, from 70% to 90.6%, depending on fraction and % DH. Results suggested that there is not a simple relationship between average peptide size and extent of % DH and ACE inactivation, but clearly the soluble fraction of the hydrolysate, containing the smallest peptides, is responsible for most of the ACE inhibition activity of the hydrolysate. Hydrolysates prepared from a pure and uniform catfish protein isolate substrate do therefore show a potential for ACE inhibition and may find use as bioactive ingredients. Copyright © 2007 Society of Chemical Industry [source] Triclosan inhibition of mycobacterial InhA in Saccharomyces cerevisiae: yeast mitochondria as a novel platform for in vivo antimycolate assaysLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2010A. Gurvitz Abstract Aims:, To demonstrate the suitability of yeast to act as a novel biotechnological platform for conducting in vivo inhibition assays using drugs with low efficacies towards their mycobacterial targets, such as occurs in the situation with triclosan and InhA. Methods and Results:, A surrogate yeast host represented by Saccharomyces cerevisiae etr1, cells lacking Etr1p, the 2- trans -enoyl-thioester reductase of mitochondrial type 2 fatty acid synthase (FASII), was designed to rely on the Mycobacterium tuberculosis FASII enzyme InhA. Although InhA is 10 000 times less sensitive to the antimicrobial drug triclosan than is bacterial FabI, the respiratory growth of yeast cells depending on InhA was severely affected on glycerol medium containing triclosan. Conclusions:, The yeast system could detect enzyme inhibition despite the use of a drug with only low efficacy. Significance and Impact of the Study:, Tuberculosis affects a third of the human population, and InhA is a major drug target for combating this disease. InhA is inhibited by isoniazid, but triclosan-derived compounds are presently being developed as antimycolates. A demonstration of triclosan inhibition of InhA in yeast represents a meaningful variation in studying this effect in mycobacteria, because it occurred without the potentially confusing aspects of perturbing protein,protein interactions which are presumed vital to mycobacterial FASII, inactivating other important enzymes or eliciting a dedicated transcriptional response in Myco. tuberculosis. [source] Toward an "omic" physiopathology of reactive chemicals: Thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compoundsMASS SPECTROMETRY REVIEWS, Issue 5 2009Federico Maria Rubino Abstract Cancer and degenerative diseases are major causes of morbidity and death, derived from the permanent modification of key biopolymers such as DNA and regulatory proteins by usually smaller, reactive molecules, present in the environment or generated from endogenous and xenobiotic components by the body's own biochemical mechanisms (molecular adducts). In particular, protein adducts with organic electrophiles have been studied for more than 30 [see, e.g., Calleman et al., 1978] years essentially for three purposes: (a) as passive monitors of the mean level of individual exposure to specific chemicals, either endogenously present in the human body or to which the subject is exposed through food or environmental contamination; (b) as quantitative indicators of the mean extent of the individual metabolic processing which converts a non-reactive chemical substance into its toxic products able to damage DNA (en route to cancer induction through genotoxic mechanisms) or key proteins (as in the case of several drugs, pesticides or otherwise biologically active substances); (c) to relate the extent of protein modification to that of biological function impairment (such as enzyme inhibition) finally causing the specific health damage. This review describes the role that contemporary mass spectrometry-based approaches employed in the qualitative and quantitative study of protein,electrophile adducts play in the discovery of the (bio)chemical mechanisms of toxic substances and highlights the future directions of research in this field. A particular emphasis is given to the measurement of often high levels of the protein adducts of several industrial and environmental pollutants in unexposed human populations, a phenomenon which highlights the possibility that a number of small organic molecules are generated in the human organism through minor metabolic processes, the imbalance of which may be the cause of "spontaneous" cases of cancer and of other degenerative diseases of still uncharacterized etiology. With all this in mind, it is foreseen that a holistic description of cellular functions will take advantage of new analytical methods based on time-integrated metabolomic measurements of a new biological compartment, the "adductome," aimed at better understanding integrated organism response to environmental and endogenous stressors. