12 research outputs found

    Evolution of Bacterial Phosphoglycerate Mutases: Non-Homologous Isofunctional Enzymes Undergoing Gene Losses, Gains and Lateral Transfers

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    The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect.Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain

    phosphoglycérate mutase indépendante et la pyruvate phosphate dikinase : potentielles cibles pour la découverte de traitements pour les filarioses

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    La majorité des maladies négligées chez l'Homme sont causées par des parasites. Les nématodes parasites ou filaires sont responsables d'un grand nombre de ces maladies, dont certaines sont transmissibles par des arthropodes piqueurs-suceurs comme les moustiques ou les mouches noires. Il est estimé que 150 millions de personnes dans le monde sont infectées par une filaire et plus d'un milliard de personnes sont à risque. Le contrôle des filarioses s'appuie actuellement sur un petit nombre de médicaments qui sont insuffisants en raison de leur faible efficacité et du développement de résistance. Il y a donc un besoin urgent de médicaments nouveaux et améliorés. Depuis quelques années, les symbiotes intracellulaires a-protéobacterien du genre Wolbachia, qui sont présents dans la plupart des filaires, ont été étudiés comme une nouvelle approche de chimiothérapie. En effet, ces bactéries sont essentielles pour le développement du ver, sa fécondité et sa survie. La découverte de médicaments basée sur des cibles précises représente une approche pour la découverte de nouveaux produits thérapeutiques. Nous avons identifié l'enzyme phosphoglycérate mutase co-facteur indépendante (PGMi) comme une cible thérapeutique potentielle chez les filaires et Wolbachia. PGMi a précédemment été reconnue comme une cible thérapeutique dans plusieurs parasites protozoaires dont Trypanosoma brucei, l'agent causal de la trypanosomiasis Africaine. PGM catalyse l'interconversion du 2 et 3-phosphoglycérate (2- et 3-PG) dans les voies glycolytique et néoglucogénique. Bien que ces voies soient très conservées entre les différents organismes, deux PGMs ont été identifiées, la PGMi et la phosphoglycérate mutase cofacteur dépendante, PGMd. Les mammifères possèdent exclusivement PGMd. Nous avons également identifié l'enzyme glycolytique /néoglucogénique pyruvate phosphate dikinase (PPDK), comme une cible potentielle contre Wolbachia. PPDK catalyse la conversion réversible de l'AMP, du phosphoénolpyruvate et du PPi en ATP, Pi et pyruvate. Dans la plupart des organismes, y compris les mammifères, cette activité est assurée par la pyruvate kinase et aucune PPDK n'est présente. Mon travail de doctorat s'est concentré sur la caractérisation moléculaire et biochimique des PGMis d'un certain nombre d'organismes et de la PPDK de Wolbachia, en vue de développer davantage ces enzymes en tant que cibles pharmaceutiques. Des procédures ont été élaborées afin d'optimiser l'expression et la purification de protéines recombinantes. Les PGMis de nématode ont montré des caractéristiques biochimiques similaires, ce qui indique qu un seul inhibiteur de l'enzyme pourrait être efficace contre toutes les enzymes de nématodes. Les PGMis de Wolbachia, T. brucei et E. coli (et PGMd) ont également été étudiées. Le but ultime de nos études sur la PGMi et la PPDK, est la découverte d'inhibiteurs spécifiques qui pourraient représenter des composés meneurs (lead) pour la poursuite du développement de composés anti-parasitaires. Plusieurs bibliothèques de phages ont été criblées et des peptides qui se lient à la PGMi ont été identifiés. Malheureusement, aucune d'eux n a montré d'activité inhibitrice.The majority of neglected diseases in humans are caused by parasites. Parasitic nematodes are responsible for a substantial number of these diseases, some of which are transmitted by blood feeding arthropods like mosquitoes or black flies. It is estimated that 150 million people in the world are infected with a filarial nematodes and more than a billion people are at risk. Control of filariasis currently relies on a small number of drugs which are inadequate because of their limited efficacy and development of drug resistance. Therefore there is an urgent need for new and improved drugs. In recent years, obligate a-proteobacterial endosymbionts of the genus Wolbachia that are present in most filarial nematodes have been investigated as a new approach to chemotherapy since these bacteria are essential for worm development, fertility and survival. Target-based drug discovery represents one approach to discovering new therapeutics. We have identified the enzyme co-factor independent phosphoglycerate mutase (iPGM) as a potential drug target in nematode parasites and Wolbachia. iPGM has previously been recognized as a drug target in several parasitic protozoa including Trypanosoma brucei, the causative agent of African trypanosomiasis. PGMs catalyze the interconversion of 2- and 3-phosphoglycerate (2-PG and 3-PG) in the glycolytic and gluconeogenic pathways. Although these pathways are highly conserved among different organisms, two distinct PGM enzymes are known to exist, iPGM and the cofactor dependent phosphoglycerate mutase, dPGM. Mammals possess exclusively dPGM. We also identified the glycolytic/gluconeogenic enzyme pyruvate phosphate dikinase (PPDK) as a potential Wolbachia drug target. PPDK catalyses the reversible conversion of AMP, PPi and phosphoenolpyruvate into ATP, Pi and pyruvate. In most organisms, including mammals, this activity is performed by pyruvate kinase and no PPDK is present. My Ph.D studies focused on the molecular and biochemical characterization of iPGM from a number of organisms and PPDK from Wolbachia, with a view to developing these enzymes further as drug targets. Procedures were developed to optimize expression and purification of recombinant proteins. The nematode iPGM enzymes were found to possess similar biochemical characteristics, indicating that a single enzyme inhibitor would likely be effective against all nematode enzymes. iPGM enzymes from Wolbachia, T. brucei and E. coli (and dPGM) were also studied. The ultimate goal of our studies on iPGM and PPDK is the discovery of specific inhibitors that may represent lead compounds for further development as anti-parasitic compounds. Several phage display libraries were screened and peptides which bind to iPGM were identified. Unfortunately, none displayed enzyme inhibitory activity

