Small-molecule inhibitors block NorA efflux by conformational trapping.

Multidrug efflux pumps are major drivers of antibiotic resistance, yet progress in understanding and inhibiting these transporters has been limited by a lack of selective chemical probes and inhibitor-bound structures. Here we report IMP-2380, a potent and selective chemical probe targeting the clinically important Staphylococcus aureus efflux pump NorA. A phenotypic high-throughput screen monitoring suppression of the ciprofloxacin-induced SOS DNA damage response identified a chemical series that selectively inhibits NorA and was optimized to yield IMP-2380. The probe restores ciprofloxacin susceptibility in methicillin-resistant S. aureus, delivering low-nanomolar potentiation in vitro and robust efficacy in an in vivo infection model. Cryo-electron microscopy at 2.52-Å resolution revealed the structure of NorA bound to a small-molecule inhibitor. IMP-2380 binds an 'outward-open' transporter conformation, occluding the cytosolic substrate-binding cavity and preventing antibiotic efflux. IMP-2380 provides a high-quality probe for dissecting multidrug efflux and establishes a structural framework for restoring antibiotic efficacy through efflux pump inhibition.