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:725,784, 2009 [source] Inhibition of cyclooxygenase (COX) enzymes by compounds from Daucus carota L. SeedsPHYTOTHERAPY RESEARCH, Issue 8 2003kali A. Momin Abstract Cyclooxygenase (COX) enzymes inhibitory assay directed investigation of Daucus carota seed extracts resulted in the isolation and characterization of compounds, 2,4,5-trimethoxybenzaldehyde (1), oleic acid (2), trans -asarone (3) and geraniol (4). Compounds 1,4 showed 3.32, 45.32, 46.15, and 3.15% of prostaglandin H endoperoxide synthase-I (COX-I) inhibitory activity and 52.69, 68.41, 64.39 and 0% prostaglandin H endoperoxide synthase-II (COX-II) inhibitory activity, respectively at 100 mg mL,1. Compound 1 showed selectivity towards COX-II enzyme inhibition at 100 µg mL,1. The COX-II/COX-I ratio for compound 1 was 17.68 at 100 µg mL,1 compared to solvent control. Ibuprofen, Naproxen, Aspirin, Celebrex and Vioxx at concentrations of 2.06, 2.52, 180, 1.67 and 1.67 µg mL,1, respectively, gave COX-II/COX-I ratios of 1.13, 0.92, 0.24, 16 and 75, respectively. The inhibition of COX-II enzymes by compounds 1 at 100 µg mL,1 was signi,cant when compared to Aspirin, Ibuprofen, Naproxen and Celebrex at concentrations studied. Copyright © 2003 John Wiley & Sons, Ltd. [source] Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosisPROTEIN SCIENCE, Issue 8 2007Nicole A. Kruh Abstract The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD+ (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein,protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer,dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present. [source] A new generation of protein display scaffolds for molecular recognitionPROTEIN SCIENCE, Issue 1 2006Ralf J. Hosse Abstract Engineered antibodies and their fragments are invaluable tools for a vast range of biotechnological and pharmaceutical applications. However, they are facing increasing competition from a new generation of protein display scaffolds, specifically selected for binding virtually any target. Some of them have already entered clinical trials. Most of these nonimmunoglobulin proteins are involved in natural binding events and have amazingly diverse origins, frameworks, and functions, including even intrinsic enzyme activity. In many respects, they are superior over antibody-derived affinity molecules and offer an ever-extending arsenal of tools for, e.g., affinity purification, protein microarray technology, bioimaging, enzyme inhibition, and potential drug delivery. As excellent supporting frameworks for the presentation of polypeptide libraries, they can be subjected to powerful in vitro or in vivo selection and evolution strategies, enabling the isolation of high-affinity binding reagents. This article reviews the generation of these novel binding reagents, describing validated and advanced alternative scaffolds as well as the most recent nonimmunoglobulin libraries. Characteristics of these protein scaffolds in terms of structural stability, tolerance to multiple substitutions, ease of expression, and subsequent applications as specific targeting molecules are discussed. Furthermore, this review shows the close linkage between these novel protein tools and the constantly developing display, selection, and evolution strategies using phage display, ribosome display, mRNA display, cell surface display, or IVC (in vitro compartmentalization). Here, we predict the important role of these novel binding reagents as a toolkit for biotechnological and biomedical applications. [source] Telomerase enzyme inhibition (TEI) and cytolytic therapy in the management of androgen independent osseous metastatic prostate cancerTHE PROSTATE, Issue 6 2010Yingming Li Abstract BACKGROUND Recurrent prostate cancer can be osseous, androgen independent and lethal. The