Investigating β-Lactam Drug Targets in Using Chemical Probes.

Tuberculosis (TB), caused by the bacterial pathogen (), infects 10 million people a year. An estimated 25% of humans harbor latent TB infections, an asymptomatic form of the disease. In both active and latent infections, relies on cell wall peptidoglycan for viability. In the current work, we synthesized fluorescent analogues of β-lactam antibiotics to study two classes of enzymes that maintain 's peptidoglycan: penicillin-binding proteins (PBPs) and l,d-transpeptidases (LDTs). This set of activity-based probes included analogues of three classes of β-lactams: a monobactam (aztreonam-Cy5), a cephalosporin (cephalexin-Cy5), and a carbapenem (meropenem-Cy5). We used these probes to profile enzyme activity in protein gel-resolved lysates of . All three out-performed the commercial reagent Bocillin-FL, a penam. Meropenem-Cy5 was used to identify β-lactam targets by mass spectrometry, including PBPs, LDTs, and the β-lactamase BlaC. New probes were also used to compare PBP and LDT activity in two metabolic states: dormancy and active replication. We provide the first direct evidence that dynamically regulates the enzymes responsible for maintaining peptidoglycan in dormancy. Lastly, we profiled drug susceptibility in lysates and found that meropenem inhibits PBPs, LDTs, and BlaC.