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质子偶联叶酸转运蛋白（PCFT）的基础上，PCFT是一种对新一代抗叶酸剂培美曲塞具有高亲和力的低pH转运蛋白，当主要叶酸/抗叶酸转运蛋白（还原叶酸载体）缺失且肿瘤细胞 对甲氨蝶呤和其他抗叶酸剂有很高的耐药性。在目前的资助期内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/抗叶酸的元素。所提出的研究重点是发展一个全面的理解PCFT功能结合实验室和计算策略的结构基础。具体目标是：（i）鉴定限定水转运途径、限定叶酸结合及其特异性以及作为质子结合和偶联的决定因素的残基。这些研究使用取代的半胱氨酸可及性方法。(ii)通过双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，并阐明第一胞内环在PCFT功能中发挥作用的基础。(iii)确定PCFT的结构改变对培美曲塞和其他抗叶酸剂体外抗肿瘤活性的影响，特别是在酸性条件下。(iv)为了利用获得的实验数据，在一个迭代的方法，通知，完善和验证PCFT的同源性模型，并进行对接分析，以确定关键元素的亲和力和选择性的叶酸/抗叶酸结合口袋。这些研究还将为开发新型抗叶酸剂提供信息，这些抗叶酸剂旨在通过PCFT在实体瘤的酸性微环境中选择性递送。在这些研究中开发的突变体PCFT的稳定性，运输到细胞膜，叶酸/抗叶酸底物的选择性，并在质子结合和质子偶联转染到一个独特的Hela细胞系，缺乏内源性叶酸转运蛋白的作用进行了评估。这是补充电生理测量在非洲爪蟾卵母细胞的电流和酸化所产生的叶酸-质子-PCFT三元复合物的运输，这也使得可能的分析，不放射性标记的抗叶酸剂的运输特性。

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.