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

Chembiochem. 2020 Feb 3;21(3):368-372. doi: 10.1002/cbic.201900379. Epub 2019 Nov 8.

Abstract

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 LdtMt2 , which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2 , 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.

Keywords: antibiotics; beta-lactams; fluorescent probes; inhibitors; tuberculosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / pharmacology*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / pharmacology*
  • High-Throughput Screening Assays
  • Microbial Sensitivity Tests
  • Molecular Structure
  • Mycobacterium tuberculosis / drug effects*
  • Mycobacterium tuberculosis / metabolism
  • Peptidyl Transferases / antagonists & inhibitors*
  • Peptidyl Transferases / metabolism
  • beta-Lactams / chemistry
  • beta-Lactams / pharmacology*

Substances

  • Anti-Bacterial Agents
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • beta-Lactams
  • Peptidyl Transferases