Molecular Mechanisms of P-Glycoprotein

描述(申请人提供)：P-糖蛋白(P-gp)是一种依赖于ATP的外排转运蛋白，在药物分布、药物相互作用和耐药性中发挥关键作用。调节P-gp活性的药物可能通过增加其他药物向中枢神经系统的输送，或通过改善其药代动力学性质而具有治疗作用。P-gp是一种高度混杂的转运蛋白，它在12-跨膜螺旋结构域(TMS)中包括一个或多个药物结合部位，该部位与核苷酸结合结构域(NbD)通信，核苷酸结合结构域与ATP结合并水解三磷酸腺苷(ATP)，从而驱动TM结构域的构象变化。由于在模型膜中研究P-糖蛋白很困难，目前还没有对P-糖蛋白反应循环中的任何基本步骤进行动力学测量，这一点仍然没有得到很好的确定。对这些过程的速率的了解对于在几个建议的方案中确定哪个机械模型最准确是至关重要的。该方案的目标是利用P-gp在脂质双层纳米盘中的应用，通过利用停流光谱、表面等离子体共振和单分子内总荧光显微镜(TIRFM)的研究来挑战P-gp的两种相互竞争的机制模型。对于后两种方法，将比较几种表面附着策略以优化数据采集。在几种P-gp底物和抑制剂的存在下，核苷酸的结合和解离速率常数将被测定。这些研究的扩展包括使用与不同药物共价加成的单个半胱氨酸P-gp突变体，以确定药物位置对核苷酸结合和解离的影响。同样，荧光药物的结合和解离将通过结合在NBD上的不同核苷酸来确定。这些研究将提供第一批P-gp配体相互作用的动力学数据，这些数据将阐明几个以前尚未阐明的机制细节。这样的研究可能导致构象特异的P-gp抑制剂，在现有药物的药代动力学特性调节中发挥作用。 公共卫生相关性：P-糖蛋白是一种大的跨膜螺旋外排转运蛋白，在癌细胞对药物的耐药性以及大多数药物在所有治疗领域的分布和清除中发挥着重要作用。P-gp的分子机制尚不清楚。这一建议的长期目标是了解外排转运体P-糖蛋白的分子机制，以便合理设计针对P-糖蛋白的药物，并改善其他药物的治疗方案。

DESCRIPTION (provided by applicant): P-glycoprotein (P-gp) is an ATP-dependent efflux transporter that plays a critical role in drug distribution, drug-drug interactions and Drug resistance. Drugs that modulate P-gp activity could have therapeutic utility by increasing delivery of other drugs to the central nervous system, or by improving their pharmacokinetic properties. P-gp is an extremely promiscuous transporter that includes a drug binding site, or sites, within a 12- transmembrane helix domain (TMs), which communicates with nucleotide binding domains (NBDs) that bind and hydrolyze ATP to drive conformational changes in the TM domain. Due to the difficulty in studying P-gp in model membranes no kinetic measurements have been made on any elementary step within the P-gp reaction cycle, which remains poorly defined. Knowledge of the rates of these processes is essential to determine which mechanistic models are most accurate, among several proposed schemes. The goals of this proposal are to exploit P-gp in lipid bilayer nanodiscs, to challenge two competing mechanistic models of P-gp with studies that utilize stopped-flow spectroscopy, surface plasmon resonance and single molecule total internal fluorescence microscopy (TIRFM). For the latter two methods, several surface attachment strategies will be compared to optimize data acquisition. The binding and dissociation rate constants of nucleotides will be determined in the presence of several P-gp substrates and inhibitors. An extension of these studies includes the use of single cysteine P-gp mutants covalently adducted with varying drugs, to determine the effect of drug location on nucleotide binding and dissociation. Similarly, binding and dissociation of fluorescent drugs will be determined with varying nucleotides bound at the NBDs. These studies will provide the first kinetic data for P-gp ligand interactions that will elucidate several mechanistic details that have not been previously clarified. Such studies could lead to conformation-specific inhibitors of P-gp with utility in the modulation of pharmacokinetic properties of existing drugs. PUBLIC HEALTH RELEVANCE: P-glycoprotein is a large transmembrane helical efflux transporter that plays a major role in cancer cell resistance to drugs and the distribution and clearance of most drugs spanning all therapeutic areas. The molecular mechanism of P-gp remains unknown. The long term goal of this proposal is to understand the molecular mechanisms of the efflux transporter P-glycoprotein in order to rationally design drugs that target P-gp and improve the therapeutic profile of other drugs.