Investigating β-lactam drug targets in Mycobacterium tuberculosis using chemical probes

Abstract

Tuberculosis is a deadly disease that requires a flexible arsenal of drugs to treat it. Although {beta}-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 {beta}-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 {beta}-lactam targets in lysates from both hypoxic and actively-replicating cultures of Mtb. We identified numerous targets, including transpeptidases, carboxypeptidases, and the {beta}-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.