Mechanism of drug transport by P-glycoprotein

描述（由申请方提供）：人质膜蛋白P-糖蛋白（Pgp）是一种ATP驱动的药物输出泵，可抵消癌细胞中的化疗，并限制治疗药物在其他组织中的生物利用度。调节Pgp的药物输出活性可以改善癌症治疗和艾滋病治疗。本课题的总体目标是阐明Pgp介导药物转运的分子机制。利用研究药物转运的新技术和详细的结构分析，产生了转运机制的分子模型。在这个建议中，这个模型将进行测试和完善，使用诱变的人Pgp和定量动力学，热力学，光谱和计算方法的酶分析。该模型假设ATP水解与药物转运的偶联涉及在核苷酸位点起始并通过细胞内结构域传递的协同构象运动，导致跨膜螺旋旋转，以及驱动药物通过蛋白质通道的重排。在具体目标1中，将在热力学和转运研究中对药物通道进行功能表征，以区分Pgp反应循环的替代模型。新的自旋标记的运输基板将被合成，以促进这些研究。在具体目标2中，将完善药物转运的水合交换模型，该模型将药物结合和偶联转运事件与构象变化相关联。使用诱变将药物结合位点映射到P-糖蛋白的结构，并对药物结合位点的功能后果和物理性质进行表征。在具体目标3动态结构/功能的关系将通过观察构象变化与耦合药物转运事件通过EPR光谱技术的应用。将测定1）参与膜界面初始药物结合的残基和2）参与膜另一侧药物释放的跨膜螺旋6（TM 6）在反应循环期间的构象变化。在这些研究中获得的知识将被用来实现一个严格的，动态的，分子描述耦合药物转运的P-gp，可以帮助药物和方法的合理设计，以克服或调节这种转运蛋白。

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.