Molecular Mechanisms of P-Glycoprotein

P-糖蛋白的分子机制

• 批准号: 8531994
• 负责人: WILLIAM M ATKINS
• 金额: $ 27.06万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 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/8531994
• 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 Interactions; Drug Kinetics; Drug Targeting; 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

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.

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-药物相互作用和耐药性中起关键作用。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传递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体，在12-跨膜螺旋结构域（TMs）内包含一个或多个药物结合位点，该转运体与核苷酸结合结构域（nbd）通信，后者结合并水解ATP以驱动TM结构域的构象变化。由于在模型膜中研究P-gp的困难，没有对P-gp反应周期内的任何基本步骤进行动力学测量，这仍然是不明确的。了解这些过程的速率对于确定在几种提出的方案中哪种机制模型是最准确的至关重要。本提案的目标是利用脂质双层纳米圆盘中的P-gp，通过利用停流光谱、表面等离子体共振和单分子全内部荧光显微镜（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底物和抑制剂的存在下确定。这些研究的延伸包括使用与不同药物共价内合的单个半胱氨酸P-gp突变体，以确定药物位置对核苷酸结合和解离的影响。同样，荧光药物的结合和解离将由不同的核苷酸结合在nbd上决定。这些研究将为P-gp配体相互作用提供第一个动力学数据，这将阐明以前尚未阐明的几个机制细节。这些研究可能会导致P-gp构象特异性抑制剂在现有药物的药代动力学特性的调节中发挥作用。