Abstract LB-222: Chemical and structural flexibility contribute to the broad substrate and modulator specificity of multidrug resistance-linked P-glycoprotein (ABCB1).
Authors: Ambudkar Suresh V. , Chufan Eduardo E. , Kapoor Khyati , Durell Stewart R. , Sim Hong-May 
Published: August 2014

Abstract
The human ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein, P-gp) mediates efflux of cytotoxic anticancer agents from cells, thereby conferring resistance to chemotherapeutic agents in most cancers. One of the most important features of ABC drug transporters, including P-gp, is their ability to recognize a variety of chemically dissimilar hydrophobic or amphipathic compounds. In this report, we elucidate the molecular basis of the promiscuous nature of this transporter. We modeled the structure of human P-gp using the X-ray structure of mouse P-gp as a template, with docked substrates (cyclosporine A, valinomycin and nilotinib) and modulators (tariquidar and 5′-Fluorosulfonylbenzonyl 5′-adenosine) in the pocket where the QZ59-peptide inhibitors bind mouse P-gp. Single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, M949, F978, V982 and A985 residues at the drug binding site with cys in a cys-less P-gp were expressed in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cys-less wild-type P-gp. Several drugs including cyclosporine A, valinomycin, nilotinib and tariquidar partially or completely lose the ability to inhibit the photolabeling of mutant P-gps with the transport substrate [125I]-Iodoarylazidoprazosin, indicating that these drugs cannot bind at their primary binding site. However, the drugs can modulate the ATP hydrolysis of the mutant P-gps, demonstrating that they bind at alternate sites. The transport of six fluorescent substrates in HeLa cells overexpressing triple mutants (Y307C/Q725C/V982 and Y307A/Q725A/Y953A) is also not significantly altered, showing that substrates bound at alternate sites are still transported. In aggregate, our results suggest that a large flexible pocket in the transmembrane domains in P-gp is able to bind chemically diverse compounds through a versatile allocation of hydrogen-bond donors and acceptors and hydrophobic residues. When residues of the primary drug-binding site are mutated, substrates and modulators bind to alternate sites on the transporter, revealing an exceptional chemical flexibility. We propose that both the chemical and structural flexibility provide the molecular basis for interaction of P-gp and possibly other ABC drug transporters with a wide range of substrates and modulators.
Citation Format: Suresh V. Ambudkar, Eduardo E. Chufan, Khyati Kapoor, Stewart R. Durell, Hong-May Sim. Chemical and structural flexibility contribute to the broad substrate and modulator specificity of multidrug resistance-linked P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2013-LB-222