. This revised proposal focuses on mechanistic and structural studies on thymidylate synthase/dihydrofolate reductase (TS-DHFR) . These two enzymes are crucial for DNA synthesis and one-carbon transfers. In many protozoan parasties, these two catalytic activities are located on a single polypeptide chain to form the bifunctional TS- DHFR. In mammals, these enzymes are separate and monofunctional. A considerable amount of mechanistic information is available for the human monofunctional TS and DHFR, since each enzyme has been successfully targeted with the anticancer drugs, 5-fluorouracil and methotrexate, respectively. Earlier work as well as preliminary transient kinetic studies from the PI's laboratory indicate substantial mechanistic differences in the bifunctional parasitic enzyme and the monofunctional human enzymes. The ultimate goal of the proposed research is to take advantage of the differences between the bifunctional parasitic enzyme and the human enzymes to develop novel drugs for the treatment of parasitic diseases. To accomplish this goal, the following specific aims are proposed: 1) Elucidate the molecular mechanism involved in enzyme catalysis and substrate channeling for the bifunctional TS-DHFR from Toxoplasma gondii, 2) Use site-directed mutagenesis as a tool for mechanistic and structural studies with the Leishmania TS-DHFR and the Tosoplasma TS-DHFR, and 3) Use combinatorial library screening in conjunction with computer modeling to identify compounds that interfere with substrate channeling.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI044630-02
Application #
6170698
Study Section
Special Emphasis Panel (ZRG1-AARR-3 (01))
Program Officer
Laughon, Barbara E
Project Start
1999-04-15
Project End
2002-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
2
Fiscal Year
2000
Total Cost
$163,026
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Martucci, W Edward; Rodriguez, Johanna M; Vargo, Melissa A et al. (2013) Exploring novel strategies for AIDS protozoal pathogens: ?-helix mimetics targeting a key allosteric protein-protein interaction in C. hominis TS-DHFR. Medchemcomm 4:
Dasgupta, Tina; Chitnumsub, Penchit; Kamchonwongpaisan, Sumalee et al. (2009) Exploiting structural analysis, in silico screening, and serendipity to identify novel inhibitors of drug-resistant falciparum malaria. ACS Chem Biol 4:29-40
Vargo, Melissa A; Martucci, W Edward; Anderson, Karen S (2009) Disruption of the crossover helix impairs dihydrofolate reductase activity in the bifunctional enzyme TS-DHFR from Cryptosporidium hominis. Biochem J 417:757-64
Martucci, W Edward; Udier-Blagovic, Marina; Atreya, Chloe et al. (2009) Novel non-active site inhibitor of Cryptosporidium hominis TS-DHFR identified by a virtual screen. Bioorg Med Chem Lett 19:418-23
Martucci, W Edward; Vargo, Melissa A; Anderson, Karen S (2008) Explaining an unusually fast parasitic enzyme: folate tail-binding residues dictate substrate positioning and catalysis in Cryptosporidium hominis thymidylate synthase. Biochemistry 47:8902-11
Dasgupta, Tina; Anderson, Karen S (2008) Probing the role of parasite-specific, distant structural regions on communication and catalysis in the bifunctional thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum. Biochemistry 47:1336-45
Doan, Lanxuan T; Martucci, W Edward; Vargo, Melissa A et al. (2007) Nonconserved residues Ala287 and Ser290 of the Cryptosporidium hominis thymidylate synthase domain facilitate its rapid rate of catalysis. Biochemistry 46:8379-91
Massimine, Kristen M; McIntosh, Michael T; Doan, Lanxuan T et al. (2006) Eosin B as a novel antimalarial agent for drug-resistant Plasmodium falciparum. Antimicrob Agents Chemother 50:3132-41
Massimine, Kristen M; Doan, Lanxuan T; Atreya, Chloe A et al. (2005) Toxoplasma gondii is capable of exogenous folate transport. A likely expansion of the BT1 family of transmembrane proteins. Mol Biochem Parasitol 144:44-54
Atreya, Chloe E; Anderson, Karen S (2004) Kinetic characterization of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis: a paradigm shift for ts activity and channeling behavior. J Biol Chem 279:18314-22

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