Molecular Regulation of Folate and Antifolate Transport

摘要 叶酸是一种阴离子分子，很难通过扩散穿过生物膜。叶酸摄取主要是 由还原型叶酸载体(RFC；SLC19A1)和质子偶联叶酸转运体(PCFT； SLC46A1)。在内化后，叶酸促进一碳(C1)代谢，导致甘氨酸的合成， 丝氨酸和蛋氨酸，以及嘌呤核苷酸和胸腺苷。C1代谢包括胞质和 通过丝氨酸、甘氨酸和甲酸之间的交换连接的线粒体通路。无处不在的 表达的RFC是细胞和组织中叶酸的主要膜转运体。RFC也是一个重要的 临床上使用的治疗癌症的C1抑制剂(如培美曲塞)以及其他适应症的转运体，以及 RFC与耐药性有关。PCFT调节上胃肠道对叶酸的吸收。 其他组织中的PCFT水平一般不高。与RFC不同的是，PCFT的传输在酸性pH下是最佳的， 接近肿瘤微环境。PCFT在人肿瘤细胞系和原代细胞中广泛表达 标本。我们发现了新的针对癌症的细胞毒性PCFT靶向C1抑制剂，并建立了一种 不同于RFC的PCFT的全面构效关系。新型吡咯嘧啶 化合物(AGF94和AGF347)对表达PCFT的肿瘤显示出强大的抗增殖活性 在酸性pH条件下被放大。在内化后，AGF94抑制了从新的嘌呤(DNP)的Biosynn- 关于β-甘氨酰胺核糖核苷酸甲酰转移酶(GARFTase)的论文，而AGF347抑制有丝分裂- 线粒体C1在丝氨酸羟甲基转移酶2(SHMT2)上的代谢，对C1有额外的影响 胞质中的代谢(GARFTase和5-氨基咪唑-4-甲酰胺生物合成DNP 核苷酸甲酰转移酶和SHMT1)。两种抑制剂都耗尽了ATP；AGF347耗尽了甘氨酸和 胞质C1池，对谷胱甘肽和活性氧水平以及mTOR的下游影响 发信号。AGF94和AGF347对早期和晚期异种肿瘤显示了良好的体内疗效。 嫁接。我们假设，我们的PCFT靶向药物为癌症治疗提供了一种全新的方法。在本R01中 更新，我们探索促进叶酸转运体和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中建议将 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控PCFT中的作用。在目标2中，我们将 描述线粒体C1抑制剂的细胞药效学，包括它们的转运和 新陈代谢。目标1和目标2的一个重要焦点将是肿瘤微环境的作用， 包括低氧和酸性pH对抗肿瘤药物生物学的影响以及这些系列药物的疗效。我们的 拟议的研究因其新颖性和对临床翻译的关注而独树一帜。

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.