Despite advances in the therapy of opportunistic infections complicating AIDS, they remain a leading cause of morbidity and mortality. Testing of potential therapeutic agents is confounded by the difficulties in the cultivation of many of these pathogens and the unreliability of animal models in the testing of new agents. In order to circumvent these problems, we plan to clone enzymes which are targets for known classes of antibiotics, expressing these proteins in large quantities in heterologous systems (e.g. E. Coli and yeast), and characterizing the enzymologic parameters of the targets. The expressed proteins and their sequences will provide the basis for the three-dimensional structure determination and molecular modeling of the targets. The structural information will provide the basis for rational drug design and identification. Classes of compounds which have the structural components necessary to be new inhibitors will be tested for their efficacy in the in vitro inhibition of the targets. The organisms that have been chosen for study are Pneumocystis carinii, Cryptococcus neoformans, and Toxoplasma gondii. These organisms cause the bulk of life-threatening infections in patients with AIDS. Our group has experience with Pneumocystis carinii and Cryptococcus neoformans. Many of targets selected for this study have already been isolated and are ready for structural determination and molecular modeling. The other targets will rapidly be isolated given our experience in this field. The targets chosen are crucial enzymes in the folate and thymidylate biosynthetic pathways. Since agents which inhibit this pathway are already in clinical use for Pneumocystis carinii and Toxoplasma gondii, we know that these pathways are appropriate targets for chemotherapeutic intervention. The targets that have been selected are dihydrofolate reductase (DHFR; the target of trimethoprim), dihydropteroate synthetase (DHPS; the sulfonamide target), and thymidylate synthase (TS; the likely target of 5-fluorocytosine). Abundant kinetic structural information is available for two these enzymes (TS and DHFR), much of which has been determined by members of this group. Together the group provides a continuous system of drug development from cloning to expression to structural determination to molecular modeling. The group has demonstrated previous productive interactions. The drug targets in opportunistic pathogens will not be an entirely new venture, but an extension of on-going projects in these fields.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project--Cooperative Agreements (U01)
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Special Emphasis Panel (SRC (76))
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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