MECHANISM OF DRUG TRANSPORT BY P-GLYCOPROTEIN

化疗作为治愈许多癌症患者的方法往往失败，因为肿瘤 细胞对多种抗癌化疗药物产生抗药性 探员们。人类质膜蛋白P-糖蛋白已被发现 在表现这种多药耐药现象的许多肿瘤中过度表达 抵抗。P-糖蛋白被认为是一种由ATP驱动的药物- 输出泵进入癌细胞，从而保护肿瘤免受化疗的伤害。 对P-糖蛋白的分子机制知之甚少 将大量且种类繁多的细胞毒性化合物输送出细胞。 这项提案的长期目标是了解 P-糖蛋白完成其主动转运机制。有了这个 信息、药物的合理设计和克服这一问题的方法 临床上重要的交通工具将近在咫尺。 P-的基本性质的详细刻画 糖蛋白将被用来理解能量是如何从 三磷酸腺苷的水解物被用来运输各种不同的药物 化学结构。我们将利用遗传学、动力学和热力学 酶分析和定点探针定位的方法。目标1是 进一步开发正常和突变的酵母表达系统 人类P-糖蛋白。这将促进基因操作 并提供了一种成本效益高的系统来生产大型 用于生化和结构分析的纯化P-糖蛋白的量 学习。我们已经发现，这个系统可以用于选拔 有害突变和功能恢复次级突变的图谱 重要残基和蛋白质序列。此外，我们还将继续 开发测试方法来测量基本的传输特性，例如 周转率、药物特异性、药物与药物之间的协同性 运输和运输之间的核苷酸位置和偶联程度和滑动 水解液。这些分析将允许进行定量分析和 了解突变的影响。目标2是测试结构 提出了P-糖蛋白的化学模型。突变分析将 用来为特定的氨基酸残基和结构域分配功能 结构。将使用随机诱变和逆转分析来寻找 新的重要残基和结构域，并绘制出 其功能结构域之间的蛋白质。定点突变将是 用于测试机械功能，这些功能被归因于 特别的残留物。目标3是通过站点获取结构信息 定向诱变。特定的半胱氨酸和色氨酸替代品将 通过放置光谱对结构进行功能赋值 在核苷酸和药物结合部位的指定位置上的探针。这 将允许距离测绘和直接监测配体的结合 以及运输过程中的构象变化。综合起来，结果是 这项研究应该会导致对分子的详细理解 P-糖蛋白转运药物的机制。

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.