Folates are essential for life and folate deficiency contributes to a range of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate and, more recently, raltitrexed and pemetrexed, continue to occupy a unique and important niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. This renewal application for a grant currently in its 19th year proposes to better understand the biology and therapeutic potential of the human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT), two of the major membrane transport systems for folates and antifolates in human cells, tissues and tumors which are widely expressed, yet are functionally and anatomically distinct. Our structure-function studies of hRFC have provided substantial insights into its membrane topology, N-glycosylation, important domains and amino acids, and three-dimensional packing associations. Most recently, we began to characterize hRFC quaternary structure and unambiguously identified hRFC homo-oligomers composed of monomeric hRFC molecules, and obtained compelling evidence for a """"""""negative dominance"""""""" of wild type hRFC by inactive hRFC mutants, involving downstream effects on intracellular trafficking and/or functional """"""""coupling"""""""" between hRFC monomers. Following identification of the low pH folate transporter, hPCFT, in 2006, we began a series of studies designed to directly assess the possible therapeutic importance of this transporter vis ` vis hRFC for cancer. Key reagents were generated including stably transfected cell lines (including Tet-regulated models), a functional """"""""cysteine-less"""""""" hPCFT construct, antibodies to the hPCFT protein, and, perhaps most exciting, the first generation of specific and potent hPCFT cytotoxic substrates that are not transported by hRFC.
Aim 1 of our proposed study will characterize the structural, functional, and regulatory features of hRFC homo- oligomers. A wide range of biochemical, cellular, and molecular approaches will be used to identify the minimum functional unit and characterize the structure of oligomeric hRFC, and to explore the implications of hRFC oligomers to transport mechanism, antifolate resistance, and biochemical modulation.
Aim 2 will establish the therapeutic potential, along with the structural and functional features of hPCFT. The focus is overtly on possibilities for therapeutic targeting tumors with cytotoxic antifolate drugs that are selective for hPCFT (and folate receptors) but are not transported by hRFC. Proposed studies include assays of hPCFT and hRFC tumor expression and activity profiles, functional stoichiometries for hRFC and hPCFT, and the therapeutic impact of concomitant tumor expression of these transport systems, as well as folate receptors. Our proposed studies are strongly supported by our preliminary and published results, and are distinctive for their novelty and likelihood of providing critical new insights into mechanism and regulation of these physiologically and pharmacologically important membrane transporters.

Public Health Relevance

Folates are essential for life and folate deficiency contributes to a range of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate and, more recently, raltitrexed and pemetrexed, continue to occupy a unique and important niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. The application proposes to better understand the biology and therapeutic potential of the human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT), two of the major membrane transport systems for folates and antifolates in human cells, tissues and tumors which are widely expressed, yet are functionally and anatomically distinct. Our proposed studies provide an important prelude for the design of new therapeutics for cancer including solid tumors and approaches for therapeutically altering transporter levels and function in the context of dietary folate supplementation and antifolate therapy of cancer and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA053535-22
Application #
8444582
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Forry, Suzanne L
Project Start
1993-02-12
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2015-02-28
Support Year
22
Fiscal Year
2013
Total Cost
$243,541
Indirect Cost
$83,317
Name
Wayne State University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
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 α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthes 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
Wilson, Mike R; Kugel, Sita; Huang, Jenny et al. (2015) Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6. Biochem J 469:33-44
Wang, Lei; Wallace, Adrianne; Raghavan, Sudhir et al. (2015) 6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors. J Med Chem 58:6938-59
Hou, Zhanjun; Matherly, Larry H (2014) Biology of the major facilitative folate transporters SLC19A1 and SLC46A1. Curr Top Membr 73:175-204
Wilson, Mike R; Hou, Zhanjun; Matherly, Larry H (2014) Substituted cysteine accessibility reveals a novel transmembrane 2-3 reentrant loop and functional role for transmembrane domain 2 in the human proton-coupled folate transporter. J Biol Chem 289:25287-95
Wang, Yiqiang; Cherian, Christina; Orr, Steven et al. (2013) Tumor-targeting with novel non-benzoyl 6-substituted straight chain pyrrolo[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis. J Med Chem 56:8684-95
Cherian, Christina; Kugel Desmoulin, Sita; Wang, Lei et al. (2013) Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter. Cancer Chemother Pharmacol 71:999-1011

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