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New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis.

Anthony KG, Strych U, Yeung KR, Shoen CS, Perez O, Krause KL, Cynamon MH, Aristoff PA, Koski RA - PLoS ONE (2011)

Bottom Line: Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity.Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors.

View Article: PubMed Central - PubMed

Affiliation: L2 Diagnostics, LLC, New Haven, Connecticut, United States of America. Karen.Anthony@L2dx.com

ABSTRACT

Background: In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.

Methodology/principal findings: To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.

Conclusions/significance: This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

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Related in: MedlinePlus

HTS assays for screening alanine racemase-specific inhibitors.(A) Coupled alanine racemase assay. (B) L-alanine dehydrogenase coupling enzyme assay.
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pone-0020374-g002: HTS assays for screening alanine racemase-specific inhibitors.(A) Coupled alanine racemase assay. (B) L-alanine dehydrogenase coupling enzyme assay.

Mentions: The two enzymatic assays developed for screening alanine racemase-specific inhibitors are shown in Figure 2. The first assay is based on the Esaki and Walsh reaction where the conversion of D-alanine to L-alanine by alanine racemase is coupled to the deamination of L-alanine to pyruvate by the NAD-dependent L-alanine dehydrogenase (Figure 2A) [33]. The formation of NADH from this reaction can be quantified fluorometrically following excitation at 340 nm and emission at 460 nm. This coupled reaction offers a convenient, cost-effective and straightforward means to screen for alanine racemase inhibitors in a high-throughput setting. One drawback of this assay however, is its inability to discriminate inhibitors of the coupling enzyme. Since the latter would result in high rates of false positives, it was necessary to develop a second assay to identify inhibitors of the coupling enzyme. Shown in Figure 2B, this assay essentially consists of the second half of the coupled reaction and directly measures the deamination of L-alanine to pyruvate.


New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis.

Anthony KG, Strych U, Yeung KR, Shoen CS, Perez O, Krause KL, Cynamon MH, Aristoff PA, Koski RA - PLoS ONE (2011)

HTS assays for screening alanine racemase-specific inhibitors.(A) Coupled alanine racemase assay. (B) L-alanine dehydrogenase coupling enzyme assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102704&req=5

pone-0020374-g002: HTS assays for screening alanine racemase-specific inhibitors.(A) Coupled alanine racemase assay. (B) L-alanine dehydrogenase coupling enzyme assay.
Mentions: The two enzymatic assays developed for screening alanine racemase-specific inhibitors are shown in Figure 2. The first assay is based on the Esaki and Walsh reaction where the conversion of D-alanine to L-alanine by alanine racemase is coupled to the deamination of L-alanine to pyruvate by the NAD-dependent L-alanine dehydrogenase (Figure 2A) [33]. The formation of NADH from this reaction can be quantified fluorometrically following excitation at 340 nm and emission at 460 nm. This coupled reaction offers a convenient, cost-effective and straightforward means to screen for alanine racemase inhibitors in a high-throughput setting. One drawback of this assay however, is its inability to discriminate inhibitors of the coupling enzyme. Since the latter would result in high rates of false positives, it was necessary to develop a second assay to identify inhibitors of the coupling enzyme. Shown in Figure 2B, this assay essentially consists of the second half of the coupled reaction and directly measures the deamination of L-alanine to pyruvate.

Bottom Line: Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity.Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors.

View Article: PubMed Central - PubMed

Affiliation: L2 Diagnostics, LLC, New Haven, Connecticut, United States of America. Karen.Anthony@L2dx.com

ABSTRACT

Background: In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.

Methodology/principal findings: To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.

Conclusions/significance: This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

Show MeSH
Related in: MedlinePlus