氮唑类抗真菌药物药效构象及其与酶活性位点对接

目的： 研究氮唑类抗真菌药物与其受体蛋白活性位点相互作用机理。方法： 用随机构象搜寻和分子动力学模拟退火法确定15个4种不同类型的氮唑类抗真菌药物最低能量构象;用活性类似物法限定药物分子药效基团之间的距离,搜寻到各化合物药效构象;将各化合物药效构象对接到白色念珠菌羊毛甾醇14α去甲基化酶活性位点中。结果： 4种结构类型的氮唑类药物在酶活性位点中有相似的对接位置;真菌和哺乳动物的活性位点结构特异性的残基分布在F螺旋C端、β6-1和β6-2区中;氮唑类抗真菌药物共同的卤代芳环结构落入相同的疏水空穴中,其中Y132的侧链羟苯基结构可与抑制剂卤代芳环形成电子迁移复合物。结论： 对接结果与已知SAR分析结论相符,阐明了氮唑类药物与活性位点的氨基酸残基作用方式,探讨结构选择性药物的结构需求。     

氮唑类抗真菌药物研究进展

氮唑类抗真菌药物的结构特点是分子中都含有一个或两个咪唑或三唑环,并且都是1位氮原子与芳烃基相连,芳烃基一般为一卤或二卤取代苯环,结构变化大多是氮唑环与芳环之间碳原子上取代基的改变。作用机理氮唑类抗真菌药物,不论是咪唑类还是三唑类都有着相似的作用机理。分子药理学研究表明,这类药物有两种主要作用方式:其主要作用机理是在低浓度时抑制真菌细胞内麦角甾醇的生物合成。氮唑类药物分子中一个氮原子(咪唑环3位和三唑环4位)可与真菌细胞内P-450分子中亚铁血红素上的亚铁离子相络合,从而抑制P-     

唑类抗真菌药进展

近些年来,真菌感染的发病率呈上升趋势,日益引起人们的重视,抗真菌药的研究也成为热点之不,目前临床应用和研究得较多的是唑类抗真菌药,相继有几代产品问世,唑类抗真菌药的结构大致可分为４类,多苯甲基唑类,α－卤代苯乙基－β,Ｎ－咪唑乙基苄醚类,α－卤代苯乙基－α－环氧戊环唑类和α－卤代苯乙基三唑类,其代表药物分别为克霉唑、咪康唑、酮康唑和氟康唑。其基本药效基团为无取代唑环（咪唑或三唑）,间位Ｎ与真菌１４     

抗真菌药物的创新设计研究

运用计算机辅助药物设计技术和分子生物学技术,建立了一套集分子设计、化学合成和分子筛选三大系统为一体的抗真菌药创新设计体系.其工作原理是:通过药效构象搜寻、3D-QSAR研究,建立了抗真菌药物与受体作用模型,预测设计高活性化合物;另一方面,根据抗真菌药靶酶--羊毛甾醇14α-去甲基化酶的已知序列,采用同源蛋白模建技术构建其三维结构,再通过药物-靶酶对接研究,确定活性位点,设计与活性位点最佳匹配的活性化合物;然后针对设计的化合物类型,研究建立一套科学的合成方法,完成化合物的合成;最后以靶酶为模型,建立一套快速准确的分子筛选方法,用筛选结果指导进一步的设计与合成,直至得到最佳的新化学单体(NCE).将该体系用于抗真菌药物设计,获得了一个广谱、高效、低毒的抗真菌新药--艾迪康唑.     

三唑类化合物的合成及其抗真菌活性Ⅰ

根据氮唑类抗真菌药物的作用机理及构效关系，设计合成了１５个１（１Ｈ１，２，４，三唑１基）２（２，４二氟苯基）３取代苯氧基２丙醇类化合物，其结构经氢核磁共振谱证实．初步体外抑菌实验表明：该类化合物对常见深部致病真菌如白念珠菌、新生隐球菌、申克氏孢子丝菌、卡氏枝孢霉菌均有一定程度的抗菌活性．化合物（Ｉ２），（Ｉ９）的体外活性优于氟康唑，与酮康唑相当     

三唑类化合物的合成及其抗真菌活性Ⅲ

为了寻找新型高效低毒的抗真菌药,根据氮唑类抗真菌药物的构效关系和作用机理,设计了２１个２,２,－双取代－４－（２,４－二氟苯基）－４－（１Ｈ－＝１,２,４－三唑－１－）基甲基－１,３二氧戊环类化合物,其结构经元素分析及氢核磁共振谱证实。初步的体外抗真菌活性试验表明：所有的目标化合物对６种常见的致病真菌都有不同程度的抗真菌活性,对浅部真菌的效果要比对深部真菌的效果好,其中化合物（Ｉ７,）（Ｉ１２）,     

