REGULATION OF SQUALENE EPOXIDASE EXPRESSION AND THE ENZYME EVOLUTION

哺乳动物脱醇酯生物合成后期酶抑制剂筛选方法的建立及应用。利用表达载体将胆固醇生物合成后期哺乳动物酶基因导入pombe裂殖酵母中，构建转化菌。哺乳动物酶抑制剂的初步筛选方法是在真菌酶特异性抑制剂存在的情况下，通过筛选针对目标哺乳动物酶的生物和化学来源的抑制剂来评价转化子的生长抑制作用。利用已知的15种抗真菌药物，建立了真菌材料对转化子生长抑制作用的标准。比较了在特比奈芬存在下表达大鼠角鲨烯环氧酶的转化子对真菌材料的生长抑制作用，并与已知的抗真菌药物进行了比较。使用570份真菌分泌物和内容物样本进行了初步筛选。有18个样品的生长抑制率超过5%，其中9个样品的生长抑制率超过20%。对9个样品的进一步分析，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卡迪酸盐化合物，正在进行中。结果与已建立的抑制转化子生长的药物的初步发现相一致，表明该方法是筛选靶向哺乳动物酶抑制剂的有效方法。我们现在正在将这种筛选方法应用于哺乳动物的羊毛甾醇合成酶抑制剂。

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.