Mechanism of drug transport by P-glycoprotein

P-糖蛋白的药物转运机制

• 批准号: 7029736
• 负责人: MARWAN KHALID AL-SHAWI
• 金额: $ 26.06万
• 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 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7029736
• 关键词: P glycoprotein; active transport; adenosine triphosphate; antineoplastics; binding sites; chemical kinetics; chemical models; computational biology; conformation; drug metabolism; electron spin resonance spectroscopy; genetic manipulation; human genetic material tag; hydrolysis; multidrug resistance; pharmacokinetics; protein structure function; protein transport; thermodynamics

DESCRIPTION (provided by applicant): The human plasma membrane protein, P-glycoprotein (Pgp) is an ATP-driven drug-exporting pump that counteracts chemotherapy in cancer cells and limits the bioavailability of therapeutic drugs in other tissues. Modulation of the drug-exporting activity of Pgp could improve cancer treatments and AIDS treatment. The overall goal of this project is to elucidate the molecular mechanism of drug transport by Pgp. Utilizing new techniques for studying drug transport and detailed structural analysis, a molecular model of the transport mechanism has been generated. In this proposal, this model will be tested and refined using mutagenesis of human Pgp and quantitative kinetic, thermodynamic, spectroscopic and computational methods of enzyme analysis. The model postulates that the coupling of ATP hydrolysis to drug transport involves concerted conformational movements initiated at the nucleotide sites and transmitted through the intracellular domains that lead to transmembrane helix rotations, and rearrangements that drive the drug through the protein channel(s). In Specific Aim 1, drug channel(s) will be functionally characterized in thermodynamic and transport studies to distinguish between alternative models for the reaction cycle of Pgp. Novel spin-labeled transport substrates will be synthesized to facilitate these studies. In Specific Aim 2, the hydration exchange model of drug transport, which associates drug binding and coupled transport events with conformational changes will be refined. Drug-binding sites will be mapped to the structure of P-glycoprotein using mutagenesis and assessment of functional consequences and physical properties of the drug-binding sites will be characterized. In Specific Aim 3 dynamic structure/function relationships will be located by observing conformational changes associated with coupled drug transport events through the application of EPR spectroscopic techniques. Conformational changes during the reaction cycle will be determined for 1) residues involved in initial drug binding at the membrane interface and 2) transmembrane-helix 6 (TM6) involved in drug release at the other side of the membrane. Knowledge acquired in these studies will be used to achieve a rigorous, dynamic, molecular description of coupled drug transport by P-gp that could aid in the rational design of drugs and methodologies to overcome or modulate this transporter.

描述（由申请人提供）：人质膜蛋白P-糖蛋白（PGP）是ATP驱动的药物外出的泵，可抵消癌细胞中的化学疗法，并限制其他组织中治疗药物的生物利用度。 PGP药物过外活性的调节可以改善癌症治疗和艾滋病治疗。该项目的总体目标是阐明PGP药物转运的分子机制。利用新技术研究药物传输和详细的结构分析，已经产生了转运机制的分子模型。在该提案中，将使用人类PGP的诱变以及定量动力学，热力学，光谱和计算方法的酶分析测试和完善该模型。该模型假定ATP水解与药物转运的耦合涉及在核苷酸部位启动的协同构型运动，并通过细胞内结构域传输，从而导致跨膜螺旋旋转，并重新安排通过蛋白质通道（S）驱动药物的重排。在特定的目标1中，药物通道在热力学和运输研究中将在功能上表征，以区分PGP反应周期的替代模型。新型自旋标记的运输底物将合成以促进这些研究。在特定的目标2中，将完善药物运输的水合交换模型，该模型将药物结合和耦合转运事件与构象变化相关联。使用诱变并评估药物结合位点的功能后果和物理特性，将映射药物结合位点为P-糖蛋白的结构。在特定目标中，3动态结构/功能关系将通过使用EPR光谱技术观察与耦合药物传输事件相关的构象变化找到。将确定反应周期期间的构象变化1）涉及膜界面处初始药物结合的残基和2）跨膜释放的跨膜螺旋6（TM6）。在这些研究中获得的知识将用于实现P-gp偶联药物转运的严格，动态的，分子的描述，这可以有助于制定药物和方法的合理设计，以克服或调节该转运蛋白。