A Fluorescence-based Assay for Screening β-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2.

Mycobacterium tuberculosis l,d-transpeptidases (Ldts), which are involved in cell-wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence-based assay for Ldt(Mt2), which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with Ldt(Mt2), thus making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of beta-lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of beta-lactams are more potent Ldt inhibitors than other beta-lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure-activity relationship studies on Ldts, thereby facilitating the identification of new antibiotics active against M. tuberculosis.