High-throughput screen with the l,d-transpeptidase Ldt(Mt2) of Mycobacterium tuberculosis reveals novel classes of covalently reacting inhibitors

Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The l,d-transpeptidase Ldt(Mt2), which is responsible for the formation of 3 → 3 cross-links in the cell wall peptidoglycan, has been identified as essential for M. tuberculosis virulence. We optimised a high-throughput assay for Ldt(Mt2), and screened a targeted library of ∼10 000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g., β-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the Ldt(Mt2) catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the Ldt(Mt2) active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC(50) value of ∼1 μM. The results provide leads for the development of new covalently reaction inhibitors of Ldt(Mt2) and other nucleophilic cysteine enzymes.