Proton-coupled folate/antifolate transport

描述(由申请人提供)：本申请建立在本实验室发现的质子偶联叶酸转运体(PCFT)的基础上，这是一种低pH转运体，对新一代抗叶酸培美曲塞具有高亲和力，当主要的叶酸/反叶酸转运体(还原的叶酸载体)缺失和肿瘤细胞时，它唯一地维持这种药物的活性 对甲氨蝶呤和其他抗叶酸具有高度抗药性。在当前供资期间的研究已经开始阐明PCFT的结构-功能，包括其拓扑结构、其功能所需的残基以及区分叶酸/反叶酸的元素。建议的研究重点是结合实验室和计算策略对PCFT功能的结构基础进行全面的理解。具体的目标是：(I)确定定义水转运途径的残基，定义叶酸结合及其特异性的残基，以及质子结合和偶联的决定因素。这些研究利用了取代半胱氨酸可及性方法。(Ii)通过二半胱氨酸突变体的化学交联，表征作为三级结构决定因素的PCFT跨膜螺旋与胞外门之间的相互作用，并阐明第一细胞内环在PCFT功能中所起作用的基础。(Iii)确定PCFT的结构变化对培美曲塞和其他抗叶酸盐体外抗肿瘤活性的影响，特别是在酸性条件下。(4)利用以迭代方式获得的实验数据，通报、改进和验证多氯联苯的同源模型，并进行对接分析，以确定对叶酸/反叶酸结合口袋的亲和力和选择性至关重要的元素。这些研究还将为新型抗叶酸的开发提供信息，该设计可在实体肿瘤的酸性微环境中通过PCFT选择性地传递。在这些研究中开发的突变PCFT通过转染缺乏内源性叶酸转运体的独特的Hela细胞系来评估它们的稳定性、向细胞膜的转运、叶酸/反叶酸底物的选择性以及在质子结合和质子偶联中的作用。此外，在非洲爪哇卵母细胞中，还可以对叶酸-质子-PCFT三元复合体运输产生的电流和酸化进行电生理测量，这也使分析不能辐射的反叶酸的运输特性成为可能。

DESCRIPTION (provided by applicant): This application builds upon this laboratory's discovery of the proton-coupled folate transporter (PCFT), a low-pH transporter with high affinity for the new-generation antifolate, pemetrexed, that uniquely sustains the activity of this agent when the major folate/antifolate transporter, the reduced folate carrier, is absent and tumor cells are highly resistance to methotrexate and other antifolates. Studies over the current funding period have begun to elucidate the structure-function of PCFT encompassing its topology, residues required for its function, and elements that discriminate among folates/antifolates. The studies proposed are focused on developing a comprehensive understanding of the structural basis for PCFT function combining laboratory and computational strategies. The specific aims are: (i) To identify residues that define the aqueous translocation pathway, that define folate binding and its specificity, and that are determinants of proton binding and coupling. These studies utilize the substituted cysteine accessibility method. (ii) To characterize the interaction between PCFT transmembrane helices that are determinants of tertiary structure, and an extracellular gate, by chemical cross-linking of di-cysteine mutants, and to clarify the basis for the role the first intracellular loop plays in PCFT function. (iii) To determine the impact of structural alterations of PCFT on the antitumor activities of pemetrexed and other antifolates in vitro, particularly under acidic conditions. (iv) To utilize the experimental data obtained, in an iterative approach, to inform, refine and validate a homology model of PCFT and to undertake docking analyses in order to identify elements critical to the avidity and selectivity of the folate/antifolate binding pocket. These studies will also inform the development of novel antifolates designed for selectively delivery by PCFT within the acidic microenvironment of solid tumors. Mutant PCFTs developed in these studies are assessed for their stability, trafficking to the cell membrane, folate/antifolate substrate selectivity, and roles in proton binding and proton coupling by transfection into a unique Hela cell line that lacks endogenous folate transporters. This is complemented by electrophysiological measurements in Xenopus oocytes of current and acidification generated by the transport of the folate-proton-PCFT ternary complex, that also make possible analyses of transport properties of antifolates that are not radiolabled.