Structural Analysis of Fungal Lanosterol Demethylase for Developing High-selective Antifungal Agents

真菌羊毛甾醇脱甲基酶的结构分析用于开发高选择性抗真菌药物

• 批准号: 03671075
• 负责人: YOSHIDA Yuzo
• 金额: $ 1.28万
• 依托单位: Mukogawa Women's Universtiy
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03671075/
• 关键词: P450; Azole anitifungal agent; Fungi; Mammalia; Target specificity; Lanosterol; Substrate recognition; Sterol demethylase inhibitor; ステロール脱メチル化酵素; 抗真菌剤; 構造活性相関; ステロール生合成阻害剤; 選択性; シトクロム P450; ラノステロール 脱メチル化酵素; ステロイド代謝; 酵母; ステロ-ル代謝; シトクロムP4

Aim of porject : Azole antifungals are potent inhibitors of fungal P450(14DM). Therefore, high selectivity for fungal P450(14DM) without any inhibitory effect on mammalian P450s is expected for them. The aim of this project is to assume structures necessary for azole antifungals to have high selectivity for fungal P450(14DM) through structural analysis of the substrate recognition site of P450(14DM).Results : (1) Fungal P450(14DM) showed structure-specific interaction with the 3 beta-hydroxyl group, DELTA8-lanostene skeleton and the terminal part of the sterol side chain. (2) The sterol ring of the substrate might be holded in the space over pyrrole C and heme iron and the side chain might be situated on the amino acid residue(s) covering pyrrole A.(3) Rat and yeast P450(14DM)s showed different recognition for the structure of the sterol side chain. These findings suggest a possibility that azole compounds interacting with the side-chain recognition site of P450(14DM) may show high selectivity for the fungal enzyme. This possibility was confirmed by the following comparative experimennts on the effects of the azole compounds having isoprenoid moiety on rat and yeast P450(14DM)s. (4) The azole compound bearing geranyl moiety that corresponds to the lanosterol side chain showed potent inhibition by interacting with the side-chain recognition site of these enzymes. (5) Decrement in the inhibitory effect by the substitution of the geranyl moiety with longer or shorter isoprenoids was far greater in the rat enzyme than in the yeast one. (6) They act weak inhibitors for the rat liver 7-ethoxycoumarin deethylase P450. These lines of evidence suggest a possibility that the azole compounds interacting with the side-chain recognition part of P450(14DM) show high selectivity for the fungal enzyme.

目的：唑类抗真菌药物是真菌P450（14 DM）的有效抑制剂。因此，预期它们对真菌P450（14 DM）具有高选择性，而对哺乳动物P450没有任何抑制作用。结果：（1）真菌P450（14 DM）与3 β-羟基、DELTA 8-羊毛甾烯骨架和甾醇侧链末端具有结构特异性的相互作用。(2)底物的甾醇环可能位于吡咯C和血红素铁上方的空间中，侧链可能位于覆盖吡咯A的氨基酸残基上。(3)大鼠和酵母P450（14 DM）对甾醇侧链的结构显示出不同的识别。这些研究结果表明，唑类化合物与P450（14 DM）的侧链识别位点相互作用可能对真菌酶表现出高选择性。这种可能性通过以下关于具有类异戊二烯部分的唑类化合物对大鼠和酵母P450（14 DM）的作用的对比实验得到证实。(4)带有相当于羊毛甾醇侧链的香叶基部分的唑类化合物通过与这些酶的侧链识别位点相互作用而表现出强效抑制作用。(5)减少的抑制作用的替代香叶基部分与较长或较短的类异戊二烯是远远大于在酵母中的酶在大鼠。(6)对大鼠肝脏7-乙氧基香豆素脱乙基酶P450有弱抑制作用。这些证据表明，与P450（14 DM）的侧链识别部分相互作用的唑类化合物可能对真菌酶显示出高选择性。

项目成果

Yoshida,Y.: "Sterol 14α-Demethylase and Its Inhibition" Biochem.,Soc.Trans.19. 778-782 (1991)

Yoshida, Y.：“甾醇 14α-脱甲基酶及其抑制”Biochem.，Soc.Trans.19 (1991)。

Aoyama, Y.: "Role of the side chain of Lanosterol in substrate recognition and catalytic activity of lanosterol 14alpha-demethylase (cytochrome P-450/14DM) of Yeast" Biochim.Biophys.Acta. 1081. 262-266 (1991)

Aoyama, Y.：“羊毛甾醇侧链在酵母羊毛甾醇 14α-脱甲基酶（细胞色素 P-450/14DM）底物识别和催化活性中的作用”Biochim.Biophys.Acta。

Aoyama, Y.: "Structural analysis of interaction between the side-chain of substrate and active site of lanosterol 14alpha-demethylase (P45014DM) of yeast" Biochim.Biophys.Acta.1122. 251-255 (1992)

Aoyama, Y.：“底物侧链与酵母羊毛甾醇 14α-脱甲基酶 (P45014DM) 活性位点之间相互作用的结构分析”Biochim.Biophys.Acta.1122。

Yoshida,Y.: "Cytochrome P450 2nd.ed." Kodansha & VCH, 292 (1993)

吉田 Y.：“细胞色素 P450 第二版”

Aoyama,Y.: "Inhibition by a Novel Azole Antifungal Agent with a Gelanyl Group on Lanosterol 14α-Demethylase of Yeast" Biochem.Pharmacol.44. 1701-1705 (1992)

Aoyama，Y.：“带有香兰基的新型唑类抗真菌剂对酵母羊毛甾醇 14α-脱甲基酶的抑制”Biochem.Pharmacol.44 1701-1705 (1992)。