Abstract
The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis. During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.
Highlights
The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs
The only compound clinically approved for treatment of tuberculosis to target this pathway is D-cycloserine (DCS), a structural analogue of D-alanine that inhibits D-alanine:D-alanine ligase (Ddl) and alanine racemase (Alr), enzymes involved in the cytoplasmic stages of PG biosynthesis [2, 3]
A more comprehensive analysis of the time dependence of inhibition of the three Alr enzymes by BCDA demonstrated that M. tuberculosis Alr (MtAlr) underwent inactivation at rates 940- and 1,700-fold lower than those seen with the BsAlr and E. coli Alr (EcAlr) orthologues, respectively, despite almost identical affinity values (Ki, 130 to 230 M) for BCDA (Fig. 1B and C; see Fig. S1B in the supplemental material)
Summary
The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the D-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development. To the best of our knowledge, this is the first report of a MurI-targeting compound with whole-cell activity against M. tuberculosis that represents a potential novel scaffold-target combination for development of new drugs against this remarkable pathogen and perhaps against other bacterial pathogens
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