Substrate Specificity Determinants in Nutrient Solute Carrier Transporters

摘要 在人类中，有超过460个溶质载体(SLC)转运体调节溶质的输入和流出， 包括神经递质、营养物质和离子。SLC正在成为各种疾病的关键治疗靶点。 疾病和无序。例如，营养SLC转运蛋白在重新编程的代谢中发挥着重要作用。 通过向细胞提供营养物质，在癌症、自身免疫性疾病和心脏缺血中发挥作用 构建生物量或作为促进细胞增殖和分化的信号分子，或调节细胞 死亡。我们的主要目标是描述营养物质中底物和抑制剂的特异性决定因素。 转运体，并开发新的策略来调节其功能。我们采取综合的方法 这包括计算生物学和有机化学，以及生化和生物物理学。 描述丙氨酸-丝氨酸-半胱氨酸转运蛋白2(ASCT2， SLC1A5)。 在这个项目的目标1中，我们将生成和提炼人类SLC1家族的同源模型 构象。我们将描述模型底物结合部位的结构特征，以及最近发现的 变构位点(S)，决定底物和抑制剂的专一性。关键残留物将通过现场定向测试 诱变和电生理方法。最后，我们将开发一个可公开访问的数据库 包括所有可建模的人类SLC转运蛋白的同源模型，并将与疾病相关的突变映射到 这些模型并提供了它们预期的功能效果。 在目标2中，我们将合理设计调节营养功能的小分子工具化合物 转运体通过不同的机制。这将扩大转运蛋白抑制剂的化学空间，并提供 对目标1中开发的模型进行测试。我们将重点介绍以下方法：(A)优化和 表征新型构象特异性抑制剂，例如可光激活和荧光化合物，其 与ASCT2的底物结合部位相互作用；(B)设计针对结构域的变构抑制剂 在ASCT2中的接口，以测试该转运体可能的升降机制；以及(C)开发具有 ASCT2和L氨基酸转运蛋白1(LAT1，SLC7A5)的双特异性，我们假设 由于它们在重新编程的代谢中的协同作用，对细胞增殖有明显的抑制作用 网络。 如果成功，这个项目将提高我们对新兴市场中结构与功能关系的理解 治疗靶点。该项目还将提供新的化学探针，以进一步表征 这些蛋白质及其在疾病中的作用，以及一个有用的公开在线资源。

SUMMARY In humans, there are over 460 Solute Carrier (SLC) transporters that mediate the import and efflux of solutes, including neurotransmitters, nutrients, and ions. The SLCs are emerging as key therapeutic targets for a variety of diseases and disorders. For example, nutrient SLC transporters play a major role in reprogrammed metabolic networks in cancer, autoimmune disease, and heart ischemia, by supplying cells with nutrients that are used to build biomass or serve as signaling molecules that enhance cell proliferation and differentiation, or regulate cell death. Our broad goal is to describe the substrate and inhibitor specificity determinants in nutrient transporters and develop novel strategies to modulate their functions. We take an integrative approach that includes computational biology and organic chemistry, coupled with biochemical and biophysical approaches, and in vitro models of disease, to characterize the Alanine-Serine-Cysteine Transporter 2 (ASCT2, SLC1A5). In Aim 1 of this project, we will generate and refine homology models for the human SLC1 family in multiple conformations. We will describe structural features of the models’ substrate binding site and recently discovered allosteric site(s), that determine substrate and inhibitor specificity. Key residues will be tested with site-directed mutagenesis and electrophysiological approaches. Finally, we will develop a publicly-accessible database that includes homology models for all modelable human SLC transporters, and maps disease-related mutations onto the models and provides their predicted functional effect. In Aim 2, we will rationally design small molecule tool compounds that modulate the function of nutrient transporters via distinct mechanisms. This will expand the chemical space of transporter inhibitors and provide a test for the models developed in Aim 1. We will focus on the following approaches: (A) optimizing and characterizing novel conformation-specific inhibitors, such as photoactivatable and fluorescent compounds that interact with the substrate binding site of ASCT2; (B) designing allosteric inhibitors targeting the domain-domain interface in ASCT2, to test the putative elevator mechanism of this transporter; and (C) developing inhibitors with dual specificity for ASCT2 and the L-type Amino Acid Transporter 1 (LAT1, SLC7A5), which we hypothesize to have a pronounced inhibitory effect on cell proliferation due to their cooperativity in reprogrammed metabolic networks. If successful, this project will improve our understanding of structure-function relationships in emerging therapeutic targets. This project will also provide novel chemical probes to further characterize the structure of these proteins and their role in disease, as well as a useful publicly available online resource.