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 ?-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.

Public Health Relevance

In this application, we explore the unique biology of the facilitative folate transporters and the role of one- carbon (C1) metabolism in cancer, with a goal of further optimizing therapeutic applications of novel targeted anti-tumor agents. The project explores therapeutic applications of the human proton-coupled folate transporter (PCFT) which occurs in many tumors and is functionally and anatomically distinct from the ubiquitous reduced folate carrier, the major tissue folate transporter. Additional focus is on essential C1 metabolic pathways that are compartmentalized and are differentially expressed in tumor cells versus normal cells as they contribute to cancer etiology and progression. Our proposed studies provide a necessary prelude for the design of a new generation of therapeutics for cancer, and how therapeutic benefit can be maximized for our novel agents, drawing from the remarkably complex physiology of tumor cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Forry, Suzanne L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Wayne State University
Schools of Medicine
United States
Zip Code
Shah, Khushbu; Raghavan, Sudhir; Hou, Zhanjun et al. (2018) Development and validation of chemical features-based proton-coupled folate transporter/activity and reduced folate carrier/activity models (pharmacophores). J Mol Graph Model 81:125-133
Matherly, Larry H; Hou, Zhanjun; Gangjee, Aleem (2018) The promise and challenges of exploiting the proton-coupled folate transporter for selective therapeutic targeting of cancer. Cancer Chemother Pharmacol 81:1-15
Ravindra, Manasa; Wilson, Mike R; Tong, Nian et al. (2018) Fluorine-Substituted Pyrrolo[2,3- d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor ? and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis. J Med Chem 61:4228-4248
Giovannetti, E; Zucali, P A; Assaraf, Y G et al. (2017) Role of proton-coupled folate transporter in pemetrexed resistance of mesothelioma: clinical evidence and new pharmacological tools. Ann Oncol 28:2725-2732
Hou, Zhanjun; Gattoc, Leda; O'Connor, Carrie et al. (2017) Dual Targeting of Epithelial Ovarian Cancer Via Folate Receptor ? and the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates. Mol Cancer Ther 16:819-830
Wilson, Mike R; Hou, Zhanjun; Yang, Si et al. (2016) Targeting Nonsquamous Nonsmall Cell Lung Cancer via the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]Pyrimidine Thienoyl Antifolates. Mol Pharmacol 89:425-34
Golani, Lalit K; Wallace-Povirk, Adrianne; Deis, Siobhan M et al. (2016) Tumor Targeting with Novel 6-Substituted Pyrrolo [2,3-d] Pyrimidine Antifolates with Heteroatom Bridge Substitutions via Cellular Uptake by Folate Receptor ? and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesi J Med Chem 59:7856-76
Wilson, Mike R; Hou, Zhanjun; Wilson, Lucas J et al. (2016) Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6-7 linker. Biochem J 473:3545-3562
Wang, Yiqiang; Mitchell-Ryan, Shermaine; Raghavan, Sudhir et al. (2015) Novel 5-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and as potential antitumor agents. J Med Chem 58:1479-93
Wang, Lei; Wallace, Adrianne; Raghavan, Sudhir et al. (2015) 6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor ? and the Proton-Coupled Folate Transporter in Human Tumors. J Med Chem 58:6938-59

Showing the most recent 10 out of 32 publications