Identification Of The Eletriptan Binding Site On P-Glycoprotein Determined From Nuclear Magnetic Resonance Spectroscopy

P-glycoprotein (Pgp) serves as the gatekeeper across the blood-brain barrier and protects the brain from chemical insults; however, this protective role reduces brain penetration of neurotherapeutics. The drug class triptans are used in the treatment of migraines and have a range of Pgp transport rates. We have chosen to work with eletriptan (ETT) because this triptan drug has a high transport rate and represents the best opportunity to decipher the mechanism of transport. The affinity and the number of binding sites to Pgp were estimated using fluorescence quenching. ATPase activity of reconstituted Pgp revealed ETT to have a monophasic curve and was fit to Michaelis-Menten equation. Using paramagnetic relaxation enhancement (PRE) NMR of cysteine-labeled Pgp, the ETT binding site was identified to lie in the central binding cavity. ETT molecules bound to Pgp were also found to be in an extended conformation by exchange-transferred NOE (etNOE) spectroscopy. The position of ETT was validated using saturati...