Investigating β-lactam drug targets in <i>Mycobacterium tuberculosis</i> using chemical probes

Abstract Tuberculosis is a deadly disease that requires a flexible arsenal of drugs to treat it. Although β-lactam antibiotics are rarely used to treat Mycobacterium tuberculosis ( Mtb ) infections today, the targets of these drugs are present in the bacterium. Moreover, the cell wall peptidoglycan of Mtb contains an abundance of unusual (3→3) cross-links. These cross-links are formed by enzymes called L,D-transpeptidases, which are susceptible to inhibition by the carbapenem class of antibiotics. We developed new small molecule probes to investigate the L,D-transpeptidases and other β-lactam drug targets in Mtb . We synthesized probes based on three classes of antibiotics, a monobactam, cephalosporin, and carbapenem. For the carbapenem, we synthesized a meropenem analogue conjugated to a far-red fluorophore. This probe was particularly useful in identifying active L,D-transpeptidases in protein gel-resolved lysates. Next we analyzed β-lactam targets in lysates from both hypoxic and actively-replicating cultures of Mtb . We identified numerous targets, including transpeptidases, carboxypeptidases, and the β-lactamase BlaC. Overall, we provide evidence that Mtb dynamically regulates the enzymes responsible for maintaining cell wall peptidoglycan and that meropenem is a good inhibitor of those enzymes.