Understanding polyspecific drug binding in P-glycoprotein

描述(由申请人提供)：由P-糖蛋白(PGP)介导的多药耐药(MDR)是许多癌症、艾滋病毒和精神疾病治疗中的一个重大问题。PGP是一种三磷酸腺苷结合盒转运体，通过三磷酸腺苷依赖的机制将许多结构上无关的药物泵出细胞。我们最近的PGP的X射线结构确定了疏水和芳香氨基酸，它们有助于两种不同的抑制剂与药物结合部位的结合。在这个提议中，我们将测试抗癌药物与蛋白质跨膜区域中定义的亚袋内不同残基结合的假设。利用色氨酸(Trp)荧光猝灭，我们将绘制出纯化的蛋白质与三种原型底物相互作用的位置 生物化学定义的和不同的结合部位，以及常见抗癌药物和新发现的抑制剂的结合部位。这项建议的新奇之处在于我们开发了一种功能性的无TRP的PGP，并在这个无TRP的背景下引入了一个或多个处于战略位置的TRP来监测药物结合。通过这种新的方法，我们将研究药物/抑制剂结合的分子机制和动力学，并确定最近发现的阻滞剂的作用机制。我们计划获得关于不同表面如何 蛋白质亚囊与抗癌药物相互作用，以及不同阻滞剂的作用方式。后者对于为高通量筛选开发基于机械和结构的潜在阻滞剂的面板将是无价的。 与公共健康相关：P-糖蛋白(PGP)是细胞的清洁机，将有害物质泵送到细胞外。在癌症化疗中，PGP会将化疗药物从原本打算杀死的肿瘤细胞中移除，从而引发问题。通过更多地了解PGP是如何识别它在细胞外进行的化学物质的，科学家们可能会设计出新的药物，以防止PGP干扰抗癌药物的宝贵作用。

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.