There is a long-standing interest to characterize the molecular events that mediate the production and processing of ergosterol biosynthesis as a strategy for selective chemical targeting of antifungals for therapeutics discovery and development. The focus of this proposal will be on ergosterol synthesis and the family of sterol methyl transferase (SMT) enzymes from Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Pneumocystis carinii. Four projects are outlined: 1) Establish the de novo pathway to ergosterol in the test fungi using 13C-labeled intermediates. 2) Evaluate the growth, sterol composition and SMT activity of the test fungi treated with a set of drugs synthesized in our laboratory to serve as single-action (inhibit SMT activity) or dual-action (inhibit SMT and 14a-demethylase activities) inhibitors of ergosterol synthesis. The kinetics of SMT activity in response to substrate analogs will be explored as a means to fashion novel inhibitors. 3) The sterol composition of P. carinii is plant-like in having 24-methyl and 24-ethyl sterols and these """"""""phytosterols"""""""" are regarded as signature lipids for diagnostic purposes. We recently cloned the P. carinii SMT by a PCR-based homology strategy from ESTs provided by a cooperator. We will determine whether the properties of the P. carinfi SMT are similar to the properties of the C. albicans SMT. Purification of the C. albicans and P. carinii SMTs will be achieved after subcloning the relevant cDNA into a Escherichia coil expression system. Chemical affinity and photoaffinity labeling will be used to identify the sterol and AdoMet-binding subdomains, respectively, in the active center. Site-directed mutagenesis followed by activity assay will be employed to probe the functional importance of select residues involved with catalysis. 4) The three-dimensional structure of a SMT will be defined with the aid of a cooperator. The results from these studies will facilitate the design of therapeutic strategies aimed to provide species-specific discrimination of inhibition in the ergosterol biosynthetic pathway.
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