Opportunistic infections are the primary cause of suffering and death in individuals with AIDS. Many of these infections are produced by parasites which rarely affect individuals who are not immunocompromised. Unfortunately, successful combination therapies against the HIV-1 virus still leave most patients susceptible to opportunistic parasitic infections. The drugs which are currently available for the treatment of these parasitic infections suffer from a lack of selectivity resulting in host toxicity and untoward side effects. Thus there is a need for novel therapeutic strategies which may be more selective and less toxic. This proposal outlines mechanistic and structural studies on a unique bifunctional enzyme which will serve as basis for the design of novel antiparasitic drugs. Two enzymes crucial for DNA synthesis and one-carbon transfers are thymidylate synthase and dihydrofolate reductase. In many protozoan parasites, these two catalytic activities are located on a single polypeptide chain to form a bifunctional thymidylate synthase (TS)/dihydrofolate reductase (DHFR) enzyme. In mammalian species, the thymidylate synthase and dihydrofolate activities occur as separate catalytic activities on mono functional enzymes. A considerable amount of mechanistic information is available for the human monofunctional thymidylate synthase and dihydrofolate reductase 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 Pl's lab indicate substantial mechanistic differences in the bifunctional parasitic and mono functional human enzymes. The three dimensional structure of the bifunctional TS-DHFR enzyme is available for computer modeling studies to identify inhibitors through docking programs. The central theme of this proposal is that an in-depth kinetic and structural evaluation of the bifunctional TS-enzyme at a molecular level will provide a crucial mechanistic understanding that can be exploited as a novel therapeutic approach. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI044630-04A1
Application #
6748300
Study Section
Special Emphasis Panel (ZRG1-AARR (03))
Program Officer
Coyne, Philip Edward
Project Start
1999-04-15
Project End
2007-12-31
Budget Start
2004-01-15
Budget End
2004-12-31
Support Year
4
Fiscal Year
2004
Total Cost
$307,950
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
043207562
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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