Comprehensive Identification of β-Lactam Antibiotic Polypharmacology in <i>Mycobacterium tuberculosis</i>

Infections with <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) cause tuberculosis (TB), which requires at least 6 months of treatment with multiple antibiotics. There is emergent interest in using β-lactam antibiotics to improve treatment outcomes for patients. These drugs target cell wall biosynthesis, but a comprehensive list of enzymes inhibited by β-lactams in <i>Mtb</i> is lacking. In the current study, we sought to identify and characterize <i>Mtb</i> enzymes inhibited by β-lactam antibiotics using physiological conditions representative of both acute and chronic TB disease. We used new activity-based probes based on the β-lactam antibiotic meropenem due to its approval by the World Health Organization for TB treatment. Activity-based probes label enzymes based on both substrate specificity and catalytic mechanism, enabling precise identification of drug targets. We identified previously undiscovered targets of meropenem in addition to known cell wall biosynthetic enzymes. We validated β-lactam binding and hydrolysis for six newly identified targets: Rv1723, Rv2257c, Rv0309, DapE (Rv1202), MurI (Rv1338), and LipD (Rv1923). Our results demonstrate that there are at least 30 enzymes in <i>Mtb</i> vulnerable to inhibition by meropenem. This is many more β-lactam targets than historically described, suggesting that efficacy in <i>Mtb</i> is a direct result of polypharmacology.