1-(1H-1,2,4-三唑-1-基)-2-(2,4-二氟苯基)-3-取代-2-丙醇的合成及抗真菌活性研究

１（１Ｈ１，２，４三唑１基）２（２，４二氟苯基）３取代２丙醇的合成及抗真菌活性研究张大志周廷森吴义杰刘超美麻铭川冯向庭（第二军医大学药学院，上海２００４３３）氮唑类化合物是目前抗真菌药物研究最为活跃的领域之一。该类化合物可分...     

烯丙胺和苄胺类抗真菌药物比较分子力场分析(CoMFA)的研究

本实验采用“活性类似物法”搜寻到烯丙胺和苄胺类抗真菌化合物的药效构象。其与药物萘替芬、特比萘芬的晶体构象相同。以此构象构建各化合物,采用rmsfit规则叠合各分子,进行CoMFA研究,得到预测能力很强的抗6种常见致病真菌的3D-QSAR模型。并用8个本实验室设计合成的新化合物和5个文献报道的化合物对模型预测能力进行验证。最后结合2D-QSAR方程首次提出了烯丙胺、苄胺类抗真菌化合物与受体蛋白相互作用模式图。     

三唑类化合物的合成及其抗真菌活性Ⅱ

为了寻找新型高效低毒的抗真菌药，根据氮唑类抗真菌类药物的构效关系和作用机理，设计合成了８个２－（取代苯氧基）甲基－４（２，４－二氟苯基）－４－（１Ｈ－１，２，４－三唑－１－基）甲基－１，３－二氧戊环类化合物。初步的体外抗真菌活性试验表明，所有的目标化合物对５种常见的深部致病真菌都有不同程度的抗真菌活性，所有目标化合物的活性与克霉唑相当或更高，特别是对白念珠菌，新生隐球菌、卡氏枝孢菌效果尤其显著。     

1-(1H-1,2,4-三唑-1-基)-2-(2,4-二氟苯基)-3-(N-甲基-N-取代苄基氨基)-2-丙醇的合成及抗真菌活性研究

根据三唑类抗真菌药物作用靶酶－羊毛甾醇14α－去甲基化酶的三维晶体结构和药物与酶活性的位点的对接结果，设计合成了11个1－（1H－1，2，4－三唑－1－基）－2（2，4－二氟苯基）－3－（N－甲基－N－取代苄基氨基）－2－丙醇化合物，11个目标化合物均系首次报道，体外抗真菌活性试验结果表明，所有目标化合物对七种致病真菌都有不同程度的抗真菌活性，而且都比氟康唑的体外抗真菌活性好，化合物11的抗菌谱最广，抗真菌活性最高，对新型隐球菌，白色念珠菌，羊毛状小孢子菌和红色毛癣菌的抗菌活性比酮康唑高，有进一步开发的价值。化合物3，4，10也表现出较高的抗真菌活性。     

A three-dimensional model of lanosterol 14alpha-demethylase of Candida albicans and its interaction with azole antifungals

The three-dimensional structure of lanosterol 14alpha-demethylase (P450(14DM), CYP51) of Candida albicans was modeled on the basis of crystallographic coordinates of four prokaryotic P450s: P450BM3, P450cam, P450terp, and P450eryF. The P450(14DM) sequence was aligned to those of known proteins using a knowledge-based alignment method. The main chain coordinates of the core regions were transferred directly from the corresponding coordinates of P450BM3. The side chain conformations of the core regions were determined by the conformations of the equivalent residues with the highest homologous scores in four crystal structures. The model was then refined using molecular mechanics and molecular dynamics. The reliability of the resulting model was assessed by Ramachandran plots, Profile-3D, hydropathy plot analysis, and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues such as the heme binding residues, the residues interacting with redox-partner protein and/or involved in electron transfer, the residues lining substrate access channel, and the substrate binding residues were identified from the model. These residues are candidates for further site-directed mutagenesis and site-specific antipeptide antibody binding experiments. The active analogue approach was employed to search the pharmacophoric conformations for 14 azole antifungals. The resulting bioactive conformations were docked into the active site of lanosterol 14alpha-demethylase of Candida albicans. All 14 azole antifungals are shown to have a similar docking mode in the active site. The halogenated phenyl group of azole inhibitors is deep in the same hydrophobic binding cleft as the 17-alkyl chain of substrate. The pi-pi stacking interaction might exist between halogenated phenyl ring of inhibitors and the aromatic ring of residue Y132. The long side chains of some inhibitors such as itraconazole and ketoconazole surpass the active site and interact with the residues in the substrate access channel. To compare with mammalian enzymes, structurally selective residues of the active site of fungal lanosterol 14alpha-demethylase are distributed in the C terminus of F helix, beta6-1 sheet and beta6-2 sheet.     