purpose is to discern the efficacy of synthetic small molecule telomerase enzyme inhibitors (TEI) alone or in combination with other cytotoxic therapies in controlling metastatic osseous prostate cancer. METHODS C4-2B was pre-treated with a match or mismatch TEI for 6 weeks and then inoculated into nude mice subcutaneously or intraosseously. In a separate experiment, untreated C4-2B was injected into femur of nude mice. The mice were divided into seven systemic "combination" treatment groups of control, Ad-BSP-E1a virus, docetaxel, mismatch and match TEI. Serum PSA was followed longitudinally. Histology analyses and histomorphometry were performed. Repeated measure analysis was applied for statistical analysis and Bonferroni method was used in multiple comparisons. RESULTS In the pre-treated study, the PSA of match treated cells in subcutaneous or intraosseous model was significantly lower than mismatch TEI or PBS treated group (P,<,0.05). Histology revealed increased fibrosis, apoptosis and decreased PSA staining in the match TEI treated subcutaneous xenografts. In the combination treatment study, the PSA was significantly lower in single/double treatment and triple treatment than control (P,<,0.05). Histology revealed that triple therapy mice had normal femur architecture. Histomorphometrics revealed that the area of femur tumor and woven bone was significantly positively correlated (P,=,0.007). CONCLUSIONS Multiple lines of data point toward the efficacy of systemically administered telomerase inhibitors. Combining cytotoxic regimens with telomerase inhibitors could be beneficial in controlling prostate cancer. Clinical trials are warranted to explore the efficacy of TEI in prostate cancer. Prostate 70: 616,629, 2010. © 2009 Wiley-Liss, Inc. [source] Photometric and Electrochemical Enzyme-Multiplied Assay Techniques Using ,-Galactosidase as Reporter EnzymeBIOTECHNOLOGY PROGRESS, Issue 3 2006Francis H. Ko ,-Galactosidase (,-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known ,-gal substrate analog, o -nitrophenyl ,- d -galactopyranoside, yields the visibly colored, o -nitrophenol product upon hydrolysis, whereas the substrate, p -aminophenyl ,- d -galactopyranoside, gives rise to an electrooxidizable product, p -aminophenol. These ,-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for ,-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, ,-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the ,-gal-estradiol conjugate up to ,75%. Reporter enzyme inhibition is relieved significantly by addition of ,2 ng/mL of estradiol (as E-BSA), which competes for binding with the antibody. Thus, the presence of analyte (E-BSA) is reported by the enzyme (,-gal), which amplifies the ligand-protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p -aminophenol, is responsive to added estradiol within minutes. These results show that ,-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems. [source] Off-Target Decoding of a Multitarget Kinase Inhibitor by Chemical ProteomicsCHEMBIOCHEM, Issue 7 2009Enrico Missner Abstract Unbiased: Chemical proteomics was used to profile compound interactions in an unbiased fashion. We present here the application of different compound-immobilization routes for decoding nonprotein kinase off-targets of the multitarget kinase inhibitor C1, which interacts with distinct compound moieties. Since the approval of the first selective tyrosine kinase inhibitor, imatinib, various drugs have been developed to target protein kinases. However, due to a high degree of structural conservation of the ATP binding site, off-target effects have been reported for several drugs. Here, we report on off-target decoding for a multitarget protein kinase inhibitor by chemical proteomics, by focusing on interactions with nonprotein kinases. We tested two different routes for the immobilization of the inhibitor on a carrier matrix, and thus identified off-targets that interact with distinct compound moieties. Besides several of the kinases known to bind to the compound, the pyridoxal kinase (PDXK), which has been described to interact with the CDK inhibitor (R)-roscovitine, was captured. The PDXK,inhibitor interaction was shown to occur at the substrate binding site rather than at the ATP binding site. In addition, carbonic anhydrase 2 (CA2) binding was demonstrated, and the