Predicting the <i>in Vivo</i> Mechanism of Action for Drug Leads Using NMR Metabolomics

New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens and to expand the dwindling supply of effective antimicrobials. A common impediment to drug development is the lack of an easy approach to determine the <i>in vivo</i> mechanism of action and efficacy of novel drug leads. Toward this end, we describe an unbiased approach to predict <i>in vivo</i> mechanisms of action from NMR metabolomics data. <i>Mycobacterium smegmatis</i>, a non-pathogenic model organism for <i>Mycobacterium tuberculosis</i>, was treated with 12 known drugs and 3 chemical leads identified from a cell-based assay. NMR analysis of drug-induced changes to the <i>M. smegmatis</i> metabolome resulted in distinct clustering patterns correlating with <i>in vivo</i> drug activity. The clustering of novel chemical leads relative to known drugs provides a mean to identify a protein target or predict <i>in vivo</i> activity

Assessment of Metabolic Changes in <i>Mycobacterium smegmatis</i> Wild-Type and <i>alr</i> Mutant Strains: Evidence of a New Pathway of d‑Alanine Biosynthesis

In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in <i>Mycobacterium tuberculosis</i> and <i>Mycobacterium smegmatis</i> for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of <i>alr</i> inactivation on the cellular metabolism of <i>M. smegmatis</i>. The <i>M. smegmatis alr</i> insertion mutant TAM23 exhibited essentially identical growth to wild-type mc²155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc²155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of <i>M. smegmatis</i> and that specific Alr inhibitors will have no bactericidal action