Small molecule inhibitors of the NorA multidrug efflux pump potentiate antibiotic activity by binding the outward-open conformation.

Antibiotic resistance is among the greatest threats of the modern era. Multidrug efflux pumps expel antibiotics from bacterial cells and present a particular challenge by conferring resistance to a broad range of antibiotic classes; however, there is currently a lack of potent and selective inhibitors. Here, we report the discovery of , a drug-like chemical probe for the multidrug efflux pump NorA that delivers low-nanomolar potentiation of ciprofloxacin activity and activity in an infection model. A phenotypic high-throughput screen for inhibitors of the ciprofloxacin-activated SOS DNA repair pathway in methicillin-resistant (MRSA) identified hit compounds targeting NorA, and subsequent optimization established as the most potent NorA inhibitor discovered to date. The structure of NorA bound to was solved by cryo-electron microscopy at 2.52 Å resolution, revealing that the small molecule locks the pump in the 'outward-open' conformation. This closes the inner face and prevents antibiotics binding from the cytosol, providing an explanation for the exceptional potency of and structure-activity relationship across the series. represents an active NorA inhibitor, functioning via a structurally defined outward-open binding mode, and will enable future exploration of NorA as a druggable target to combat antibiotic resistance.