Molecular Regulation of Folate and Antifolate Transport

摘要

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.