High-throughput screen with the -transpeptidase Ldt of 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. 10 000 electrophilic compounds were screened for inhibition of the essential l,d -transpeptidase (Ldt Mt2 ) of Mycobacterium tuberculosis . Potent covalently reacting inhibitors were identified and characterised by protein X-ray and MS studies.