Understanding polyspecific drug binding in P-glycoprotein

描述（由申请人提供）：p -糖蛋白（Pgp）介导的多药耐药（MDR）是许多癌症、HIV和精神疾病治疗中的一个重要问题。Pgp是一种atp结合盒转运体，通过atp依赖机制将许多结构无关的药物泵出细胞。我们最近的Pgp X射线结构鉴定了疏水性和芳香氨基酸，它们有助于两种不同抑制剂与药物结合位点的结合。在这个提议中，我们将测试一个假设，即抗癌药物结合到蛋白质跨膜区域中定义的亚袋内的不同残基亚群。利用色氨酸（Trp）荧光猝灭，我们将绘制出纯化蛋白与占据的三个原型底物相互作用的位点

DESCRIPTION (provided by applicant): Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) is a significant problem in the treatment of many cancers, HIV, and psychiatric illnesses. Pgp is an ATP-binding cassette transporter that pumps many structurally unrelated drugs out of the cell through an ATP-dependent mechanism. Our recent X- ray structure of Pgp identified hydrophobic and aromatic amino acids that contribute to binding of two different inhibitors to the drug-binding site. In this proposal, we will test the hypothesis tht anticancer drugs bind to different subsets of residues within defined subpockets in the transmembrane regions of the protein. Using tryptophan (Trp) fluorescence quenching, we will map out sites of interaction of the purified protein with three prototypical substrates that occupy biochemically defined and distinct binding sites, as well as those of common anticancer drugs and newly identified inhibitors. The novelty of this proposal is our development of a functional Trp-free Pgp, and the introduction into this Trp-free background of one or more Trps at strategic positions to monitor drug binding. With this new approach, we will address the molecular mechanism and kinetics of drug/inhibitor binding and determine the mechanisms of action of the recently-identified blockers. We plan to obtain direct information on how different surfaces of the protein subpockets interact with anticancer drugs, and how different blockers work. The latter will be invaluable to develop mechanistically- and structurally-based panels of potential blockers for high-throughput screening. PUBLIC HEALTH RELEVANCE: P-glycoprotein (Pgp) is the cell's cleaning machine, pumping harmful substances to the outside of the cell. In cancer chemotherapy, Pgp can cause problems by removing chemotherapy drugs from the tumor cells that they were intended to kill. By learning more about how Pgp recognizes the chemicals that it carries out of the cell, scientists may devise new drugs to prevent Pgp from interfering with the valuable effects of anticancer drugs.