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bioRxiv · 10.64898/2026.02.05.698683

A Peptide-Based Assay for Profiling Activity and Inhibitors of Mycobacterial L,D-Transpeptidases

Ocius, K. L.·Sanborn, R. E.·Naick, A.·Basta, L. A. B.·Pires, M.
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Abstract

Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM). L,D-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of {beta}-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.

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Ocius, K. L., Sanborn, R. E., Naick, A., Basta, L. A. B., Pires, M.. 2026-02-06. A Peptide-Based Assay for Profiling Activity and Inhibitors of Mycobacterial L,D-Transpeptidases. https://doi.org/10.64898/2026.02.05.698683

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