Substrate Specificity of the Choroid Plexus and Kidney Transporter Oat1

描述(申请人提供)：除了ABC超家族的成员外，有机阴离子转运体，如Oat1，最早由PI的实验室发现，很可能是体内最重要的异源和多特异性代谢物转运体。燕麦被认为是至关重要的 用于调节药物、毒素和代谢物在脑脊液、血液、尿液和其他体液之间的分布。在这个重新提交的申请中，我们建议在电子计算机、体外、结构生物学和体内方法中使用来提供底物-转运体相互作用的全面图像，并使用PI‘s组相对独特的分析来验证这一点。因此，该小组有多种在硅胶、体外和体内工作的方法，而我们的合作者(张博士)是迄今为止药物转运领域中最重要的结构生物学家之一，对任何非ABC哺乳动物药物的硅胶-体外-体内分析。 运输商还没有完成，我们认为我们处于这样做的理想位置。我们计划使用基于配体和转运体的计算模型，我们有丰富的经验，以确定目标底物通过Oat1(SA1，将与圣地亚哥超级计算机的合作者一起完成)摄取和处理的分子决定因素。然后将使用药效团对化学结构文库进行虚拟筛选，然后进行化合物的湿法实验室验证。我们还将与Chang博士(包括他的实验室和NIH蛋白质结构倡议的一个组成部分TransportPDB)合作，结晶和确定Oat1的高分辨率X射线结构，以获得对其底物结合和运输(SA2)的结构要求的关键见解。在SA3中，我们建议通过测试体内药代动力学分析对燕麦转运抑制的预测来进一步验证我们对底物-转运体相互作用的分析(来自SA1和SA2)(与我们之前显示系统代谢异常以及药物和毒素处理缺陷的OAT1基因敲除相比)。这些研究的结果也可能对理解燕麦1在全身和组织特异性新陈代谢中的作用(除了潜在地导致延长药物半衰期的抑制剂)具有广泛的意义。我们试图解决所有先前的批评，重点澄清“意义”、“创新”和“方法”，我们已经为结晶目标提供了初步数据。我们还在2010-2011年间发表了许多相关的“计算-湿实验论文”。在其中一篇论文中，我们描述了使用药效团建模和虚拟筛选来鉴定一种新的高亲和力抑制剂，该抑制剂在转运试验中得到了初步验证；这种高亲和力抑制剂也可能有助于获得燕麦1底物结合结构。PI可能是该领域独一无二的，它汇集了世界级的专家(都在加州大学圣地亚哥分校或斯克里普斯大学)，他们的实验室经常使用建议的方法。还讨论了“替代的”计算和湿实验室方法，以防主要方法不如预期的成功。 公共卫生相关性：一些常见药物、毒素和生理代谢物的消除依赖于肾脏中的转运蛋白。这些底物和转运体在消除过程中如何相互作用尚不清楚。在这里，我们试图调查这一点，以及确定这些转运蛋白之一的特定抑制剂。

DESCRIPTION (provided by applicant): Apart from members of the ABC superfamily, organic anion transporters such as Oat1, first identified by the PI's lab, may well be the most important xenobiotic and multi-specific metabolite transporters in the body. Oats are believed to be critical for regulating the distribution of drugs, toxins and metabolites between the CSF, blood, urine and other body fluids. In this RESUBMISSION application, we propose to employ in silico, in vitro, structural biological and in vivo methods to provide a comprehensive picture of substrate-transporter interactions and to validate this using assays that are relatively unique to the PI's group. Thus the group has multiple in silico, in vitro and in vivo approaches working, and our collaborator (Dr. Chang) is one of the premier structural biologists in the field of drug transport So far a "full circuit" of in silico-in vitro-in vivo analysis of any of the non-ABC mammalian drug transporters has not been performed, and we argue that we are in an ideal position to do this. We plan to use ligand and transporter-based computational modeling, with which we have extensive experience, to identify the molecular determinants which target substrates for uptake and handling via Oat1 (SA1, to be done with collaborators at the San Diego Supercomputer). Virtual screening of chemical structural libraries using pharmacophores will then be done, followed by wet lab validation of compounds. We will also crystallize and determine the high-resolution x-ray structure of Oat1 in collaboration with Dr. Chang (involving both his laboratory and TransportPDB, a component of the NIH Protein Structural Initiative) to gain key insights into the structural requirements for its substrate binding and transport (SA2). In SA3, we propose to further validate our analysis of substrate-transporter interactions (from SA1 and SA2) by testing predictions of inhibition of Oat1 transport by in vivo pharmacokinetic analysis (in comparison with the OAT1 knockout in which we have previously shown both aberrant systemic metabolism as well as defective drug and toxin handling). The results of these studies could also have broad implications for understanding the role of Oat1 in whole body and tissue-specific metabolism (quite apart from potentially leading to inhibitors that prolong drug half-life). We have tried to address all the prior criticisms, focusing on clarifying the "Significance," "Innovation" and "Approach," and we have provided preliminary data for the crystallization aim. We have also published many relevant "computational-wet lab papers" in 2010-2011. In one of these papers, we describe the use of pharmacophore modeling and virtual screening to identify a novel high-affinity inhibitor that was preliminarily validated in a transport assay; this high-affinity inhibior may also aid in obtaining an Oat1 substrate-bound structure. Perhaps unique for this field, the PI has brought together world-class experts (all at UCSD or Scripps) whose labs regularly use the proposed methods. Also discussed are "alternative" computational and wet lab approaches in case the primary approach is less successful than anticipated. PUBLIC HEALTH RELEVANCE: The elimination of a number of common drugs, toxins and physiological metabolites depends upon transporters in the kidney. How these substrates and the transporter interact in the process of elimination is not known. Here we seek to investigate this, as well as identify specific inhibitors of one of these transporters.