.) CR, a coccidian parasite, causes diarrheal disease in 15 percent of acquired immunodeficiency syndrome (AIDS) patients in the United States, and up to 50 percent of AIDS in developing nations. Symptoms may include profuse watery diarrhea with dehydration and malnutrition; extraintestinal complications include involvement of the biliary tract, pancreas, and lungs. In spite of the severity and prevalence of this infection in AIDS, no effective therapy has been identified. DHFR catalyzes an essential enzymatic reaction in the folate biosynthetic pathway in all organisms. Through evolution this enzyme has diverged, it has maintained essential catalytic activity, but its underlying three-dimensional structure has changed. This principle has been used to identify inhibitors of the enzyme which differentially bind to DHFRs of mammalian hosts and their bacterial or protozoan pathogens and appear to be effective chemotherapeutic agents. However, DHFR also varies greatly among protozoa and the available inhibitors (pyrimethamine, trimethoprim, proguanil) bind poorly to some protozoan DHFRs (Leishmania, Trypanosoma, and Eimeria) and very well to others (Plasmodium) with corresponding variation in effectiveness as therapeutic agents. Molecular cloning and expression of catalytically-active Leishmania DHFR recently lead to assessment of the inhibitory potential of 5-benzyl-2,4-diaminopyrimidines and the identification of a vastly improved inhibitor of Leishmania DHFR which is the prototype for the development of new chemotherapeutics for leishmaniasis. The marked differences in sensitivity of DHFR among protozoa suggests that the CR DHFR, like the Leishmania enzyme, may be relatively insensitive to classical DHFR inhibitors. It is proposed to use a similar approach to develop new therapy for cryptosporidiosis. During the two year course of this project it is planned to: (1) Identify, isolate and sequence genomic clones encoding CR DHFR, (2) Engineer the DHFR gene for expression of catalytically-active recombinant enzyme in an heterologous expression system and, (3) Determine the kinetic constants of purified recombinant DHFR and quantitatively assay its inhibition by pyrimethamine, trimethoprim and proguanil, methotrexate, tirmetrexate, piritrexim, and a collection of 5-(substituted)-benzyl-2,4-diaminopyrimidines. This information will help focus future laboratory and clinical investigations among anti-cryptosporidial agents; if the Cryptosporidium enzyme is exquisitely sensitive to clinically common DHFR-targeted drugs (pyrimethamine, trimethoprim and proguanil) further research on drug delivery or parasite metabolism would be suggested, whereas if the enzyme's inhibition profile is similar to that of the host enzyme, the testing of clinically novel DHFR inhibitors would be more appropriate and may, following the Leishmania paradigm, lead to the identification of effective anti-cryptosporidial therapy.
