Opportunistic infections caused by Cryptosporidium parvum and Toxoplasma gondii represent life threatening diseases for immuno-compromised patients, children and the elderly. There are currently no effective treatments available for cryptosporidiosis and treatments for toxoplasmosis require the coadministration of sulfadioxine, a compound to which many patients have severe adverse reactions. The development of novel therapeutics that are highly potent and highly selective for the pathogen is of immediate importance. Crystal structures of the validated drug target, dihydrofolate reductase-thymidylate synthase (DHFR-TS), a bifunctional enzyme in protozoa, from C. parvum and T. gondii, provide essential evidence for structure-based drug design against these targets. In the first Aim of this proposal, we will design trimethoprim analogs to interact favorably with species-selective elements of the C. parvum DHFRTS structure. Species-selective elements will be determined by comparing crystal structures of pathogenic DHFR-TS and human DHFR. New inhibitors will be modeled into the structure of the enzyme, while accounting for ligand-induced conformational changes, and binding modes predicted. Crystal structures of trimethoprim analogs will guide future design in an iterative cycle. In the second Aim, we will improve the potency and selectivity of a promising T. gondii DHFR-TS inhibitor, using information from crystal structures of T. gondii DHFR-TS bound to lipophilic inhibitors. Designs for the improvement of T. gondii DHFR-TS inhibitors will take advantage of species-selective elements. In the third Aim, we will elucidate the structural basis of pyrimethamine resistance in DHFR and design potency and selectivity into a novel inhibitor of pyrimethamine-resistant T. gondii DHFR-TS. DHFR resistance to pyrimethamine and other antimicrobials is a threatening problem and novel therapeutics capable of inhibiting the resistant enzymes are desperately needed. We will solve a crystal structure of pyrimethamine-resistant DHFR-TS bound to a novel and exciting inhibitor that shows an inhibition constant of 350 nM against the resistant enzyme in preliminary studies. Using the structural information we will elucidate the structural basis of resistance and modify the novel inhibitor for greater potency and selectivity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067542-03
Application #
6891305
Study Section
AIDS and Related Research 8 (AARR)
Program Officer
Jones, Warren
Project Start
2003-05-01
Project End
2005-12-31
Budget Start
2005-05-01
Budget End
2005-12-31
Support Year
3
Fiscal Year
2005
Total Cost
$76,361
Indirect Cost
Name
Dartmouth College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
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Paulsen, Janet L; Viswanathan, Kishore; Wright, Dennis L et al. (2013) Structural analysis of the active sites of dihydrofolate reductase from two species of Candida uncovers ligand-induced conformational changes shared among species. Bioorg Med Chem Lett 23:1279-84
Anderson, Amy C (2012) Winning the arms race by improving drug discovery against mutating targets. ACS Chem Biol 7:278-88
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Anderson, Amy C (2012) Structure-based functional design of drugs: from target to lead compound. Methods Mol Biol 823:359-66
Algul, Oztekin; Paulsen, Janet L; Anderson, Amy C (2011) 2,4-Diamino-5-(2'-arylpropargyl)pyrimidine derivatives as new nonclassical antifolates for human dihydrofolate reductase inhibition. J Mol Graph Model 29:608-13
Anderson, Amy C; Pollastri, Michael P; Schiffer, Celia A et al. (2011) The challenge of developing robust drugs to overcome resistance. Drug Discov Today 16:755-61
Paulsen, Janet L; Bendel, Stephen D; Anderson, Amy C (2011) Crystal structures of Candida albicans dihydrofolate reductase bound to propargyl-linked antifolates reveal the flexibility of active site loop residues critical for ligand potency and selectivity. Chem Biol Drug Des 78:505-12

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