MECHANISM OF DRUG TRANSPORT BY P-GLYCOPROTEIN

化疗往往不能治愈许多癌症患者，因为肿瘤 细胞变得对广泛的抗癌化疗药物具有抗性， 剂.人的质膜蛋白，P-糖蛋白，已被发现， 在许多表现出这种多药耐药现象的肿瘤中过度表达， 阻力P-糖蛋白被认为是一种ATP驱动的药物， 在癌细胞中输出泵，从而保护肿瘤免受化疗。 目前对P-糖蛋白在细胞内表达的分子机制知之甚少， 将大量不同数量的细胞毒性化合物转运出细胞。 本提案的长期目标是了解 P-糖蛋白完成其主动转运机制。与此 信息、合理设计药物和克服这一问题的方法 临床上重要的运输将更近。 详细描述了P- 糖蛋白将进行了解如何能源来自 ATP的水解被用于运输各种各样的药物 化学结构。我们将运用遗传学，动力学和热力学 酶分析方法和位点特异性探针放置。 目标1是 为了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 这将有助于基因操作， 并提供了一种成本有效系统， 大量纯化的P-糖蛋白用于生化和结构分析， 问题研究我们已经发现这个系统可以用于选择 有害突变和功能恢复的二次突变 重要的残基和蛋白质序列。此外，我们将继续 开发测定基本传输特性的分析方法， 周转率、药物特异性、药物和 核苷酸位点以及转运和 水解这些试验将允许定量分析和 了解突变的影响。 目标2是测试结构 和P-糖蛋白的化学模型。 突变分析将 用于将功能分配给特定的氨基酸残基和结构域 结构.随机诱变和回复突变分析将用于发现 新的重要残基和结构域，并映射内的相互作用 蛋白质的功能结构域之间。定点诱变将是 用于测试机械功能， 特别的残留物。目的3是通过位点获得结构信息 定向诱变特异性半胱氨酸和色氨酸替代物将 通过将光谱分析法用于结构的功能分配， 在核苷酸和药物结合位点中的限定位置处的探针。这 将允许距离映射和直接监测配体的结合 以及运输过程中构象的变化。综合来看， 这项研究的结果应该会导致对分子生物学的详细了解。 P-糖蛋白的药物转运机制。

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.