MECHANISM OF DRUG TRANSPORT BY P-GLYCOPROTEIN

描述：(逐字摘自申请者摘要)人的质膜 蛋白，P-糖蛋白(P-gp)是一种由ATP驱动的药物输出泵 中和癌细胞中的化疗并限制其生物利用度 体内的其他治疗药物。P-糖蛋白的作用机制 毒品的运输仍然是个谜，因此这项提案的长期目标 就是阐明这一机制。人P-gp基因的诱变及其定量 酶分析的动力学、热力学和光谱方法将是 用于研究ATP水解和药物转运的偶联作用。这个 第一个目标是产生突变形式的P-糖蛋白，并筛选 转运/三磷酸腺苷水解偶联突变。突变分析也将是 用于测试建议的传输机制并将功能分配给 特别是氨基酸残基。有关进一步将函数分配给 结构，定位半胱氨酸残基将被放置在药物和 核苷酸结合位点。这将允许放置光谱 位于指定位置的探针，将提供对结合的直接观察 配基和运输过程中的构象变化。第二个目标是 定量测量功能和耦合的相互作用 药物结合部位和核苷酸结合部位。药物结合和转运 将与ATP水解机制并行进行研究，以及 它们之间相互作用的热力学测量将是 已经成立了。接下来，将研究ATP的水解反应，以揭示 催化反应的部分步骤及其与药物的关系 有约束力的。第三个目的是研究药物的结构和动力学。 通过应用定点定向自旋标记运输位点。 总而言之，这些结果将导致对如何 P-糖蛋白转运药物并提供药物的结构模型 结合部位。这些知识将有助于药物和药物的合理设计 克服或调节这种转运体的方法，从而增强 化疗和改善癌症治疗以及改善艾滋病 治疗。

DESCRIPTION: (Verbatim from the Applicant's Abstract)The human plasma membrane protein, P-glycoprotein (P-gp) is an ATP driven drug exporting pump that counteracts chemotherapy in cancer cells and limits the bioavailability of other therapeutic drugs in the body. The mechanism by which P-glycoprotein transports drugs remains enigmatic, thus the long range goal of this proposal is to elucidate this mechanism. Mutagenesis of human P-gp and quantitative kinetic, thermodynamic and spectroscopic methods of enzyme analysis will be employed to investigate the coupling of ATP hydrolysis and drug transport. The first aim will be to generate mutant forms of P-glycoprotein and screen for transport/ATP hydrolysis coupling mutations. Mutational analysis will also be used to test proposed transport mechanisms and to assign functions to particular amino acid residues. For further assignments of function to structure, site-directed cysteine residues will be placed in the drug and nucleotide binding sites. This will allow for the placement of spectroscopic probes at defined locations that will provide direct observation of binding of ligands and changes in conformation during transport. The second aim is to quantitatively measure the functional and coupling interactions between the drug binding sites and nucleotide binding sites. Drug binding and transport will be examined in parallel with the ATP hydrolytic mechanism, and thermodynamic measurements of the interactions between them will be established. Next, the ATP hydrolytic reaction will be investigated to reveal the partial reaction steps involved in catalysis and how they relate to drug binding. The third aim is to study the structure and dynamics of the drug transport sites through the application of site-directed spin labeling. Together the results will lead to a detailed understanding of how P-glycoprotein transports drugs and provide a structural model of the drug binding sites. Such knowledge will aid in the rational design of drugs and methodologies to overcome or modulate this transporter, thus enhancing chemotherapy and improving cancer treatment as well as improving AIDS treatment.