Pneumocystis carinii (pc) and toxoplasma gondii (tg) are major causes of opportunistic infection and mortality in immuno-suppressed patients, particularly those with AIDS. Antifolate drugs, usually consisting of a sulfonamide in combination with trimethoprim, an inhibitor of the enzyme dihydrofolate reductase (DHFR), have been the most effective drugs in clinical use to date. However, their use has been limited by problems of toxicity and resistance. The major goal of this project is determination of the three-dimensional crystal structures of dihydrofolate reductase (DHFR) from fungal (Pneuntocystis carinii, pc), protozoal (toxoplasma gondii, tg), and mammalian (rat liver, mouse and human) sources as complexes with antifolates that show selectivity and specificity for the pc or tg enzyme.
The aim i s to compare structural details of antifolate-enzyme interactions in order to design more selective and potent agents as effective treatment for opportunistic infections that cause pneumonia, a major cause of mortality among AIDS patients. As part of this protocol we plan to exploit these structural data for the design and synthesis of new selective antifolates.
Six specific aims are proposed to test the hypothesis that efficacy of antifolate use in combating opportunistic infections from Pneumocystis carinii or toxoplasma gondii organisms is a result of specific enzyme-inhibitor interactions. We will analyze: (1) the first human-derived pcDHFR inhibitor complexes to examine the effects on ligand binding that result from the significant sequence changes between species, (2) the first rat liver DHFR complexes to validate correlations of inhibition with human DHFR, (3) the first tgDHFR complexes, (4) mouse DHFR for comparison to human DHFR, (5) DHFR complexes with novel antifolates, and (6) homology modeling data to understand features that control antifolate selectivity. The knowledge gained by these studies will be utilized in the design and synthesis of new antifolates. Appropriate targets have been selected for study with various DHFR enzymes. Analysis of these data will provide molecular level details of inhibitor-enzyme geometry, hydrogen bonding, conformation and the role of specific active site residues, especially the contribution by the substitution at positions 31 and 64 between human and pcDHFR in modulating pc selectivity. Since selectivity apparently requires only small changes in enzyme-inhibitor geometry, we propose to look for subtle differences in a series of carefully determined crystal structures of DHFR complexes with antifolates that show selectivity to a particular species of DHFR. Thus knowledge of the three dimensional structure of enzyme-inhibitor complexes are required to define the mechanism of DHFR selectivity and action. Dr. Sherry Queener, Indiana University, will measure inhibitory activity of selected antifolates.
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