Browse
Submit Data
Databases
API
Help

Dataset Information

24 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Selective inhibitors of bacterial phosphopantothenoylcysteine synthetase.

ABSTRACT: Bacterial phosphopantothenolycysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine (PPC) from (R)-phosphopantothenate, l-cysteine, and cytidine-5'-triphosphate (CTP) and has been shown to be essential for growth and survival. The reaction proceeds through a phosphopantothenoyl cytidylate, mixed anhydride intermediate. Both structural and kinetic characterization studies on PPCS have shown differences in the nucleobase binding site between the bacterial and human enzyme. We report for the first time the design and synthesis of mimics of the phosphopantothenoyl cytidylate, which proved to be potent inhibitors of PPCS. These compounds were evaluated in vitro against PPCS from human and several species of bacteria and showed marked selectivity (up to 1000-fold) toward the bacterial enzymes. A phosphodiester intermediate mimic was the most potent of the compounds synthesized and displayed slow-onset, tight-binding kinetics toward E. faecalis PPCS.

SUBMITTER: Patrone JD

PROVIDER: S-EPMC2787235 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Selective inhibitors of bacterial phosphopantothenoylcysteine synthetase.

Patrone James D JD Yao Jiangwei J Scott Nicole E NE Dotson Garry D GD

Journal of the American Chemical Society 20091101 45

Bacterial phosphopantothenolycysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine (PPC) from (R)-phosphopantothenate, l-cysteine, and cytidine-5'-triphosphate (CTP) and has been shown to be essential for growth and survival. The reaction proceeds through a phosphopantothenoyl cytidylate, mixed anhydride intermediate. Both structural and kinetic characterization studies on PPCS have shown differences in the nucleobase binding site between the bacterial and human enzym ...[more]

PMID: 19902973

Similar Datasets

Pantethine rescues phosphopantothenoylcysteine synthetase and phosphopantothenoylcysteine decarboxylase deficiency in Escherichia coli but not in Pseudomonas aeruginosa.

Project description:Coenzyme A (CoA) plays a central and essential role in all living organisms. The pathway leading to CoA biosynthesis has been considered an attractive target for developing new antimicrobial agents with novel mechanisms of action. By using an arabinose-regulated expression system, the essentiality of coaBC, a single gene encoding a bifunctional protein catalyzing two consecutive steps in the CoA pathway converting 4'-phosphopantothenate to 4'-phosphopantetheine, was confirmed in Escherichia coli. Utilizing this regulated coaBC strain, it was further demonstrated that E. coli can effectively metabolize pantethine to bypass the requirement for coaBC. Interestingly, pantethine cannot be used by Pseudomonas aeruginosa to obviate coaBC. Through reciprocal complementation studies in combination with biochemical characterization, it was demonstrated that the differential characteristics of pantethine utilization in these two microorganisms are due to the different substrate specificities associated with endogenous pantothenate kinase, the first enzyme in the CoA biosynthetic pathway encoded by coaA in E. coli and coaX in P. aeruginosa.

| S-EPMC3133259 | biostudies-literature

Characterization and kinetics of phosphopantothenoylcysteine synthetase from Enterococcus faecalis.

Project description:The enzyme phosphopantothenoylcysteine synthetase (PPCS) catalyzes the nucleotide-dependent formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and L-cysteine in the biosynthetic pathway leading to the formation of the essential biomolecule, coenzyme A. The Enterococcus faecalis gene coaB encodes a novel monofunctional PPCS which has been cloned into pET23a and expressed in Escherichia coli BL21 AI. The heterologous expression system yielded 30 mg of purified PPCS per liter of cell culture. The purified enzyme chromatographed as a homodimer of 28 kDa subunits on Superdex HR 200 gel filtration resin. The monofunctional protein displayed a nucleotide specificity for cytidine 5'-triphosphate (CTP) analogous to that seen for bifunctional PPCS expressed by most prokaryotes. Kinetic characterization, utilizing initial velocity and product inhibition studies, found the mechanism of PPCS to be Bi Uni Uni Bi Ping-Pong, with the nucleotide CTP binding first and CMP released last. Michaelis constants were 156, 17, and 86 microM for CTP, (R)-phosphopantothenate, and L-cysteine, respectively, and the k(cat) was 2.9 s(-1). [carboxyl-(18)O]Phosphopantothenate was prepared by hydrolysis of methyl pantothenate with Na(18)OH, followed by enzymatic phosphorylation with E. faecalis pantothenate kinase (PanK). The fate of the carboxylate oxygen of labeled phosphopantothenate, during the course of the PPCS-catalyzed reaction with CTP and L-cysteine, was monitored by (31)P NMR spectroscopy. The results show that the carboxylate oxygen of the phosphopantothenate is recovered with the CMP formed during the reaction, indicative of the formation of a phosphopantothenoyl cytidylate catalytic intermediate, which is consistent with the kinetic mechanism.

| S-EPMC2892170 | biostudies-literature

Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis.

Project description:BackgroundCoenzyme A (CoA) is an essential metabolite, synthesized from vitamin B5 by the subsequent action of five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK. Mutations in Drosophila dPPCS disrupt female fecundity and in this study we analyzed the female sterile phenotype of dPPCS mutants in detail.ResultsWe demonstrate that dPPCS is required for various processes that occur during oogenesis including chorion patterning. Our analysis demonstrates that a mutation in dPPCS disrupts the organization of the somatic and germ line cells, affects F-actin organization and results in abnormal PtdIns(4,5)P2 localization. Improper cell organization coincides with aberrant localization of the membrane molecules Gurken (Grk) and Notch, whose activities are required for specification of the follicle cells that pattern the eggshell. Mutations in dPPCS also induce alterations in scutellar patterning and cause wing vein abnormalities. Interestingly, mutations in dPANK and dPPAT-DPCK result in similar patterning defects.ConclusionTogether, our results demonstrate that de novo CoA biosynthesis is required for proper tissue morphogenesis.

| S-EPMC2542404 | biostudies-literature

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Project description:Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

| S-EPMC5992122 | biostudies-literature

Virtual screening to identify lead inhibitors for bacterial NAD synthetase (NADs).

Project description:Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 microM (7.6% hit rate), and two were selected for future SAR studies.

| S-EPMC2666046 | biostudies-literature

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections.

Project description:Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

| S-EPMC5655057 | biostudies-literature

Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes.

Project description:As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.

| S-EPMC155832 | biostudies-literature

Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis.

Project description:Menaquinone (vitamin K(2)) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an IC(50) value of 5.7microM.

| S-EPMC2628629 | biostudies-literature

Selective inhibitors of methionyl-tRNA synthetase have potent activity against Trypanosoma brucei Infection in Mice.

Project description:Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 μM, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

| S-EPMC3088252 | biostudies-literature

Indole/triazole conjugates are selective inhibitors and inducers of bacterial biofilms.

Project description:Herein is described a method of accessing indole/triazole and benzothiophene/triazole analogues that selectively promote or inhibit biofilm formation by Gram-positive and Gram-negative bacteria. Structure/function studies revealed that the addition of a bromine atom at the 2-position of the indole/triazole scaffold altered activity against both Gram-negative and Gram-positive bacteria and could transform a biofilm inhibitor into a biofilm inducer. Isosteric replacement of the indole core by a benzothiophene significantly impaired anti-biofilm activity. A competition assay exposing Escherichia coli to the most potent biofilm inducer and an inhibitor of E. coli biofilm formation was performed. The inducer exhibited the ability to mute the effect of the anti-biofilm compound for this targeted bacterial population.

| S-EPMC3733276 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data