MECHANISM OF DRUG TRANSPORT BY P-GLYCOPROTEIN

P-糖蛋白的药物转运机制

• 批准号: 6879891
• 负责人: MARWAN KHALID AL-SHAWI
• 金额: $ 7.98万
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879891
• 关键词: P glycoprotein; active transport; adenosinetriphosphatase; binding sites; chemical models; conformation; drug metabolism; electron spin resonance spectroscopy; fluorescent dye /probe; liposomes; multidrug resistance; mutant; pharmacokinetics; protein purification; protein structure function; protein transport; site directed mutagenesis; thermodynamics; yeasts

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.

描述：（逐字研究申请人的摘要）人质膜 蛋白质，P-糖蛋白（P-GP）是ATP驱动的药物导出泵 抵消癌细胞中的化疗，并限制 体内其他治疗药物。 P-糖蛋白的机制 运输药物仍然神秘，因此该提案的远距离目标 是阐明这种机制。人类P-gp的诱变和定量 动力学，热力学和光谱法分析将是 用于研究ATP水解和药物转运的偶联。这 第一个目的是生成突变形式的P-糖蛋白和筛选 转运/ATP水解耦合突变。突变分析也将是 用于测试提议的运输机制，并将功能分配给 特定的氨基酸残基。为了进一步分配功能 结构，位于位置的半胱氨酸残基将放置在药物中， 核苷酸结合位点。这将允许光谱学的放置 定义位置的探针将直接观察到结合 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相互作用 药物结合位点和核苷酸结合位点。毒品结合和运输 将与ATP水解机制并行检查 它们之间相互作用的热力学测量将是 已确立的。接下来，将研究ATP水解反应以揭示 催化涉及的部分反应步骤及其与药物的关系 结合。第三个目的是研究药物的结构和动力学 通过应用定向的自旋标记运输位点。 结果在一起将导致对如何详细了解 P-糖蛋白运输药物并提供药物的结构模型 绑定位点。这种知识将有助于毒品的合理设计和 克服或调节该转运蛋白的方法学，从而增强 化学疗法和改善癌症治疗以及改善艾滋病 治疗。