Molecular Mechanisms of P-Glycoprotein

性状（由申请方提供）：P-糖蛋白（P-gp）是一种ATP依赖性外排转运蛋白，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活性的药物可以通过增加其他药物向中枢神经系统的递送或通过改善其药代动力学特性而具有治疗效用。P-gp是一种极其混杂的转运蛋白，其在12-跨膜螺旋结构域（TM）内包括一个或多个药物结合位点，其与结合并水解ATP以驱动TM结构域中的构象变化的核苷酸结合结构域（NBD）通信。由于在模型膜中研究P-gp的困难，在P-gp反应周期内的任何基本步骤上都没有进行动力学测量，这仍然是不明确的。这些过程的速率的知识是必不可少的，以确定哪些机械模型是最准确的，在几个建议的计划。该提案的目标是利用脂质双层纳米盘中的P-gp，通过利用停流光谱、表面等离子体共振和单分子全内荧光显微镜（TIRFM）的研究来挑战P-gp的两种竞争机制模型。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。将在存在几种P-gp底物和抑制剂的情况下测定核苷酸的结合和解离速率常数。这些研究的扩展包括使用与不同药物共价加合的单个半胱氨酸P-gp突变体，以确定药物位置对核苷酸结合和解离的影响。类似地，荧光药物的结合和解离将用结合在NBD处的不同核苷酸来确定。这些研究将提供P-gp配体相互作用的第一个动力学数据，这将阐明先前尚未阐明的几个机制细节。这些研究可能导致构象特异性抑制剂的P-gp与效用在现有药物的药代动力学特性的调制。 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跨越所有治疗领域的大多数药物的分布和清除中起主要作用。P-gp的分子机制尚不清楚。该提案的长期目标是了解外排转运蛋白P-糖蛋白的分子机制，以便合理设计靶向P-gp的药物并改善其他药物的治疗特征。

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.