Protists cause numerous human diseases, including malaria, African sleeping sickness, Chagas' disease, leishmaniasis, giardiasis, and amebic meningitis. Treatments for each of these is inadequate, as few pharmaceutically exploitable metabolic differences have been found between humans and protists. Sterol biosynthesis is a promising but underexplored area for finding vital protist enzymes that lack human homologs, and the primary goal of this proposal is identifying novel drug targets in protist sterol metabolism. Animals and fungi synthesize the sterol ring system in one step using lanosterol synthase, which cyclizes oxidosqualene to lanosterol. The overwhelming majority of protists in which sterol biosynthesis has been examined synthesize that ring system in two steps: cycloartenol synthase cyclizes oxidosqualene to cycloartenol, which is then converted to lanosterol by cyclopropyl isomerase. These organisms should be susceptible to specific inhibitors of either cycloartenol synthase or cyclopropyl isomerase. Presented in this proposal are experiments to characterize these enzymes from several protists. A series of compounds designed to specifically inhibit cycloartenol synthase is described. Recombinant organisms that express the protist cycloartenol synthases and cyclopropyl isomerases will be constructed to provide enzymes with which to test the drug candidates. Metabolic research in pathogenic protists is hindered by difficulties in obtaining sufficient biomass for classical experiments with radiolabeled tracers. The approach described here uses molecular biological techniques to clone and express protist genes responsible for forming the sterol ring and to determine which pathway various protists use. This laboratory has constructed several metabolically engineered yeast strains in which the normal sterol pathway (which goes through lanosterol) is supplemented with components of the plant sterol biosynthetic pathway (which goes through cycloartenol). These strains can utilize either lanosterol or cycloartenol as sterol precursors, and will serve as hosts to clone cycloartenol synthase, cyclopropyl isomerase, or lanosterol synthase by genetic complementation.
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