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ATP依赖的外排运输蛋白，在药物分布，药物 - 药物相互作用和耐药性中起着至关重要的作用。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改善其药代动力学特性来具有治疗效用。 P-gp是一种极其闻名的转运蛋白，其中包括12个跨膜螺旋结构域（TMS）内的药物结合位点或位点，该转运蛋白（TMS）与核苷酸结合结构域（NBD）通信，该核苷酸结合结构域（NBD）结合和水解ATP以驱动TM域中的构象变化。由于难以研究模型膜中的P-gp，在P-gp反应周期内的任何基本步骤中都没有进行动力学测量，该步骤的定义较差。在拟议的几个方案中，了解这些过程速率的知识对于确定哪些机械模型最准确是必不可少的。该提案的目标是利用脂质双层纳米盘中的P-gp，通过利用止损流光谱，表面等离子体的总体内部荧光显微镜（TIRFM）的研究来挑战P-gp的两个相互竞争的机械模型。对于后两种方法，将比较几种表面依恋策略，以优化数据获取。核苷酸的结合和解离速率常数将在存在几种P-gp底物和抑制剂的情况下确定。这些研究的扩展包括使用单半胱氨酸P-gp突变体与不同药物共价加入，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，这些数据将阐明以前尚未阐明的几个机械细节。这样的研究可能导致P-gp的构象特异性抑制剂，并在调节现有药物的药代动力学特性时实用。公共卫生相关性：P-糖蛋白是一种大型的跨膜外排外排运输蛋白，在癌细胞对药物的耐药性以及大多数跨越所有治疗区域的药物的分布和清除中起着重要作用。 P-gp的分子机制仍然未知。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，以便对靶向P-gp的合理设计药物，并改善其他药物的治疗特征。