determination of the IC50 revealed an enzyme inhibition in the submicromolar range. The data demonstrate that different compound immobilization routes for chemical proteomics approaches are a valuable method to improve the knowledge about the off-target profile of a compound. [source] A Mass Spectrometry Plate Reader: Monitoring Enzyme Activity and Inhibition with a Desorption/Ionization on Silicon (DIOS) PlatformCHEMBIOCHEM, Issue 7 2004Zhouxin Shen Dr. Abstract A surface-based laser desorption/ionization mass spectrometry assay that makes use of Desorption/Ionization on Silicon Mass Spectrometry (DIOS-MS) has been developed to monitor enzyme activity and enzyme inhibition. DIOS-MS has been used to characterize inhibitors from a library and then to monitor their activity against selected enzyme targets, including proteases, glycotransferase, and acetylcholinesterase. An automated DIOS-MS system was also used as a high-throughput screen for the activity of novel enzymes and enzyme inhibitors. On two different commercially available instruments, a sampling rate of up to 38 inhibitors per minute was accomplished, with thousands of inhibitors being monitored. The ease of applying mass spectrometry toward developing enzyme assays and the speed of surface-based assays such as DIOS for monitoring inhibitor effectiveness and enzyme activity makes it attractive for a broad range of screening applications. [source] The Biochemistry of Drug Metabolism , An IntroductionCHEMISTRY & BIODIVERSITY, Issue 10 2009Abstract This review on intra-individual factors affecting drug metabolism completes our series on the biochemistry of drug metabolism. The article presents the molecular mechanisms causing intra-individual differences in enzyme expression and activity. They include enzyme induction by transcriptional activation and enzyme inhibition on the protein level. The influencing factors are of physiological, pathological, or external origin. Tissue characteristics and developmental age strongly influence enzyme-expression patterns. Further influencing factors are pregnancy, disease, or biological rhythms. Xenobiotics, drugs, constituents of herbal remedies, food constituents, ethanol, and tobacco can all influence enzyme expression or activity and, hence, affect drug metabolism. [source] [Fe-Fe]-hydrogenase reactivated by residue mutations as bridging carbonyl rearranges: A QM/MM studyINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 14 2010Stefan Motiu Abstract In this work, we found aqueous enzyme phase reaction pathways for the reactivation of the exogenously inhibited [Fe-Fe]-hydrogenases by O2, or OH,, which metabolizes to H2O (Dogaru et al., Int J Quantum Chem 2008, 108; Motiu et al., Int J Quantum Chem 2007, 107, 1248). We used the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study the reactivation pathways of the exogenously inhibited enzyme matrix. The ONIOM calculations performed on the enzyme agree with experimental results (Liu et al., J Am Chem Soc 2002, 124, 5175), that is, wild-type [Fe-Fe]-hydrogenase H-cluster is inhibited by oxygen metabolites. An enzyme spherical region with a radius of 8 Å (from the distal iron, Fed) has been screened for residues that prevent H2O from leaving the catalytic site and reactivate the [Fe-Fe]-hydrogenase H-cluster. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in the Gibbs' energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs' energy decrease, further residue substitutions have been carried out. Following each substitution, geometry optimization and frequency calculations have been performed to assess the change in the Gibbs' energy for the elimination of H2O. Favorable thermodynamic results have been obtained for both single residue removal (,G,Glu374 = ,1.6 kcal/mol), single substitution (,GGlu374His = ,3.1 kcal/mol), and combined residue substitutions (,GArg111Glu;Thr145Val;Glu374His;Tyr375Phe = ,7.5 kcal/mol). Because the wild-type enzyme has only an endergonic step to overcome, that is, for H2O removal, by eliminating several residues, one at a time, the endergonic step was made to proceed spontaneously. Thus, the most promising residue deletions which enhance H2O elimination are ,Arg111, ,Thr145, ,Ser177, ,Glu240, ,Glu374, and ,Tyr375. The thermodynamics and electronic structure analyses show that the bridging carbonyl (COb) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, COb shifts towards Fed to compensate for the electron density donated to oxygen upon the elimination of H2O. However, this is not possible in the wild-type enzyme because the protein matrix hinders the displacement of COb towards Fed, which leads to enzyme inhibition. Nevertheless, enzyme reactivation can be achieved by means of appropriate amino acid substitutions. