Janine L Gray, Elizabeth V K Ledger, Tiffany Suwatthee, Thomas J Burden, Konstantina Arvaniti, Priyanka Mishra, Amber Sefton, Lydia E Papagora, Thomas B Clarke, Jennifer Riley, Erika G Pinto, Fraser Cunningham, Ian H Gilbert, David Gray, Da-Neng Wang, Kevin D Read, Thomas Lanyon-Hogg, Nathaniel J Traaseth, Andrew M Edwards, Edward W Tate
Here we report IMP-2380, a potent and selective chemical probe targeting the clinically important Staphylococcus aureus efflux pump NorA.
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.