Abstract

This project is concerned with the relation of the structure to the function of dihydrofolate reductase (dihydrofolate+ NADPH + H+ - tetrahydrofolate + NADP+). Using a number of techniques of protein chemistry, the problems to be investigated include: (a) obtaining protein sequence information on a methotrexate (MTX)-insensitive enzyme from MTX-resistant L5178Y cells and from Walker 256 carcinosarcoma cells primarily by HPLC-mapping of derived peptides and amino acid analysis; (b) chemical modification of specific amino acid residues of the protein and measurements of the effect of these modifications on the substrate, cofactor and inhibitor binding and on enzymic activity; (c) equilibrium binding studies of substrate, cofactor and inhibitors using reductases from human, L5178Y and Walker 256 cells and employing fluorescence, circular dichroism and equilibrium dialysis techniques; (d) evaluation of MTX analogues as affinity and photoaffinity labels of dihydrofolate reductases; (e) examination of selected, substituted quinazolines, triazines and pteridines as enzyme inhibitors and a determination of their transport characteristics in MTX-sensitive and -resistant cells.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA041461-03
Application #
3181956
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1985-05-01
Project End
1988-04-30
Budget Start
1987-05-01
Budget End
1988-04-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
University of Toledo
Department
Type
Schools of Medicine
DUNS #
807418939
City
Toledo
State
OH
Country
United States
Zip Code
43614

  Publications

Cody, V; Wojtczak, A; Kalman, T I et al. (1993) Conformational analysis of human dihydrofolate reductase inhibitor complexes: crystal structure determination of wild type and F31 mutant binary and ternary inhibitor complexes. Adv Exp Med Biol 338:481-6
Blakley, R L; Appleman, J R; Freisheim, J H et al. (1993) 13C and 15N nuclear magnetic resonance evidence that the active site carboxyl group of dihydrofolate reductase is not involved in the relay of a proton to substrate. Arch Biochem Biophys 306:501-9
Rosowsky, A; Mota, C E; Wright, J E et al. (1993) 2,4-Diaminothieno[2,3-d]pyrimidine analogues of trimetrexate and piritrexim as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. J Med Chem 36:3103-12
Wang, Y; Dias, J A; Nimec, Z et al. (1993) The properties and function of gamma-glutamyl hydrolase and poly-gamma-glutamate. Adv Enzyme Regul 33:207-18
Wang, X; Shen, F; Freisheim, J H et al. (1992) Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates. Biochem Pharmacol 44:1898-901
Cody, V; Luft, J R; Ciszak, E et al. (1992) Crystal structure determination at 2.3 A of recombinant human dihydrofolate reductase ternary complex with NADPH and methotrexate-gamma-tetrazole. Anticancer Drug Des 7:483-91
Freisheim, J H; Ratnam, M; McAlinden, T P et al. (1992) Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines. Adv Enzyme Regul 32:17-31
Rosowsky, A; Forsch, R A; Reich, V E et al. (1992) Side chain modified 5-deazafolate and 5-deazatetrahydrofolate analogues as mammalian folylpolyglutamate synthetase and glycinamide ribonucleotide formyltransferase inhibitors: synthesis and in vitro biological evaluation. J Med Chem 35:1578-88
Appleman, J R; Tsay, J T; Freisheim, J H et al. (1992) Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency. Biochemistry 31:3709-15
Bullerjahn, A M; Freisheim, J H (1992) Site-directed deletion mutants of a carboxyl-terminal region of human dihydrofolate reductase. J Biol Chem 267:864-70

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