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010 [source] Metabolic effects of p -coumaric acid in the perfused rat liverJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2006Leonardo C.N. Lima The p -coumaric acid, a phenolic acid, occurs in several plant species and, consequently, in many foods and beverages of vegetable origin. Its antioxidant activity is well documented, but there is also a single report about an inhibitory action on the monocarboxylate carrier, which operates in the plasma and mitochondrial membranes. The latter observation suggests that p -coumaric acid could be able to inhibit gluconeogenesis and related parameters. The present investigation was planned to test this hypothesis in the isolated and hemoglobin-free perfused rat liver. Transformation of lactate and alanine into glucose (gluconeogenesis) in the liver was inhibited by p -coumaric acid (IC50 values of 92.5 and 75.6 ,M, respectively). Transformation of fructose into glucose was inhibited to a considerably lower degree (maximally 28%). The oxygen uptake increase accompanying gluconeogenesis from lactate was also inhibited. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC50 = 160.1 ,M); no such effect was observed in freeze,thawing disrupted mitochondria. Glucose 6-phosphatase and fructose 1,6-bisphosphatase were not inhibited. In isolated intact mitochondria, p -coumaric acid inhibited respiration dependent on pyruvate oxidation but was ineffective on respiration driven by succinate and ,-hydroxybutyrate. It can be concluded that inhibition of pyruvate transport into the mitochondria is the most prominent primary effect of p -coumaric acid and also the main cause for gluconeogenesis inhibition. The existence of additional actions of p -coumaric acid, such as enzyme inhibitions and interference with regulatory mechanisms, cannot be excluded. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:18,26, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20114 [source] QSAR for Inhibition of Pseudomonas Species Lipase by 1-Acyloxy-3- N-n -octylcarbamyl-benzenesMOLECULAR INFORMATICS, Issue 3 2009Shyh-Ying Chiou Abstract 1-Acyloxy-3- N-n -octylcarbamyl-benzenes (1,9) are synthesized to characterize the Quantitative Structure,Activity Relationship (QSAR) for the Third Acyl Group Binding Site (TACS) of Pseudomonas species lipase. Inhibitors 1,9 are characterized as pseudo or alternate substrate inhibitors of the enzyme. The inhibition constant (Ki) and carbamylation constant (k2) for the enzyme inhibitions by inhibitors 1,9 are determined. The carbamate carbons of the n -octylcarbamyl moieties of inhibitors 1,9 are nucleophilically attacked by the active site serine of the enzyme and the n -octylcarbamyl groups of inhibitors 1,9 are bound to the Acyl Group Binding Site (ACS) of the enzyme. Both pKi and log,k2 values are linearly corrected with the Hansch hydrophobicity , values of the substituents of the acyl moieties of inhibitors 1,7. The slopes for these corrections are 0.13 and 0.02, respectively. This result suggests that the enzyme inhibitions by inhibitors 1,7 have a common mechanism. Thus, all acyl moieties of inhibitors 1,7 should bind to the TACS of the enzyme since the acyl and carbamyl moieties of inhibitors 1,7 are meta to each other. This result also indicates that the major interaction between the acyl moiety of inhibitors 1,7 and the TACS of the enzyme is primarily the hydrophobic interaction. The more hydrophobic characters of inhibitors 1,7 are, the more tightly these inhibitors bind to the enzyme. In contrast, 1-triphenylacetoxy-3- N-n -octylcarbamyl-benzene (8) and 1-trimethylacetoxy-3- N-n -octylcarbamyl-benzene (9) do not bind to the TACS of the enzyme due to the fact that the inhibitions by both inhibitors are not linearly correlated with ,. It is possible that these two inhibitors are too bulky to fit into the TACS of the enzyme. [source] | ||||||||||||||||||||||||||||