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