A new series of natural antifungals that inhibit P450 lanosterol C-14 demethylase. II. Mode of action.

From a Penicillium sp. we identified a new series of antifungals having a tetrahydropyran skeleton with an alkenyl side chain. We elucidated the mode of action of Ro 09-1470, the most active compound of the series. Treatment of Candida albicans with the compound caused an accumulation of C-14 methyl intermediates of ergosterol at concentrations of which no significant interference with the biosyntheses of other macromolecules and respiration was observed. P450 lanosterol C-14 demethylase (P450(14DM)) activity was inhibited and furthermore, the binding of Ro 09-1470 to the heme of the enzyme was demonstrated by a difference spectrum. We conclude that Ro 09-1470 is the first natural antifungal that inhibits the P450(14DM) of fungi.     

Structure-based de novo design,synthesis, and biological evaluation of non-azole inhibitors specific for lanosterol 14alpha-demethylase of fungi

The active site of lanosterol 14alpha-demethylase (CYP51) was investigated via MCSS functional group mapping and LUDI calculations. Several non-azole lead molecules were obtained by coupling structure-based de novo design with chemical synthesis and biological evaluation. All of the lead molecules exhibited a strong inhibitory effect on CYP51 of Candida albicans. They occupy the substrate-binding site and interfere with the binding of azole antifungal agents in a competitive manner. The mode of action of the lead molecules was validated by spectrophotomeric analysis and SAR studies. This is the first successful example reported for the inhibitor design of the cytochrome P450 superfamily using the de novo design strategy. Because the affinity of the lead molecules for CYP51 was mainly attributed to their nonbonding interaction with the apoprotein, the studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s.     

Antifungal agents. 10. New derivatives of 1-[(aryl)[4-aryl-1H-pyrrol-3-yl]methyl]-1H-imidazole,synthesis, anti-candida activity,and quantitative structure-analysis relationship studies

The synthesis, anti-Candida activity, and quantitative structure-activity relationship (QSAR) studies of a series of 2,4-dichlorobenzylimidazole derivatives having a phenylpyrrole moiety (related to the antibiotic pyrrolnitrin) in the alpha-position are reported. A number of substituents on the phenyl ring, ranging from hydrophobic (tert-butyl, phenyl, or 1-pyrrolyl moiety) to basic (NH(2)), polar (CF(3), CN, SCH(3), NO(2)), or hydrogen bond donors and acceptor (OH) groups, were chosen to better understand the interaction of these compounds with cytochrome P450 14-alpha-lanosterol demethylase (P450(14DM)). Finally, the triazole counterpart of one of the imidazole compounds was synthesized and tested to investigate influence of the heterocyclic ring on biological activity. The in vitro antifungal activities of the newly synthesized azoles 10p-v,x-c' were tested against Candida albicans and Candida spp. at pH 7.2 and pH 5.6. A CoMFA model, previously derived for a series of antifungal agents belonging to chemically diverse families related to bifonazole, was applied to the new products. Because the results produced by this approach were not encouraging, Catalyst software was chosen to perform a new 3D-QSAR study. Catalyst was preferred this time because of the possibility of considering each compound as a collection of energetically reasonable conformations and of considering alternative stereoisomers. The pharmacophore model developed by Catalyst, named HYPO1, showed good performances in predicting the biological activity data, although it did not exhibit an unequivocal preference for one enantiomeric series of inhibitors relative to the other. One aromatic nitrogen with a lone pair in the ring plane (mapped by all of the considered compounds) and three aromatic ring features were recognized to have pharmacophoric relevance, whereas neither hydrogen bond acceptor nor hydrophobic features were found. These findings confirmed that the key interaction of azole antifungals with the demethylase enzyme is the coordination bond to the iron ion of the porphyrin system, while interactions with amino acids localized in proximity of heme could modulate the biological activity of diverse antifungal agents. In conclusion, HYPO1 conveys important information in an intuitive manner and can provide predictive capability for evaluating new compounds.     

