Substrate Specificity Determinants in Nutrient Solute Carrier Transporters

摘要 细胞代谢的改变支持细胞的快速生长和增殖，在癌症、金黄色葡萄球菌等病理中，细胞代谢的改变支持细胞的快速生长和增殖。 免疫疾病和心脏缺血，导致对氨基酸代谢的依赖增加，如 谷氨酰胺和亮氨酸。营养溶质载体(SLC)转运蛋白在代谢过程中起着重要作用。 通过向细胞提供营养物质来构建生物量，作为信号分子 促进细胞增殖和分化，或调节细胞死亡。我们的广泛目标是描述子- 疾病相关营养物质SLC转运蛋白的特异性决定因素及其研究进展 独特的策略来调节它们的功能。我们采取了一种综合的方法，包括计算 化学方法，结合生化和生物物理方法以及与疾病相关的细胞系，以表征- 描述在快速生长的细胞的新陈代谢中起关键作用的两种氨基酸转运体：丙氨酸- 丝氨酸-半胱氨酸转运体(SLC1A5，ASCT2)，是一种钠依赖的氨基酸交换分子，调节细胞内蛋白质的合成。 细胞谷氨酰胺水平与中性和阳离子氨基酸转运蛋白B0(ATB0，SLC6A14) 酸转运蛋白，由Na和Cl-共转运驱动。 在本项目的目标1中，我们将继续表征ASCT2，它是一种有效的治疗各种疾病的药物靶点。 GES(如三阴性乳腺癌和前列腺癌)。尽管我们最近在理解上取得了进步 关于ASCT2的结构和功能，其生物学方面的许多方面都是高度未知的。我们 将合理地设计化学工具，利用独特的机制调节ASCT2的活性，包括： (A)与最近确定的变构位点相互作用的变构抑制剂；。(B)靶向于 ASCT2底物结合部位的独特半胱氨酸残基；和(C)构象特异性小分子 以底物结合部位的特定亚口袋为靶点的调节剂。在目标2中，我们将描述SLC6A14， 与癌症和代谢性疾病有关的未被充分研究的转运蛋白。我们将开发结构模型 SLC6A14具有不同的构象。我们将描述模型底物结合位置的生物物理特征， 包括静电势、大小、形状和疏水性，以开发关于衬底和 SLC6A14中的抑制物特异性决定因素。我们将利用这些知识来指导抑制剂的开发 以及底物，包括可光活化化合物，以直接测试抑制剂与结合位点的相互作用(S)。 这一项目的成功完成将使人们更好地了解运输和运输的机制。 营养转运蛋白的野心，以及进一步确定其在疾病中的作用的新的化学工具。值得注意的是， 我们将测试一种针对变构调节的新兴和创新的转运蛋白药物发现方法 以及通过小分子的共价抑制，剥夺过度增殖的细胞的营养，潜在地扩张 未来应用于治疗其他涉及系统性红斑狼疮的疾病。

SUMMARY Alterations in cell metabolism support rapid growth and proliferation of cells in pathologies such as cancer, au- toimmune disease, and heart ischemia, resulting in increased reliance on the metabolism of amino acids such as glutamine and leucine. Nutrient Solute Carrier (SLC) transporters play a major role in reprogrammed meta- bolic networks by supplying cells with nutrients that are used to build biomass, serve as signaling molecules that enhance cell proliferation and differentiation, or regulate cell death. Our broad goal is to describe the sub- strate and inhibitor specificity determinants in disease-related nutrient SLC transporters and develop unique strategies to modulate their functions. We take an integrative approach that includes computational chemistry methods, coupled with biochemical and biophysical approaches and disease-related cell lines, to char- acterize two amino acid transporters that play a key role in metabolism of rapidly-growing cells: the Alanine- Serine-Cysteine Transporter (SLC1A5, ASCT2), a Na+-dependent amino acid exchanger that modulates intra- cellular glutamine levels, and the Amino Acid Transporter B0+ (ATB0+, SLC6A14), a neutral and cationic amino acid transporter, driven by Na+ and Cl- co-transport. In Aim 1 of this project, we will continue characterizing ASCT2, a well-validated drug target for various patholo- gies (eg triple negative breast cancer and prostate cancer). Despite recent advancements in our understanding of ASCT2 structure and function made by us and others, many aspects of its biology are highly unexplored. We will rationally design chemical tools that modulate the activity of ASCT2 using unique mechanisms, including: (A) allosteric inhibitors interacting with a recently identified allosteric site; (B) covalent inhibitors targeting a unique cysteine residue in the substrate binding site of ASCT2; and (C) conformation-specific small molecule modulators targeting specific subpockets in the substrate binding site. In Aim 2, we will characterize SLC6A14, an understudied transporter involved in cancer and metabolic diseases. We will develop structural models of SLC6A14 in different conformations. We will describe biophysical features of the models’ substrate binding site, including electrostatic potential, size, shape, and hydrophobicity, to develop hypotheses for the substrate and inhibitor specificity determinants in SLC6A14. We will use this knowledge to guide the development of inhibitors and substrates, including photoactivatable compounds, to directly test inhibitor-binding site interaction(s). Successful completion of this project will provide a greater understanding of mechanisms of transport and inhi- bition of nutrient transporters, as well as novel chemical tools to further characterize their role in disease. Notably, we will test an emerging and innovative approach to transporter drug discovery that targets allosteric modulation and covalent inhibition via small molecules, to deprive hyper-proliferating cells of nutrients, potentially expanding future applications to treat other diseases that involve SLCs.