    phosphoglycérate mutase indépendante et la pyruvate phosphate dikinase (Potentielles cibles pour la découverte de traitements pour les filarioses)

    No full text
    La majorité des maladies négligées chez l'Homme sont causées par des parasites. Les nématodes parasites ou filaires sont responsables d'un grand nombre de ces maladies, dont certaines sont transmissibles par des arthropodes piqueurs-suceurs comme les moustiques ou les mouches noires. Il est estimé que 150 millions de personnes dans le monde sont infectées par une filaire et plus d'un milliard de personnes sont à risque. Le contrôle des filarioses s'appuie actuellement sur un petit nombre de médicaments qui sont insuffisants en raison de leur faible efficacité et du développement de résistance. Il y a donc un besoin urgent de médicaments nouveaux et améliorés. Depuis quelques années, les symbiotes intracellulaires a-protéobacterien du genre Wolbachia, qui sont présents dans la plupart des filaires, ont été étudiés comme une nouvelle approche de chimiothérapie. En effet, ces bactéries sont essentielles pour le développement du ver, sa fécondité et sa survie. La découverte de médicaments basée sur des cibles précises représente une approche pour la découverte de nouveaux produits thérapeutiques. Nous avons identifié l'enzyme phosphoglycérate mutase co-facteur indépendante (PGMi) comme une cible thérapeutique potentielle chez les filaires et Wolbachia. PGMi a précédemment été reconnue comme une cible thérapeutique dans plusieurs parasites protozoaires dont Trypanosoma brucei, l'agent causal de la trypanosomiasis Africaine. PGM catalyse l'interconversion du 2 et 3-phosphoglycérate (2- et 3-PG) dans les voies glycolytique et néoglucogénique. Bien que ces voies soient très conservées entre les différents organismes, deux PGMs ont été identifiées, la PGMi et la phosphoglycérate mutase cofacteur dépendante, PGMd. Les mammifères possèdent exclusivement PGMd. Nous avons également identifié l enzyme glycolytique /néoglucogénique pyruvate phosphate dikinase (PPDK), comme une cible potentielle contre Wolbachia. PPDK catalyse la conversion réversible de l'AMP, du phosphoénolpyruvate et du PPi en ATP, Pi et pyruvate. Dans la plupart des organismes, y compris les mammifères, cette activité est assurée par la pyruvate kinase et aucune PPDK n'est présente. Mon travail de doctorat s est concentré sur la caractérisation moléculaire et biochimique des PGMis d'un certain nombre d'organismes et de la PPDK de Wolbachia, en vue de développer davantage ces enzymes en tant que cibles pharmaceutiques. Des procédures ont été élaborées afin d'optimiser l'expression et la purification de protéines recombinantes. Les PGMis de nématode ont montré des caractéristiques biochimiques similaires, ce qui indique qu un seul inhibiteur de l'enzyme pourrait être efficace contre toutes les enzymes de nématodes. Les PGMis de Wolbachia, T. brucei et E. coli (et PGMd) ont également été étudiées. Le but ultime de nos études sur la PGMi et la PPDK, est la découverte d'inhibiteurs spécifiques qui pourraient représenter des composés meneurs (lead) pour la poursuite du développement de composés anti-parasitaires. Plusieurs bibliothèques de phages ont été criblées et des peptides qui se lient à la PGMi ont été identifiés. Malheureusement, aucune d eux n a montré d activité inhibitrice.The majority of neglected diseases in humans are caused by parasites. Parasitic nematodes are responsible for a substantial number of these diseases, some of which are transmitted by blood feeding arthropods like mosquitoes or black flies. It is estimated that 150 million people in the world are infected with a filarial nematodes and more than a billion people are at risk. Control of filariasis currently relies on a small number of drugs which are inadequate because of their limited efficacy and development of drug resistance. Therefore there is an urgent need for new and improved drugs. In