Molecular Characterization of Hepatic Organic Anion Transporting Polypeptides

描述（申请人提供）：有机阴离子转运多肽OATP1B1和OATP1B3是在人肝细胞中表达的两种主要药物转运蛋白。由于它们的多特异性，这两种转运蛋白是不良药物相互作用的潜在位点。在之前的资助期间，我们表征了底物依赖性调节，确定了底物结合位点的分子特征，并确定了对 OATP1B1 和 OATP1B3 介导的底物转运重要的几个氨基酸残基。然而，导致其多特异性的分子机制仍不清楚。我们的长期研究目标是详细了解 OATP 介导的转运机制，这是理解、预测和预防 OATP 相关的不良药物相互作用的重要先决条件。拟议研究的基本原理是，由于 OATP1B1 和 OATP1B3 与肝脏从血液中清除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了解将为改进预测和预防不良药物相互作用以及未来设计其转运活性的特定底物和/或抑制剂提供强大的科学框架。该应用的目的是识别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易位途径的中心孔转运其底物，并且 OATP1B1 和 OATP1B3 作为不对称交换器。我们计划通过两个具体目标来检验该假设：1）识别并表征 OATP1B1 和 OATP1B3 的易位途径； 2) 确定 OATP 介导的运输机制。这些具体目标的完成将通过识别不同跨膜域中影响底物易位的氨基酸来解释底物依赖性效应，并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，识别生理反阴离子，并解释可能由于转运蛋白寡聚状态引起的变构效应。这一贡献意义重大，因为其结果将提供预测 OATP 介导的药物转运所需的基本理解，除了从根本上推进有机阴离子转运领域之外，最终还会带来更有效和高效的治疗方法。 公共健康相关性：拟议的研究与公共健康相关，因为对 OATP1B1 和 OATP1B3 的结构/功能关系的基本了解将为预测 OATP 介导的药物转运提供所需的基本了解，最终导致更有效和高效的治疗。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because a fundamental understanding of the structure/function relationships of OATP1B1 and OATP1B3 will provide the fundamental understanding required to predict OATP-mediated drug transport, ultimately leading to more effective and efficient therapies.