Molecular efflux pumps belonging to the ATP-binding cassette (ABC) superfamily such as P-glycoprotein use energy from ATP hydrolysis to drive drug export across cell membranes back to the extracellular space. The Pgp transporter is widely recognized for its unusual ability to bind many clinically relevant drugs that are chemically and functionally diverse. This drug promiscuity poses challenges in the clinic because Pgp confers multidrug resistance (MDR) in cancer cells and mediates drug-drug interactions (DDIs). One of the most recognized DDIs with the Pgp transporter is between the two cardiovascular ion channel inhibitors verapamil and digoxin. Despite many in vitro and in vivo studies, the mechanistic and structural basis of verapamil and digoxin interactions with the P-glycoprotein transporter and how drug binding is coupled to ATP hydrolysis is quite elusive. To shed light on this Pgp-mediated DDI, we probed verapamil and digoxin interactions with Pgp using several NMR techniques (STDD NMR, PRE NMR), fluorescence and the kinetic fitting software COPASI. From this work, we generated a comprehensive molecular model of the DDI between verapamil and digoxin with Pgp encompassing competitive and noncompetitive inhibition of digoxin transport by verapamil. We also showed that coupling between verapamil and ATP binding to the transporter and verapamil-induced ATP hydrolysis occurs in a cooperative fashion. Excitingly, a model of digoxin bound to Pgp was determined using PRE NMR derived distance restraints to drive molecular docking in the HADDOCK software. To our knowledge, this will be the first model demonstrating the bound location of a drug to the Pgp transporter. This information will guide the rational design of effective cancer drugs and Pgp inhibitors and guide better predictions of safe multidrug regimens that bypass Pgp-mediated DDIs in the clinic.
