Structure Function Analysis of the Multi-specific Drug Transporter OCT1

描述（由申请方提供）：新生儿器官中表达最高的药物转运蛋白是有机阳离子转运蛋白（Octs），其转运药物、神经递质、代谢物和毒素。许多药物相互作用和药物代谢紊乱被认为发生在转运蛋白水平，某些多态性可导致药物毒性。Oct1/SLC22 A1是SLC22 A转运蛋白家族的原型成员，并且沿着某些ABC转运蛋白（例如，MDR），燕麦和燕麦，是体内最重要的异生物质（III期药物转运蛋白）转运蛋白之一。PI的实验室鉴定了有机阴离子转运蛋白1（Oat 1/NKT/Slc 22 a6），它与Oct1一起沿着最初帮助定义了SLC 22转运蛋白家族。我们研究了几种SLC22转运蛋白的体内功能，并进行了详细的体外分析和底物-转运蛋白相互作用的计算机模拟。然而，底物转运蛋白相互作用的详细分子分析-这是必要的，采取合理的方法来减少药物的不良影响，通过10月1日运输-是有限的，缺乏精确的结构信息。在这里，我们建议联合收割机结合我们的知识SLC22转运蛋白生物学（和技能，在体外，离体和体内测定）与一流的研究人员在X射线晶体学，蛋白质和底物建模的专业知识，以调查的关键底物转运蛋白相互作用的分子机制，涉及Oct1介导的有机阳离子外源性处理。虽然在本提案中，我们专注于Oct1，但结果也将提供与其他SLC 22药物转运蛋白相关的重要信息。据我们所知，我们是该领域中极少数几个具有联合收割机结合不同专业领域的能力的小组之一，包括计算生物学，结构生物学和体内/体外湿实验室生物学，并且是拟议研究中最具创新性的方面之一。我们将结晶和确定Oct1（SA1）的高分辨率X射线结构，与Geoffrey Chang博士（涉及他的实验室和TransportPDB，NIH蛋白质结构倡议的一个组成部分，他是创始成员）继续合作，以获得对底物结合和运输的结构基础的关键见解。我们将利用我们在基于配体和基于转运蛋白的计算方法（SA2）中的专业知识来确定底物的分子决定因素和介导其相互作用的Oct1蛋白。将对化学结构库进行基于药效团的虚拟筛选，并将确定的结构用于分子动态分析，以确定化合物的优先级。将使用PI组（SA 3）中确立的体外、离体和体内方法，在湿实验室研究中获得并检测已鉴别的化合物。这种多方面的结构-计算-湿实验室策略将在理解儿童和成人的药物不良反应方面取得重大进展，并有助于推动药物转运体领域向前发展。

DESCRIPTION (provided by applicant): Among the most highly expressed drug transporters in neonatal organs are the organic cation transporters (Octs), which transport drugs, neurotransmitters, metabolites and toxins. Many drug-drug interactions and metabolic derangements due to drugs are thought to occur at the level of the transporter, and certain polymorphisms can lead to drug toxicity. Oct1/SLC22A1 is the prototypical member of the SLC22A family of transporters and along with certain ABC transporters (e.g., MDR), Oats and Oatps, is one of the most important xenobiotic (phase III drug transporter) transporters in the body. The PI's lab identified Organic Anion Transporter 1 (Oat1/NKT/Slc22a6), which, along with Oct1 originally helped define the SLC22 transporter family. We have studied the in vivo function of several SLC22 transporters and performed detailed in vitro analysis and in silico modeling of substrate-transporter interactions. Nevertheless, detailed molecular analyses of substrate-transporter interactions-which are necessary to take a rational approach to diminishing adverse affects of drugs transported by Oct1-are limited by the absence of precise structural information. Here we propose to combine our knowledge of SLC22 transporter biology (and skill with in vitro, ex vivo and in vivo assays) together with the expertise of top-notch investigators in x-ray crystallography, protein and substrate modeling to investigate the molecular mechanisms of the key substrate-transporter interactions involved in Oct1-mediated organic cation xenobiotic handling. Although in this proposal we focus on Oct1, the results will also provide important information relevant to other SLC22 drug transporters. To our knowledge, we are among the very few groups in this field that has a proven ability to combine such diverse areas of expertise, including computationally biology, structural biology and in vivo/in vitro wet-lab biology and is one of the most innovative aspects of the proposed studies. We will crystallize and determine the high-resolution x-ray structure of Oct1 (SA1) in a continuing collaboration with Dr. Geoffrey Chang (involving both his laboratory and TransportPDB, a component of the NIH Protein Structural Initiative of which he is a founding member) to gain key insights into the structural basis of substrate binding and transport. We will use our documented expertise in ligand-based and transporter-based computational approaches (SA2) to identify the molecular determinants of substrates and the Oct1 protein mediating their interactions. Pharmacophore-based virtual screening of chemical structure libraries will be done, and identified structures will be used for molecular dynamic analyses to prioritize compounds. Identified compounds will be obtained and tested in wet-lab studies using in vitro, ex vivo and in vivo approaches well established in the PI's group (SA3). This multifaceted structural- computational-wet-lab strategy will result in major advances in understanding adverse drug reactions in children as well as adults and help move the drug transporter field forward.