Molecular Mechanisms of P-Glycoprotein

P-糖蛋白的分子机制

基本信息

批准号：
8162138
负责人：
WILLIAM M ATKINS
金额：
$ 29.51万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-21 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8162138
关键词：
ATP Hydrolysis ATP-Binding Cassette Transporters Address Adenylyl Imidodiphosphate Antineoplastic Agents Area Behavior Binding Binding Sites Biological Assay Biological Models Cells Complex Coupling Cysteine Data Dissociation Doxorubicin Drug Binding Site Drug Controls Drug Delivery Systems Drug Interactions Drug Kinetics Drug resistance Enzymes Equilibrium Flow Cytometry Fluorescence Fluorescence Microscopy Fluorescence Spectroscopy Goals Heterogeneity Hydrolysis Individual Kinetics Knowledge Label Lead Ligands Lipid Bilayers Lipids Location Measurement Measures Methods Modeling Molecular Molecular Conformation Neuraxis Nucleotides P-Glycoprotein Paclitaxel Pharmaceutical Preparations Play Prazosin Process Property Proteins Reaction Relative (related person)Research Role Scheme Series Site Spectrum Analysis Staging Sulfhydryl Compounds Surface Surface Plasmon Resonance Technology Testing Therapeutic Time Toxic effect Transmembrane Domain Tryptophan Vanadates Verapamil absorption adduct analog cancer cell data acquisition design drug distribution improved inhibitor/antagonist membrane model mutant nanodisk new technology nile red nucleotide analog residence single molecule

项目摘要

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.

描述（由申请人提供）：P-糖蛋白（P-gp）是一种ATP依赖性外排转运蛋白，在药物分布、药物间相互作用和耐药性中发挥着关键作用。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来具有治疗效用。 P-gp 是一种极其混杂的转运蛋白，在 12 跨膜螺旋结构域 (TM) 内包含一个或多个药物结合位点，该位点与结合并水解 ATP 的核苷酸结合结构域 (NBD) 通信，以驱动 TM 的构象变化领域。由于研究模型膜中的 P-gp 很困难，因此尚未对 P-gp 反应循环中的任何基本步骤进行动力学测量，该反应循环的定义仍然很差。了解这些过程的速率对于确定几种提议的方案中哪种机械模型最准确至关重要。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。核苷酸的结合和解离速率常数将在几种 P-gp 底物和抑制剂存在下测定。这些研究的延伸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通过结合在 NBD 上的不同核苷酸来确定。这些研究将为 P-gp 配体相互作用提供第一个动力学数据，从而阐明先前尚未阐明的几个机制细节。此类研究可能会产生 P-gp 构象特异性抑制剂，可用于调节现有药物的药代动力学特性。公共健康相关性：P-糖蛋白是一种大型跨膜螺旋外排转运蛋白，在癌细胞对药物的耐药性以及跨所有治疗领域的大多数药物的分布和清除中发挥着重要作用。 P-gp 的分子机制仍不清楚。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效果。