New high-throughput test speeds tuberculosis drug discovery

Tuberculosis and related non-tuberculous mycobacteria (NTM) infections remain difficult to treat because the bacteria build a tough, protective cell wall. A family of enzymes called l,d-Transpeptidases (Ldts) plays an essential role in making that cell wall by crosslinking peptidoglycan stem peptides. Because Ldts are important for cell wall integrity, they are attractive drug targets, but until now studies of Ldt activity and inhibitor testing have been hampered by low-throughput methods and limited sensitivity. Marcos Pires and his team set out to solve that bottleneck by creating a versatile, bead-based platform that can analyze Ldt activity on a much larger scale. Their approach uses peptidoglycan stem peptides that are either naturally harvested or synthetically immobilized on abiotic surfaces. These substrates are incubated with Ldts together with a fluorescent acyl acceptor so that enzyme-catalyzed crosslinking can be monitored quantitatively. By optimizing assay parameters, the researchers were able to profile multiple Ldt enzymes and to bring higher sensitivity and throughput to studies of these key bacterial enzymes.

The new platform is a bead-based, high-throughput assay that combines immobilized peptidoglycan stem peptides with a fluorescent acyl acceptor to directly measure crosslinking activity. Using this format, the team profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt using chemically defined substrate sequences. They tested a series of acyl acceptors and showed which modifications are tolerated by mycobacterial Ldts, giving insight into substrate flexibility. The assay was also used to screen β-lactam antibiotics: results showed potent inhibition by (carba)penems, whereas cephalosporins, monobactams and penams showed negligible activity against the Ldts tested. The bead-based method delivered excellent performance metrics and the researchers successfully adapted it to ELISA and 96-well formats, demonstrating that the assay can be scaled for larger inhibitor discovery campaigns and automated screening efforts.

This work provides a practical tool for accelerating discovery of compounds that target Ldts, a validated route to compromise mycobacterial cell walls. By making it possible to run many tests quickly and quantitatively, the bead-based platform should help researchers compare Ldt paralogs, test chemically defined substrates such as those used to study the class 6 Ldt, and evaluate diverse acyl acceptor designs. The clear finding that (carba)penems potently inhibit these enzymes, while cephalosporins, monobactams and penams do not, gives a focused direction for medicinal chemistry and drug repurposing efforts. The assay’s compatibility with ELISA and 96-well formats means academic labs and industry screening centers can adopt it without specialized equipment, speeding the path from target validation to candidate selection for tuberculosis and NTM therapy development.