During the period of support requested in this application, we plan to investigate further the mechanism of action of two enzymes, folypoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) and gamma-glutamyl hydrolase (GH, EC 3.4.22.17), involved in the biosynthesis and hydrolysis of folypoly-gamma-glutamates; i.e., the folate """"""""conjugates"""""""". Recently synthesized substrates and inhibitors, together with selected phosphapeptides and fluoroamino acid containing peptides to be synthesized during this period of support, will be used as mechanistic probes and/or inhibitors of FGPS and GH. In collaboration with Drs. John McGuire and John Galivan, we will use these newly synthesized molecules to study the role of intracellular poly-gamma-glutamate formation in folate one-carbon biochemistry and in antifolate chemotherapy.
The specific aims are as follows: 1. Using recombinant human FPGS, evaluate the kinetics of the reaction under pre-steady-state conditions in order to distinguish between a processive and non-processive kinetic mechanism. 2. Determine the mechanistic basis for the """"""""position-dependent"""""""" enhancement or termination of FPGS-catalyzed ligation using 3,3-difluoroglutamate-containing oligopeptides. 3. Using purified GH isolated rat hepatoma cells, determine the metal content and mechanism of GH-catalyzed hydrolysis of olgi-gamma-glutamyl peptides. Using newly synthesized fluoroglutamate-containing dipeptides, determine if tetrahedral intermediates accumulate during catalysis. 4. Complete the synthesis of phosphapeptide analogs of two similar tetrahedral intermediates postulated to form during the FPGS- and GH-catalyzed reaction. Evaluate these new compounds as FPGS and/or GH inhibitors and, if found to be effective in cell-free assays, investigate the action of these compounds as modulators of polyglutamate biosynthesis and/or polyglutamate hydrolysis in intact mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA028097-22
Application #
6124477
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Lees, Robert G
Project Start
1987-01-01
Project End
2001-11-30
Budget Start
1999-12-01
Budget End
2001-11-30
Support Year
22
Fiscal Year
2000
Total Cost
$216,780
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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
Majumdar, Debatosh; Alexander, Matthew D; Coward, James K (2009) Synthesis of isopeptide epoxide peptidomimetics. J Org Chem 74:617-27
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
Tomsho, John W; Moran, Richard G; Coward, James K (2008) Concentration-dependent processivity of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase. Biochemistry 47:9040-50
Alexander, Jessica P; Ryan, Thomas J; Ballou, David P et al. (2008) Gamma-glutamyl hydrolase: kinetic characterization of isopeptide hydrolysis using fluorogenic substrates. Biochemistry 47:1228-39
Yang, Yonghong; Coward, James K (2007) Synthesis of p-aminophenyl aryl H-phosphinic acids and esters via cross-coupling reactions: elaboration to phosphinic acid pseudopeptide analogues of pteroyl glutamic acid and related antifolates. J Org Chem 72:5748-58
Bartley, David M; Coward, James K (2006) Regioselective synthesis of alpha-methyl 2-methyleneglutarate via a novel lactonization-elimination rearrangement. J Org Chem 71:372-4
Feng, Yan; Coward, James K (2006) Prodrug forms of N-[(4-deoxy-4-amino-10-methyl)pteroyl]glutamate-gamma-[psiP(O)(OH)]-glutarate, a potent inhibitor of folylpoly-gamma-glutamate synthetase: synthesis and hydrolytic stability. J Med Chem 49:770-88
Bartley, David M; Coward, James K (2005) A stereoselective synthesis of phosphinic acid phosphapeptides corresponding to glutamyl-gamma-glutamate and incorporation into potent inhibitors of folylpoly-gamma-glutamyl synthetase. J Org Chem 70:6757-74
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

Showing the most recent 10 out of 39 publications