MECHANISM OF DRUG TRANSPORT BY P-GLYCOPROTEIN

描述：（申请人摘要中的原文）人质膜 蛋白，P-糖蛋白（P-gp）是ATP驱动的药物输出泵， 抵消癌细胞中的化疗并限制 体内的其他治疗药物。P-糖蛋白 运输毒品仍然是个谜，因此，这项提案的长期目标 就是阐明这个机制人P-gp的突变和定量 酶分析的动力学、热力学和光谱方法将 用于研究ATP水解和药物转运的耦合。的 第一个目标将是产生P-糖蛋白的突变形式，并筛选 转运/ATP水解偶联突变。突变分析也将 用于测试拟议的传输机制，并将功能分配给 特定的氨基酸残基。为进一步分配职能， 结构，定点半胱氨酸残基将被放置在药物中， 核苷酸结合位点。这将允许放置光谱 在规定位置的探针，将提供直接观察的结合 配体和运输过程中构象的变化。第二个目标是 定量测量功能和耦合之间的相互作用， 药物结合位点和核苷酸结合位点。药物结合和转运 将与ATP水解机制平行检查， 它们之间相互作用的热力学测量将是 确立了习接下来，将研究ATP水解反应以揭示 参与催化的部分反应步骤以及它们与药物的关系 约束力第三个目的是研究药物的结构和动力学 通过应用定点自旋标记来运输位点。 这些结果将有助于详细了解 P-糖蛋白转运药物并提供药物的结构模型 结合位点。这些知识将有助于药物的合理设计， 克服或调节这种转运蛋白的方法，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.