Favre B, Ryder N S
Department of General Dermatology, SANDOZ Research Institute, Vienna, Austria.
Antimicrob Agents Chemother. 1996 Feb;40(2):443-7. doi: 10.1128/AAC.40.2.443.
Squalene epoxidase (SE) is the primary target of the allylamine antimycotic agents terbinafine and naftifine and also of the thiocarbamates. Although all of these drugs are employed primarily in dermatological therapy, SE from dermatophyte fungi has not been previously investigated. We report here the biochemical characterization of SE activity from Trichophyton rubrum and the effects of terbinafine and other inhibitors. Microsomal SE activity from T. rubrum was not dependent on soluble cytoplasmic factors but had an absolute requirement for NADPH or NADH and was stimulated by flavin adenine dinucleotide. Kinetic analyses revealed that under optimal conditions the Km for squalene was 13 microM and its Vmax was 0.71 nmol/h/mg of protein. Terbinafine was the most potent inhibitor tested, with a 50% inhibitory concentration (IC50) of 15.8 nM. This inhibition was noncompetitive with regard to the substrate squalene. A structure-activity relationship study with some analogs of terbinafine indicated that the tertiary amino structure of terbinafine was crucial for its high potency, as well as the tert-alkyl side chain. Naftifine had a lower potency (IC50, 114.6 nM) than terbinafine. Inhibition was also demonstrated by the thiocarbamates tolciclate (IC50, 28.0 nM) and tolnaftate (IC50, 51.5 nM). Interestingly, the morpholine amorolfine also displayed a weak but significant effect (IC50, 30 microM). T. rubrum SE was only slightly more sensitive (approximately twofold) to terbinafine inhibition than was the Candida albicans enzyme. Therefore, this difference cannot fully explain the much higher susceptibility (> or = 100-fold) of dermatophytes than of yeasts to this drug. The sensitivity to terbinafine of ergosterol biosynthesis in whole cells of T. rubrum (IC50, 1.5 nM) is 10-fold higher than that of SE activity, suggesting that the drug accumulates in the fungus.
角鲨烯环氧酶（SE）是烯丙胺类抗真菌药特比萘芬和萘替芬以及硫代氨基甲酸盐类药物的主要作用靶点。尽管所有这些药物主要用于皮肤病治疗，但此前尚未对角质形成层真菌中的SE进行过研究。我们在此报告红色毛癣菌SE活性的生化特性以及特比萘芬和其他抑制剂的作用。红色毛癣菌的微粒体SE活性不依赖于可溶性细胞质因子，但绝对需要NADPH或NADH，并受到黄素腺嘌呤二核苷酸的刺激。动力学分析表明，在最佳条件下，角鲨烯的Km为13μM，其Vmax为0.71 nmol/h/mg蛋白质。特比萘芬是所测试的最有效抑制剂，50%抑制浓度（IC50）为15.8 nM。这种抑制作用对于底物角鲨烯而言是非竞争性的。一项对特比萘芬一些类似物的构效关系研究表明，特比萘芬的叔氨基结构及其叔烷基侧链对其高效性至关重要。萘替芬的效力（IC50，114.6 nM）低于特比萘芬。硫代氨基甲酸盐类药物托萘酯（IC50，28.0 nM）和联苯苄唑（IC50，51.5 nM）也显示出抑制作用。有趣的是，吗啉类药物阿莫罗芬也表现出微弱但显著的作用（IC50，30μM）。红色毛癣菌SE对特比萘芬抑制作用的敏感性仅比对白色念珠菌酶的敏感性略高（约两倍）。因此，这种差异不能完全解释皮肤癣菌比酵母菌对该药物高得多的敏感性（≥100倍）。红色毛癣菌全细胞中麦角固醇生物合成对特比萘芬的敏感性（IC50，1.5 nM）比对SE活性的敏感性高10倍，这表明该药物在真菌中蓄积。
