ELECTRON POOR AROMATICS AS POTENTIAL CYCLASE INHIBITORS

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Oxidosqualene cyclase enzymes catalyze remarkably complex and diverse cyclization/rearrangement processes during the course of sterol and triterpene biosynthesis. These enzymes are of interest from the point of view of mechanistic enzymology and as potential targets for antifungal and anticholesterol agents. Previous work from our laboratory provided the first examples of DNA and predicted amino acid sequences for oxidosqualene cyclase enzymes, in the form of cloned cyclase genes from the yeasts Candida albicans and Saccharomyces cerevisiae. These and other (oxido)squalene cyclase sequences are rich in tryptophan and tyrosine residues, which bear electron-rich aromatic sidechains. This led us to formulate an aromatic hypothesis for active site structure, wherein the indole and phenol sidechains from tryptophan and tyrosine, respectively, serve to direct the formation of and stabilize specific cationic transition states and high energy intermediates along the pathway for cyclization/rearrangement. We are probing the aromatic hypothesis by directed mutagenesis experiments, and also by the development of synthetic probes for the presence of electron-rich aromatics in the cyclizing active site. We have shown that electron-poor aromatic pyridinium ions, which mimic a partially cyclized state, are potent inhibitors of fungal cyclase enzymes and exhibit promising antifungal activity. We plan to expand the range of compounds for synthesis and testing. Mass spectrometry, especially FAB-MS and related techniques, is an important tool for characterization of these nonvolatile synthetic materials.