Identification of Structural Motifs in P-Glycoprotein Responsible for the Drug-Mediated Inhibition of ATP Hydrolysis

Abstract

P-glycoprotein (P-gp, ABCB1) is the most studied member of the ATP-Binding Cassette (ABC) transporter superfamily. This transporter utilizes energy from ATP hydrolysis for the efflux of a great variety of compounds, including anticancer drugs. P-gp is expressed at the apical surface of epithelial cells in the intestines, kidney, liver, adrenal gland, blood-brain barrier, and placenta, and therefore affects the pharmacokinetics of many drugs. The expression of P-gp at the surface of tumor cells is also one of the mechanisms of multidrug resistance, which occurs in many cancers. The development of an effective inhibitor requires first the understanding of the mechanism of action of P-gp. In our recent work, mutagenesis and molecular modeling studies led to the identification of a pair of phenylalanine-tyrosine structural motifs that mediate the inhibition of ATP hydrolysis by drugs such as zosuquidar, tariquidar and elacridar. Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues interact with the tyrosines in an edge-to-face conformation, helping the tyrosines to adopt the proper orientation to effectively establish hydrogen-bond contact with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation of tyrosine or phenylalanine residues that disrupts the structural motifs, they bind at lower affinity sites, leading to stimulation of ATP hydrolysis and poor inhibition of transport.