MECHANISM OF DRUG TRANSPORT BY P-GLYCOPROTEIN

P-糖蛋白的药物转运机制

• 批准号: 2685058
• 负责人: MARWAN KHALID AL-SHAWI
• 金额: $ 21.43万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2685058
• 关键词: P glycoprotein; active transport; adenosine triphosphate; chemical models; drug metabolism; fluorescent dye /probe; hydrolysis; liposomes; molecular cloning; multidrug resistance; protein purification; protein sequence; protein structure function; protein transport; site directed mutagenesis; thermodynamics; yeasts

Chemotherapy often fails as a cure for many cancer patients because tumor cells become resistant to a wide range of anti-cancer chemotherapeutic agents. The human plasma membrane protein, P-glycoprotein, has been found to be overexpressed in many tumors exhibiting this phenomenon of multidrug resistance. P-glycoprotein is thought to function as an ATP-driven drug- exporting pump in cancer cells, thus protecting tumors from chemotherapy. Little is known about the molecular mechanism by which P-glycoprotein transports a large and diverse number of cytotoxic compounds out of cells. The long range goal of this proposal is to understand how the structure of P-glycoprotein accomplishes its active transport mechanism. With this information, rational design of drugs and methodologies to overcome this clinically important transport will be much closer at hand. A detailed characterization of the fundamental properties of P- glycoprotein will be undertaken to understand how energy derived from the hydrolysis of ATP is used to transport drugs of a wide range of diverse chemical structures. We will employ genetic, kinetic and thermodynamic methods of enzyme analysis, and site specific probe placements. Aim 1 is to further develop yeast expression systems for normal and mutated forms of human P-glycoprotein. This will facilitate genetic manipulations of the protein and provide a cost effective system for producing large quantities of purified P-glycoprotein for biochemical and structural studies. We have already found that this system can be used for selection of deleterious mutations and return-of-function secondary mutations to map important residues and protein sequences. In addition, we will continue to develop assays to measure fundamental transport properties such as turnover rates, drug specificities, cooperativity between drug and nucleotide sites and extent of coupling and slippage between transport and hydrolysis. These assays will allow quantitative analyses and an understanding of the effects of mutations. Aim 2 is to test structural and chemical models proposed for P-glycoprotein. Mutational analysis will be used to assign functions to particular amino acid residues and domain structures. Random mutagenesis and reversion analysis will be used to find new important residues and domains, and to map the interactions within the protein between its functional domains. Site directed mutagenesis will be used to test for mechanistic functions that have been attributed to particular residues. Aim 3 is to elicit structural information by site directed mutagenesis. Specific cysteine and tryptophan replacements will be made for assignments of function to structure by placing spectroscopic probes at defined locations in the nucleotide and drug binding sites. This will allow distance mapping and direct monitoring of binding of ligands and changes in conformation during transport. Taken together, the results of this research should lead to a detailed understanding of the molecular mechanisms underlying drug transport by P-glycoprotein.

对于许多癌症患者而言，化学疗法通常是治愈的，因为肿瘤 细胞对广泛的抗癌化学治疗具有抗性 代理商。已发现人质膜蛋白P-糖蛋白 在许多肿瘤中表现出过度表达的多种肿瘤 反抗。 P-糖蛋白被认为是ATP驱动的药物 - 导出癌细胞中的泵，从而保护肿瘤免受化疗。 关于P-糖蛋白的分子机制知之甚少 从细胞中输送出大量多样的细胞毒性化合物。 该提议的远距离目标是了解 P-糖蛋白可以实现其主动运输机制。与此 信息，毒品和方法的合理设计，以克服这一点 临床上重要的运输将更加紧密。 p-的基本特性的详细表征 将采取糖蛋白，以了解能源如何从 ATP的水解用于运输各种各样的药物 化学结构。我们将采用遗传，动力学和热力学 酶分析的方法和特定位置探针位置。 目标1是 进一步开发正常和突变形式的酵母表达系统 人类p-糖蛋白。 这将促进 该蛋白质并提供了一种具有成本效益的系统来生产大型 用于生化和结构的纯化P-糖蛋白 研究。我们已经发现该系统可用于选择 有害突变和功能恢复次要突变 重要的残基和蛋白质序列。此外，我们将继续 开发测定方法以衡量基本运输特性，例如 周转率，药物特异性，药物之间的合作性 核苷酸位点以及运输和运输之间的耦合和滑倒的程度 水解。这些测定将允许定量分析和 了解突变的影响。 目标2是测试结构 和针对P-糖蛋白提出的化学模型。 突变分析将 用于将功能分配给特定的氨基酸残基和域 结构。随机诱变和回归分析将用于查找 新的重要残留物和域，并绘制 其功能域之间的蛋白质。站点定向诱变将是 用于测试已归因于机械功能 特定残留物。 AIM 3是按场地启动结构信息 定向诱变。特定的半胱氨酸和色氨酸替代物将 通过放置光谱镜来使功能分配为结构 核苷酸和药物结合位点的定义位置的探针。这 将允许距离映射并直接监视配体的结合 和运输过程中构象的变化。总之，结果 这项研究应导致对分子的详细理解 P-糖蛋白传输的药物基础机制。