High-throughput screen with the l,d-transpeptidase LdtMt2 of Mycobacterium tuberculosis reveals novel classes of covalently reacting inhibitors

• Published in: Chemical science (Cambridge) Vol. 14; no. 26; pp. 7262 - 7278
• Main Authors: de Munnik, Mariska, Lang, Pauline A, De Dios Anton, Francisco, Cacho, Mónica, Bates, Robert H, Brem, Jürgen, Beatriz Rodríguez Miquel, Schofield, Christopher J
• Format: Journal Article
• Language: English
• Published: Cambridge Royal Society of Chemistry 05.07.2023; The Royal Society of Chemistry
• Subjects: Biosynthesis; Chemistry; Covalence; Crystallography; Cyanamides; Cysteine; Inhibitors; Macrophages; Spectrometry; Tuberculosis
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/d2sc06858c

Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The l,d-transpeptidase LdtMt2, 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 LdtMt2, 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 LdtMt2 catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2 active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC50 value of ∼1 μM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2 and other nucleophilic cysteine enzymes.