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Elucidating the Catalytic Power of Glutamate Racemase by Investigating a Series of Covalent Inhibitors.

ABSTRACT: The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to decrease off-target liabilities. Herein, we present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase, discovered through structure-based virtual screening. A combination of enzyme kinetics, mass spectrometry, and surface-plasmon resonance experiments details a highly specific 1,4-conjugate addition of a small-molecule inhibitor with a catalytic cysteine of glutamate racemase. Molecular dynamics simulations and quantum mechanics-molecular mechanics geometry optimizations reveal the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency similar to ?-lactam antibiotics, with significant activity against methicillin-resistant S.?aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action against a target in the cell wall biosynthesis pathway.

SUBMITTER: Vance NR

PROVIDER: S-EPMC6581216 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Elucidating the Catalytic Power of Glutamate Racemase by Investigating a Series of Covalent Inhibitors.

Vance Nicholas R NR Witkin Katie R KR Rooney Patrick W PW Li Yalan Y Pope Marshall M Spies M Ashley MA

ChemMedChem 20181121 23

The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to decrease off-target liabilities. Herein, we present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase, discovered through structure-based virtual screening. A combination ...[more]

PMID: 30264520

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Project description:Project description: In this study, we have characterized with distinct biochemical assays the activity of a predicted glutamate racemase from an uncultivated bacterium (Candidatus Saccharimonas aalborgenesis), classified taxonomically within the Candidate Phyla Radiation (CPR). This racemase was produced in a Salmonella auxotrophic mutant defective in its endogenous glutamate racemase. Despite the low identity of the predicated racemase from this uncultivated bacterium compared to that of Salmonella (MurI), ~32.0%, it exhibited high specificity for glutamate as substrate in in vitro racemization assays among the rest of canonical amino acids. Moreover, the expression of this novel racemase complemented the D-Glu auxotrophy of the host bacterium used as chassis (Salmonella). Besides these physiological analyses, the study also characterized the composition and structure of the peptidoglycan produced by bacteria expressing each of the two glutamate racemases, exogenous and endogenous. Data processing protocol: Three biological replicates of two Salmonella strains expressing either the predicted glutamate racemase from the uncultivated bacterium Candidatus Saccharimonas aalborgenesis (Delta-murI-CPR, R1-R3) or the endogenous glutamate racemase (MurI) (Delta-murI-SAL, R1-R3) were grown in the nutrient rich medium LB to reach the exponential phase. At this time, bacteria were collected by centrifugation, washed and lysed by boiling in a 4% SDS-containing buffer. From this material, peptidoglycan was purified by standard protocol. Briefly, after high-speed centrifugation, the pellet containing peptidoglycan as the only SDS-insoluble material was subjected to successive enzymatic treatments with alpha-amylase, pronase (as protease) and, muramidase to cleave the glycan chains and to yield individual uncross-linked and cross-linked muropeptides. The muropeptide mixtures were analysed by LC-MS/MS to obtain a list of parental masses representing the complexity of the sample, i.e. the diversity of co-existing muropeptide classes. The parental masses were assigned as "muropeptides" when a mass of 204.3, corresponding to the N-acetyl-glucosamine molecule, was detected in the fragmentation spectra. The complete lists of parental masses were examined using FreeStyle (Thermo) software and compared among samples (biological replicas and control-problem pairs) by statistical parameters using XCMS software.

Dataset Information

Elucidating the Catalytic Power of Glutamate Racemase by Investigating a Series of Covalent Inhibitors.

Publications

Elucidating the Catalytic Power of Glutamate Racemase by Investigating a Series of Covalent Inhibitors.

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