REGULATION OF SQUALENE EPOXIDASE EXPRESSION AND THE ENZYME EVOLUTION

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Development of inhibitors screening method for the mamalian enzymes on the late stages of Deolesterol biosynthesis and its application.The transformants of Schizosaccharomyces pombe was constructed in which the mammalian enzyme cDNA of the late stages of cholesterol biosynthesis was introduced into it with expression vector. The primary screening method of mammalian enzyme inhibitors has been designed to evaluate growth inhibition of the transformants as an indicator by screening inhibitors of biological and chemical sources for the target mammalian enzyme in the presence of the inhibitor, specific for fungal enzyme. Using fifteen already-known antifungal drugs, we have established the criterion of inhibitory effect of fungal materials on the transformant growth. The growth inhibition of transformants expressing rat squalene epoxidase in the presence of terbinafine to fungal materials was compared with the alreadyknown antifungal drugs. Primary screening was carried out using 570 samples of fungal exsudates and constutuents. Eighteen samples showed over 5% growth inhibition in which nine samples showed over 20% growth inhibition. The further analysis of nine samples to determine whether they contained the cadidate compounds of inhibitors specific for mamalian squalene epoxidase is in progress. The results confirm the preliminary findings with the established drugs against transformants growth, indicating the method is efficient to screen target mammalian enzyme inhibitors. We are now applying this screening method to mammalian lanosterol synthase inhibitors.