REGULATION OF SQUALENE EPOXIDASE EXPRESSION AND THE ENZYME EVOLUTION

哺乳动物胆固醇生物合成后期酶抑制剂筛选方法的建立及应用将哺乳动物胆固醇生物合成后期酶cDNA通过表达载体导入粟酒裂殖酵母（Schizosophyomycespombe），构建了转化子。哺乳动物酶抑制剂的初步筛选方法已被设计为通过在存在对真菌酶具有特异性的抑制剂的情况下筛选生物和化学来源的靶哺乳动物酶的抑制剂来评价转化体的生长抑制作为指标。利用15种已知的抗真菌药物，我们建立了真菌材料对真菌生长抑制作用的标准。将表达大鼠角鲨烯环氧酶的转化体在特比萘芬存在下对真菌材料的生长抑制与已知的抗真菌药物进行比较。使用570份真菌渗出液和成分样品进行初步筛选。18个样品显示超过5%的生长抑制，其中9个样品显示超过20%的生长抑制。目前正在进一步分析9个样本，以确定它们是否含有哺乳动物角鲨烯环氧酶特异性抑制剂的cadidate化合物。结果证实了初步的研究结果与建立的药物对转化子的生长，表明该方法是有效的筛选靶哺乳动物酶抑制剂。我们现在正在将这种筛选方法应用于哺乳动物羊毛固醇合成酶抑制剂。

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.