Abstract

This project is concerned with various aspects of the relation of the structure to the function of dihydrofolate reductase (DHFR). The proposed problems to be investigated include: (a) site- directed mutagenesis studies of human DHFR Using recombinant DNA techniques to evaluate the functional role of various NADPH and methotrexate (MTX) or dihydrofolate (FAH2) binding amino acid residues. These studies will involve construction of recombinant plasmids and high level DHFR expression in E. coli, purification and characterization of mutant forms of human DHFR by kinetic, equilibrium binding and x-ray crystallographic studies; (b) mapping of the immunogenic determinants of human DHFR using antibodies to peptides produced by proteolysis or chemical cleavage of intact human DHFR or produced by automated peptide synthesis. These sequence-specific antibodies will also be used to detect MTX-insensitive DHFRs from human acute myelogenous leukemic cells and other tumors; (c) to obtain sequence information on a MTX-insensitive DHFR from L5178Y cells; (d) evaluation of newly synthesized folate antagonists as inhibitors of DHFRs and thymidylate synthase and as cytotoxic agents; (e) molecular characterization of the one-carbon FAH4/MTX transport system in transport-competent and - defective L1210 cells. These experiments include photoaffinity labeling of transport proteins with an MTX analogue under various conditions, a determination of the relatedness of the membrane and cytoplasmic MTX binding components, possible expression of components in transport-defective cells and sequencing of a transport protein in L1210 cells which overproduce this protein.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA041461-04
Application #
3181954
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1985-05-01
Project End
1993-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
4
Fiscal Year
1988
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

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
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
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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