Mechanism of drug transport by P-glycoprotein

描述(申请人提供)：人的质膜蛋白，P-糖蛋白(PGP)是一种由ATP驱动的药物输出泵，在癌细胞中抵消化疗，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和艾滋病的治疗。本项目的总体目标是阐明PGP转运药物的分子机制。利用研究药物转运的新技术和详细的结构分析，已经产生了转运机制的分子模型。在这项提议中，将使用人PGP的诱变以及酶分析的定量动力学、热力学、光谱和计算方法来测试和改进这一模型。该模型假设，三磷酸腺苷的水解与药物转运的耦合涉及在核苷酸位置启动的协调构象运动，并通过导致跨膜螺旋旋转的细胞内结构域传递，以及驱动药物通过蛋白质通道的重排(S)。在具体目标1中，药物通道(S)将在热力学和转运研究中进行功能表征，以区分PGP反应周期的不同模型。为了促进这些研究，将合成新型的自旋标记转运底物。在具体目标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运输事件与构象变化联系起来。药物结合位点将通过突变被映射到P-糖蛋白的结构上，并将表征药物结合位点的功能后果和物理性质的评估。在特定的目标中，将通过应用EPR波谱技术观察与耦合药物转运事件相关的构象变化来定位动态结构/功能关系。在反应周期中，将确定1)参与初始药物结合的膜界面残基和2)参与膜另一侧药物释放的跨膜螺旋6(TM6)的构象变化。在这些研究中获得的知识将被用来实现对P-gp偶联药物运输的严格、动态的分子描述，这可能有助于合理设计药物和方法来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.