The long-term goal of this program is to improve treatment of human cancer by exploiting aspects of folyl- (or antifolyl-) polyglutamate synthesis. Folylpolyglutamates are essential for cell growth, while polyglutamates of classical antifolates are implicated in their cytotoxic action and resistance and may have a role in selectivity. Detailed understanding of the synthesis and function of (anti)folylpolyglutamates may thus allow design of new agents or strategies to exploit this critical process. This long-term goal will be addressed through three specific aims: 1. Exploration of folylpolyglutamate synthetase (FPGS), the enzyme responsible for polyglutamate synthesis, as a drug target. Mutational inactivation of FPGS is lethal, thus FPGS is a potential cancer chemotherapy target. Inhibitor design is based on enzyme mechanism and structure-activity data of the applicant using recombinant human FPGS. After synthesis by expert folate chemists in collaborating laboratories, antifolates will be studied in the applicant's laboratory. Of primary interest will be the optimization of mechanism-based phosphorous-containing inhibitors found during the last grant period. Analogs that inhibit both purified FPGS and polyglutamylation in intact cells (i.e., are transported) will be studied to clarify their specificity and cellular effects. Promising drugs will undergo initial toxicity and therapeutic testing in vivo to define whether FPGS is a useful therapeutic target. 2. Determine the functional significance of the apparent physico-chemical difference between mitochondrial (mFPGS) and cytosolic FPGS (cFPGS). Although encoded by one gene, mFPGS and cFPGS differ in SDS-PAGE mobility, which may reflect a functional difference, particularly as related to antifolate resistance (Aim 3). The applicant will explore the hypothesis that this physico-chemical variance, discovered under this grant, is reflected in kinetic, substrate specificity, and/or regulation alterations. Also, since the structural difference may be reflective of function, he will examine hypotheses that it is caused by mitochondrial leader sequence truncation or by post-translational modification. 3. Elucidate the role of cFPGS and mFPGS in MTX resistance. Clinically, MTX is often given as a bolus or short (24 hr) infusion at high dose. The applicant has shown that this regimen selects for resistance via FPGS deficiency. FPGS is expressed in both cytosol and mitochondria. MTX cannot enter mitochondria, while reduced folate monoglutamates can. It is known (Shane et al.) that expression of mFPGS alone can establish folylpolyglutamate pools in both cytosol and mitochondria and allow normal cell growth. Thus, he hypothesizes that a differential decrease in cFPGS, rather than a parallel decrease in both isoforms, can contribute to resistance to pulse MTX exposure. This may explain why high-level resistance to pulse MTX can occur in the absence of a large decrease in total FPGS activity or a rise in glutamyl hydrolase activity.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Forry, Suzanne L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Roswell Park Cancer Institute Corp
United States
Zip Code
McGuire, John J; Bartley, David M; Tomsho, John W et al. (2009) Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate. Arch Biochem Biophys 488:140-5
Coward, James K; McGuire, John J (2008) Mechanism-based inhibitors of folylpoly-gamma-glutamate synthetase and gamma-glutamyl hydrolase: control of folylpoly-gamma-glutamate homeostasis as a drug target. Vitam Horm 79:347-73
Gangjee, Aleem; Zeng, Yibin; Talreja, Tina et al. (2007) Design and synthesis of classical and nonclassical 6-arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as antifolates. J Med Chem 50:3046-53
McGuire, John J; Haile, William H; Yeh, Chen-Chen (2006) 5-amino-4-imidazolecarboxamide riboside potentiates both transport of reduced folates and antifolates by the human reduced folate carrier and their subsequent metabolism. Cancer Res 66:3836-44
Gangjee, Aleem; Yang, Jie; McGuire, John J et al. (2006) Synthesis and evaluation of a classical 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine as antifolates. Bioorg Med Chem 14:8590-8
Liang, Ping; Nair, Jayakumar R; Song, Lei et al. (2005) Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene. BMC Genomics 6:125
Gangjee, Aleem; Lin, Xin; Kisliuk, Roy L et al. (2005) Synthesis of N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid as dual inhibitors of dihydrofol J Med Chem 48:7215-22
Tomsho, John W; McGuire, John J; Coward, James K (2005) Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-gamma-glutamates: kinetics of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase. Org Biomol Chem 3:3388-98
Nair, Jayakumar R; McGuire, John J (2005) Submitochondrial localization of the mitochondrial isoform of folylpolyglutamate synthetase in CCRF-CEM human T-lymphoblastic leukemia cells. Biochim Biophys Acta 1746:38-44
Gangjee, Aleem; Jain, Hiteshkumar D; McGuire, John J et al. (2004) Benzoyl ring halogenated classical 2-amino-6-methyl-3,4-dihydro-4-oxo-5-substituted thiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine antifolates as inhibitors of thymidylate synthase and as antitumor agents. J Med Chem 47:6730-9

Showing the most recent 10 out of 50 publications