Molecular Characterization of Hepatic Organic Anion Transporting Polypeptides

描述（由申请人提供）：有机阴离子转运多肽OATP1B1和OATP1B3是人类肝细胞中表达的两种主要药物转运体。由于它们的多特异性，这两种转运蛋白是药物-药物相互作用的潜在不良位点。在之前的授权期内，我们表征了底物依赖性调节，鉴定了底物结合位点的分子特征，并鉴定了对OATP1B1-和oatp1b3介导的底物运输重要的几个氨基酸残基。然而，导致其多特异性的分子机制尚不清楚。我们的长期研究目标是详细了解ooatp介导的转运机制，这是理解、预测和预防ooatp相关的药物-药物不良相互作用的必要前提。该研究的基本原理是，由于OATP1B1和OATP1B3与肝脏清除血液中多种化学物质的能力密切相关，因此对其个体和重叠转运蛋白功能的改进机制见解将为预测和预防不良药物-药物相互作用以及未来设计特定底物和/或其转运活性抑制剂提供强有力的科学框架。本应用程序的目的是确定对底物易位和刺激重要的结构域和氨基酸，并表征运输的驱动力。我们的中心假设是OATP1B1和OATP1B3通过包含底物特异性转运途径的中心孔运输底物，并且OATP1B1和OATP1B3作为不对称交换器起作用。我们计划以两个具体目标来验证这一假设：1)确定和表征OATP1B1和OATP1B3的易位途径；2)确定ooatp介导的转运机制。这些特定目标的完成将通过识别影响底物易位的不同跨膜结构域中的氨基酸来解释底物依赖效应，并将使我们更接近这些膜转运蛋白的三维结构。此外，我们将解决转运机制的争议，确定生理上的反阴离子，并解释可能由于转运蛋白的低聚状态而产生的变构效应。这一贡献是重要的，因为它的结果将提供预测OATP介导的药物运输所需的基本理解，最终导致更有效和高效的治疗，并从根本上推进有机阴离子运输领域。

DESCRIPTION (provided by applicant): The organic anion transporting polypeptides OATP1B1 and OATP1B3 are two major drug transporters expressed in human hepatocytes. Because of their multispecificity, these two transporters are potential sites of adverse drug-drug interactions. In the previous grant period, we characterized substrate dependent modulation, identified molecular characteristics of the substrate binding sites, and identified several amino acid residues important for OATP1B1- and OATP1B3-mediated substrate transport. However, the molecular mechanisms responsible for their multispecificities remain unclear. Our long term research goal is to understand in detail the mechanism of OATP-mediated transport as an essential prerequisite to understanding, predicting, and preventing OATP-related adverse drug-drug interactions. The rationale for the proposed research is that, because OATP1B1 and OATP1B3 are critically involved in the liver's ability to clear numerous chemicals from the blood, improved mechanistic insights into their individual and overlapping transporter functions will provide a strong scientific framework for improvements in the prediction and prevention of adverse drug-drug interactions, as well as the future design of specific substrates and/or inhibitors of their transport activity. The objective of this application is to identify domains and amino acids that are important for substrate translocation and stimulation, and to characterize the driving force(s) for transport. Our central hypothesis is that OATP1B1 and OATP1B3 transport their substrates via a central pore that includes substrate-specific translocation pathways, and that OATP1B1 and OATP1B3 work as asymmetrical exchangers. We plan to test the hypothesis with two specific aims: 1) Identify and characterize the translocation pathways of OATP1B1 and OATP1B3; and 2) Determine the mechanism of OATP-mediated transport. Completion of these specific aims will explain the substrate dependent effects by identifying amino acids in the different transmembrane domains that influence substrate translocation and will lead us a step closer to the three dimensional structure of these membrane transporters. Furthermore, we will resolve the controversies of the transport mechanism, identify a physiological counter-anion, and explain the allosteric effects that might be due to the oligomeric state of the transporters. This contribution is significant because its results will provide the fundamental understanding required to predict OATP- mediated drug transport, ultimately leading to more effective and efficient therapies in addition to fundamentally advancing the field of organic anion transport.