Lanomycin and glucolanomycin, antifungal agents produced by Pycnidiophora dispersa. I. Discovery, isolation and biological activity.

The antifungal agents lanomycin and glucolanomycin were isolated from Pycnidiophora dispersa. The compounds were active against species of Candida and dermatophytes but were inactive against Aspergillus fumigatus and Gram-positive and Gram-negative bacteria. The compounds inhibited the cytochrome P-450 enzyme lanosterol 14 alpha-demethylase, and are believed, therefore, to have a mode of action similar to the azole and bis-triazole class of antifungal agents.     

2-(2,4-二氟苯基)-3-(N-甲基-N-取代基酰胺基)-1-(1H 1,2,4-三唑-1-基)-2-丙醇类化合物的合成及抗真菌活性的研究

目的:寻找新的高效、低毒、广谱的抗真菌药物。方法:设计合成了21 个三唑类化合物作为真菌细胞色素P450 14α-去甲基化酶的抑制剂,并通过体外抗真菌实验测定其抗真菌活性。结果:21 个化合物均为新化合物。体外抗真菌试验表明所有目标化合物对试验真菌均有不同程度的抑制作用,特别是对白色念珠菌和近平滑念珠菌具有很好的活性。结论:所有化合物都不同程度地对真菌细胞色素P450 14α-去甲基化酶有抑制作用,化合物15 对8 种不同真菌均显示了较高的活性,有进一步研究价值。     

Resistance of clinically important yeasts to antifungal agents

Resistance of yeasts to antifungal agents was a relatively minor clinical problem for many years. Recently Candida albicans isolates resistant to fluconazole have been reported with increasing frequency in the setting of oral infections in HIV-positive patients. Improved standardization of fluconazole susceptibility testing has resulted in demonstrable correlations between yeast resistance in vitro and in vivo for this agent in the AIDS setting. Known resistance mechanisms for azole antifungals include reduced access of the drug to the intracellular sterol demethylase target, probably because of the action of multidrug resistance efflux pumps, and overproduction of that target. Management and prevention of future resistance development requires greater vigilance for surveillance than has been the practice in the past.     

Design, synthesis, and microbiological evaluation of new Candida albicans CYP51 inhibitors

In a program aimed at the design and synthesis of novel azole inhibitors of Candida albicans CYP51 (CA-CYP51), a series of azole 1,4-benzothiazines (BT) and 1,4-benzoxazines (BO) were recently synthesized. A morphological study of the enzyme active site highlighted a hydrophobic access channel, and a docking study pointed out that the C-2 position of the BT or BO nucleus was oriented toward the access channel. Here, we report the design, synthesis, and microbiological evaluation of C-2-alkyl BT and BO compounds. In both series, introduction of the alkyl chain maintained and in some cases improved the anti-Candida in vitro activity; however, there was not always a strict correlation between in vitro and in vivo activity for several compounds.     

Antifungals targeted to protein modification: focus on protein N-myristoyltransferase

Invasive fungal infections have increased dramatically in recent years to become important causes of morbidity and mortality in hospitalised patients. Currently available antifungal drugs for such infections essentially have three molecular targets: 14α demethylase (azoles), ergosterol (polyenes) and β-1,3-glucan synthase (echinocandins). The first is a fungistatic target vulnerable to resistance development; the second, while a fungicidal target, is not sufficiently different from the host to ensure high selectivity; the third, a fungistatic (Aspergillus) or fungicidal (Candida) target, has limited activity spectrum (gaps: Cryptococcus, emerging fungi) and potential host toxicity that might preclude dose escalation. Drugs aimed at totally new targets are thus needed to increase our chemotherapeutic options and to forestall, alone or in combination chemotherapy, the emergence of drug resistance. Protein N-myristoylation, the cotranslational transfer of the 14-carbon saturated fatty acid myristate from CoA to the amino-terminal glycine of several fungal proteins such as the ADP-ribosylation factor (ARF), presents such an attractive new target. The reaction, catalysed by myristoyl-CoA:protein N-myristoyltransferase (NMT), is essential for viability, is biochemically tractable and has proven potential for selectivity. In the past five years, a number of selective inhibitors of the fungal enzyme, some with potent, broad spectrum antifungal activity, have been reported: myristate analogues, myristoylpeptide derivatives, histidine analogues (peptidomimetics), aminobenzothiazoles, quinolines and benzofurans. A major development has been the publication of the crystal structure of Candida albicans and Saccharomyces cerevisiae NMTs, which has allowed virtual docking of inhibitors on the enzyme and refinement of structure–activity relationships of lead compounds.