Molecular Regulation of Folate and Antifolate Transport

摘要 叶酸是阴离子分子，通过扩散穿过生物膜很差。叶酸的摄取主要是 由还原叶酸载体（RFC; SLC 19 A1）和质子偶联叶酸转运蛋白（PCFT; SLC46A1）。在内化后，叶酸促进一碳（C1）代谢，导致甘氨酸的合成， 丝氨酸和甲硫氨酸，以及嘌呤核苷酸和胸苷酸。C1代谢包括细胞溶质和 通过丝氨酸、甘氨酸和甲酸盐之间的交换连接的线粒体途径。无处不在的 表达的RFC是细胞和组织中叶酸的主要膜转运蛋白。RFC也是重要的 临床使用的C1抑制剂的转运蛋白（例如，培美曲塞）用于癌症，以及其他适应症， RFC与耐药性有关。PCFT介导叶酸在上消化道的吸收。 其他组织中的PCFT水平通常适中。与RFC不同，PCFT转运在酸性pH下最佳， 接近肿瘤微环境。PCFT广泛表达于人类肿瘤细胞系和原发性肿瘤细胞系中。 标本我们发现了新的细胞毒性PCFT靶向的C1抑制剂，并建立了一种新的治疗癌症的方法。 与RFC不同的PCFT的全面的结构-活性关系。新型吡咯并嘧啶 化合物（AGF 94和AGF 347）对表达PCFT的肿瘤显示出有效的抗增殖活性 内化后，AGF 94抑制从头嘌呤（DNP）生物合成， 在β-甘氨酰胺核糖核苷酸甲酰转移酶（GARFT酶）的论文，而AGF 347抑制线粒体， 丝氨酸羟甲基转移酶2（SHMT 2）的C1代谢，对C1有额外影响 细胞溶质中的代谢（GARFT酶和5-氨基咪唑-4-甲酰胺处的DNP生物合成 核糖核苷酸甲酰基转移酶和SHMT 1）。两种抑制剂均耗尽ATP; AGF 347耗尽甘氨酸， 胞质C1池，对谷胱甘肽和活性氧水平以及mTOR有下游效应 发信号。AGF 94和AGF 347显示出对早期和晚期肿瘤异种的有希望的体内功效。 移植物我们认为，我们的PCFT靶向药物为治疗癌症提供了一种全新的方法。在此R 01 更新，我们探索促进叶酸转运蛋白和C1代谢的独特生物学，目标是 进一步优化我们的新型药物的治疗应用。我们在目标1中提出， 与PCFT靶向治疗相关的PCFT的细胞药效学和分子调控，包括 转录机制和蛋白质-蛋白质相互作用在调节PCFT中的作用。在目标2中，我们将 表征线粒体C1抑制剂的细胞药效学，包括其转运， 新陈代谢.目标1和2的一个重要焦点是肿瘤微环境的作用， 包括缺氧和酸性pH对抗肿瘤药物生物学和药效的影响等系列研究。我们 拟议的研究因其新奇而与众不同，并侧重于临床翻译。

ABSTRACT Folates are anionic molecules that cross biological membranes poorly by diffusion. Folate uptake is principally mediated by the reduced folate carrier (RFC; SLC19A1) and the proton-coupled folate transporter (PCFT; SLC46A1). Upon internalization, folates facilitate one-carbon (C1) metabolism, leading to synthesis of glycine, serine and methionine, and purine nucleotides and thymidylate. C1 metabolism encompasses cytosolic and mitochondrial pathways connected by an interchange between serine, glycine and formate. The ubiquitously expressed RFC is the major membrane transporter for folates in cells and tissues. RFC is also an important transporter of clinically used C1 inhibitors (e.g., pemetrexed) for cancer, as well as other indications, and loss of RFC is associated with drug resistance. PCFT mediates folate absorption in the upper gastrointestinal tract. PCFT levels in other tissues are generally modest. Unlike RFC, PCFT transport is optimal at acidic pH, approximating the tumor microenvironment. PCFT is widely expressed in human tumor cell lines and primary specimens. We discovered novel cytotoxic PCFT-targeted C1 inhibitors for cancer and established a comprehensive structure-activity relationship for PCFT that is distinct from RFC. Novel pyrrolopyrimidine compounds (AGF94 & AGF347) showed potent anti-proliferative activities toward PCFT-expressing tumors that were augmented at acid pH. Following internalization, AGF94 inhibited de novo purine (DNP) biosyn- thesis at β-glycinamide ribonucleotide formyltransferase (GARFTase), whereas AGF347 inhibited mito- chondrial C1 metabolism at serine hydroxymethyltransferase 2 (SHMT2), with additional effects on C1 metabolism in the cytosol (DNP biosynthesis at GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and at SHMT1). Both inhibitors depleted ATP; AGF347 depleted glycine and cytosolic C1 pools, with downstream effects on glutathione and levels of reactive oxygen species, and on mTOR signaling. AGF94 and AGF347 showed promising in vivo efficacies toward early and upstage tumor xeno- grafts. We posit that our PCFT-targeted agents offer an entirely new approach for treating cancer. In this R01 renewal, we explore the unique biology of the facilitative folate transporters and C1 metabolism, with a goal of further optimizing therapeutic applications of our novel agents. We propose in Aim 1 to characterize the cellular pharmacodynamics and molecular regulation of PCFT in relation to PCFT-targeted therapies, including transcriptional mechanisms and the role of protein-protein interactions in regulating PCFT. In Aim 2, we will characterize the cellular pharmacodynamics of mitochondrial C1 inhibitors including their transport and metabolism. An important focus of both Aims 1 and 2 will be on the role of the tumor microenvironment, including the impact of hypoxia and acid pH on anti-tumor drug biology and efficacy of these series. Our proposed studies are distinctive for their novelty and focus on clinical translation.