A Fluorescence-Based Assay for Screening β-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase Ldt Mt2

• Published in: Chembiochem : a European journal of chemical biology 2020-02, Vol.21 (3), p.368-372
• Main Authors: de Munnik, Mariska, Lohans, Christopher T, Langley, Gareth W, Bon, Corentin, Brem, Jürgen, Schofield, Christopher J
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacology; beta-Lactams - chemistry; beta-Lactams - pharmacology; Chemical Sciences; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - pharmacology; High-Throughput Screening Assays; Life Sciences; Microbial Sensitivity Tests; Molecular Structure; Mycobacterium tuberculosis - drug effects; Mycobacterium tuberculosis - metabolism; Peptidyl Transferases - antagonists & inhibitors; Peptidyl Transferases - metabolism
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201900379

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 , which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with Ldt , 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 β-lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β-lactams are more potent Ldt inhibitors than other β-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.