recent years, obligate a-proteobacterial endosymbionts of the genus Wolbachia that are present in most filarial nematodes have been investigated as a new approach to chemotherapy since these bacteria are essential for worm development, fertility and survival. Target-based drug discovery represents one approach to discovering new therapeutics. We have identified the enzyme co-factor independent phosphoglycerate mutase (iPGM) as a potential drug target in nematode parasites and Wolbachia. iPGM has previously been recognized as a drug target in several parasitic protozoa including Trypanosoma brucei, the causative agent of African trypanosomiasis. PGMs catalyze the interconversion of 2- and 3-phosphoglycerate (2-PG and 3-PG) in the glycolytic and gluconeogenic pathways. Although these pathways are highly conserved among different organisms, two distinct PGM enzymes are known to exist, iPGM and the cofactor dependent phosphoglycerate mutase, dPGM. Mammals possess exclusively dPGM. We also identified the glycolytic/gluconeogenic enzyme pyruvate phosphate dikinase (PPDK) as a potential Wolbachia drug target. PPDK catalyses the reversible conversion of AMP, PPi and phosphoenolpyruvate into ATP, Pi and pyruvate. In most organisms, including mammals, this activity is performed by pyruvate kinase and no PPDK is present. My Ph.D studies focused on the molecular and biochemical characterization of iPGM from a number of organisms and PPDK from Wolbachia, with a view to developing these enzymes further as drug targets. Procedures were developed to optimize expression and purification of recombinant proteins. The nematode iPGM enzymes were found to possess similar biochemical characteristics, indicating that a single enzyme inhibitor would likely be effective against all nematode enzymes. iPGM enzymes from Wolbachia, T. brucei and E. coli (and dPGM) were also studied. The ultimate goal of our studies on iPGM and PPDK is the discovery of specific inhibitors that may represent lead compounds for further development as anti-parasitic compounds. Several phage display libraries were screened and peptides which bind to iPGM were identified. Unfortunately, none displayed enzyme inhibitory activity.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

    Cofactor-independent phosphoglycerate mutase from nematodes has limited druggability, as revealed by two high-throughput screens.

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    Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data

    Robustness of the HTS assays performed at Genzyme (top) and NCDS (bottom).

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    <p>TOP: scattergram of percent inhibition data from all plates in the Genzyme HTS. Green: 100% inhibition control; red: 50% inhibition control; black: 0% inhibition control; blue: data for test compounds screened at 10 µM. BOTTOM: scattergram of NCDS data representing uninhibited iPGAM (2% DMSO, shown in red) and inhibited iPGAM (200 µM tannic acid, shown in black). 1120 wells for each condition, spread over seventy 384-well plates, are shown. Dashed lines indicate means ±3× standard deviation (SD).</p

    Dose-response curve of the confirmed hit NCDS-1. NCDS-1 and control inhibitor tannic acid were tested at concentrations from 0.78 µM to 400 µM.

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    <p>Note that tannic acid may inhibit iPGAM via nonspecific aggregation <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002628#pntd.0002628-Pohjala1" target="_blank">[40]</a>; this would render it unsuitable as a chemical probe, but does not prevent its use as a control inhibitor for comparison of inhibited and uninhibited samples. Nonlinear regression and IC<sub>50</sub> value were analyzed with GraphPad Prism software (GraphPad, San Diego, CA, USA). Data are expressed as means ± SEM of at least 3 independent experiments.</p
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