A target engagement assay to assess uptake, potency and retention of antibiotics in living bacteria

A major challenge in antibiotics drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetics binding parameters of antibiotics to their molecular targets in live bacteria. We validated this approach for a known antibiotic target, dihydrofolate reductase, using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of similar target engagement assays to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity.