Analysis of Interaction between Cytochrome P-450 and Antifungal Agents

利用酵母细胞色素P450/ 14dm（羊甾醇14-去甲基酶）的纯化制备，研究了细胞色素P450与干扰真菌甾醇生物合成的唑类抗真菌药物相互作用的分子机制。唑类抗真菌药物能有效结合细胞色素P450/14DM，抑制细胞色素羊毛甾醇去甲基化酶活性。这一发现证实了抗真菌药物的作用机制，它们的抗真菌作用可能是由于麦角甾醇的降解和14-甲基甾醇的积累。通过比较不同抗真菌药物对细胞色素P450/14DM的亲和力，可以推断出与细胞色素相互作用所需的结构。这一发现为唑类抗真菌药物的构效关系研究提供了基础概念。通过比较光学活性抗真菌药物对映体的亲和力和抑制效果，发现只有一种对映体与细胞色素P450/14DM具有极高的亲和力。这一发现表明，评估单个对映体对细胞色素P-450/14DM的抑制作用对于开发更多改进的唑类抗真菌药物具有重要意义。对酵母菌酶亲和力较低的抗真菌药物对酵母菌酶和大鼠肝酶的抑制浓度几乎相同。另一方面，发现唑类抗真菌药物与酵母细胞色素P450/14DM具有非常高的亲和力，对酵母菌酶具有一定的选择性。

Molecular mechanism of interaction between cytochrome P450 and azole antifungal agents that interfere with the fungal sterol bio-synthesis was studied by using purified preparation of cytochrome P450/14DM (lanosterol 14-demethylase) of yeast.Azole antifungal agents avidly bind to cytochrome P450/14DM and inhibited the lanosterol demethylase activity of the cytochrome. This finding confirmed the proposed mechanism of action of the anti-fungal agents that their antifungal action might be due to depretion of ergosterol and accumulation of 14-methylsterols.By comparing affinities of various antifungal agents for cyto-chrome P450/14DM with one another, the structures necessary for interaction with the cytchrome could be deduced. This finding provided fundamental concept for structure-function relationship of azole antifungal agents.By comparing affinities and inhibitory effects of enantiomers of optically active antifungal agents, it was found that only one enantiomer interacted with cytochrome P450/14DM with extremely high affinity. This finding indicated that assesment of the inhibitory effects of individual enantiomers on cytochrome P-450/14DM should be important for developing more improved azole antifungal agents.The antifungal agents which showed relatively low affinity for the yeast enzyme inhibited both the yeast and rat liver enzymes at nearly the same concentration. On the other hand, it was found that the azole antifungal agents that interacted with yeast cytochrome P450/14DM with very high affinity showed some selectivity for the yeast enzyme.