This proposal involves a multi-disciplinary research effort directed at studies of a) enzymes in folate metabolism in protozoa and b) mechanisms of drug resistance in these organisms. The organisms to be studied are Leishmania and Plasmodium, casual agents of diseases (Leishmaniasis and malaria, respectively) of worldwide importance. Our objectives are aimed at obtaining fundamental biochemical information, with the belief that such knowledge will provide insight into how to exploit what is found and assist in controlling these organisms. We will continue studies on the structure, function and inhibition of the bifunctional thymidylate synthase (TS)-dihydrofolate reductase (DHFR) which is uniquely found in protozoa. We intend to express the enzyme from Leishmania, and study its higher order structure in detail; studies on computer assisted structure modeling and drug design will be initiated. We will continue studies on the amplified DNA found in anti-folate resistant Leishmania. We will further characterize the structure of these DNAs and delineate differences observed in different cell lines. In Plasmodium falciparum, we will isolate the TS-DHFR gene, characterize it and express it in E. coli or yeast; we will study molecular aspects of drug resistance towards anti-folates and initiate other studies of enzymes in folate metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019358-08
Application #
3128739
Study Section
Biochemistry Study Section (BIO)
Project Start
1982-09-01
Project End
1992-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
8
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Pharmacy
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Trujillo, M; Duncan, R; Santi, D V (1997) Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme. Protein Eng 10:567-73
Sirawaraporn, W; Sathitkul, T; Sirawaraporn, R et al. (1997) Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase. Proc Natl Acad Sci U S A 94:1124-9
Yu, P L; Zhao, J; Yu, M et al. (1996) Functional expression of the dihydrofolate reductase domain of Leishmania major dihydrofolate reductase-thymidylate synthase bifunctional protein. Protein Expr Purif 8:23-7
Prapunwattana, P; Sirawaraporn, W; Yuthavong, Y et al. (1996) Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Mol Biochem Parasitol 83:93-106
Reche, P; Arrebola, R; Santi, D V et al. (1996) Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain. Mol Biochem Parasitol 76:175-85
Carreras, C W; Santi, D V (1995) The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem 64:721-62
Reche, P; Arrebola, R; Olmo, A et al. (1994) Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi. Mol Biochem Parasitol 65:247-58
Arrebola, R; Olmo, A; Reche, P et al. (1994) Isolation and characterization of a mutant dihydrofolate reductase-thymidylate synthase from methotrexate-resistant Leishmania cells. J Biol Chem 269:10590-6
Sirawaraporn, W; Prapunwattana, P; Sirawaraporn, R et al. (1993) The dihydrofolate reductase domain of Plasmodium falciparum thymidylate synthase-dihydrofolate reductase. Gene synthesis, expression, and anti-folate-resistant mutants. J Biol Chem 268:21637-44
Ivanetich, K M; Santi, D V (1992) 5,6-dihydropyrimidine adducts in the reactions and interactions of pyrimidines with proteins. Prog Nucleic Acid Res Mol Biol 42:127-56

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