# Molecular Regulation of Folate and Antifolate Transport

> **NIH NIH R01** · WAYNE STATE UNIVERSITY · 2020 · $292,600

## Abstract

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...

## Key facts

- **NIH application ID:** 10071673
- **Project number:** 2R01CA053535-28
- **Recipient organization:** WAYNE STATE UNIVERSITY
- **Principal Investigator:** Zhanjun Hou
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $292,600
- **Award type:** 2
- **Project period:** 1993-02-12 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10071673

## Citation

> US National Institutes of Health, RePORTER application 10071673, Molecular Regulation of Folate and Antifolate Transport (2R01CA053535-28). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10071673. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*