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.     

新型含哌嗪侧链三唑类化合物的设计、合成和抗真菌活性研究

目的 基于唑类药物与靶酶相互作用模式,设计新型三唑类化合物,并测试其对常见致病真菌的抑制活性.方法 采用酰化反应合成目标化合物,通过1H NMR和MS确证其化学结构,经微量液基稀释法测试体外抗真菌活性.结果 合成了12个含C1甲基和哌嗪侧链的新型唑类化合物,它们均显示了中度至良好的抗真菌活性.结论 部分目标化合物对白色念珠菌的活性优于对照药氟康唑,值得进一步深入构效关系研究.     

三唑类抗真菌新药Ravuconazole

现已有酮康唑、咪康唑、氟康唑及依曲康唑等四个唑类抗真菌药物在临床上使用,其作用机制是抑制细胞色素P-450单氧化酶及羊毛甾醇14-α-脱甲基酶,后者是真菌麦角甾醇生物合成的关键酶.     

3种抗真菌药治疗甲真菌病药物经济学分析

目的对伊曲康唑、特比萘芬、氟康唑3种不同抗真菌药物治疗120例甲真菌病患者的成本效果分析。方法选择120例病例,随机分为3组,分别给予伊曲康唑、特比萘芬、氟康唑治疗,观察各组疗效并运用药物经济学方法进行分析。结果3种药物均有较好的抗真菌疗效,特比萘芬疗效优于伊曲康唑及氟康唑,伊曲康唑疗效优于氟康唑。平均成本-效果比分别为8.9,8.0,11.7。结论药物经济学分析结果为特比萘芬治疗甲真菌病最优。     

对1例肺隐球菌感染患者的药学监护

1例42岁女性患者,因咳嗽、咳痰伴胸闷1月余,咯血3d入院。结合肺隐球菌的病理结果,先后给予抗真菌药物氟康唑和伊曲康唑治疗。住院期间药师及时评估治疗方案,并对患者的肝肾功能监测、抗真菌药物潜在药物相互作用以及药物配制注意事项等提供药学监护。患者首次静脉滴注伊曲康唑结束后,出现一过性视物模糊,11d后再次静脉滴注自觉症状加重,第13天停药后未再出现视物模糊,考虑为伊曲康唑的不良反应。接受抗真菌药物治疗32d后,因症状较前改善,患者出院继续接受抗真菌治疗。鉴于肺隐球菌病长期正规治疗的重要性,提示患者关注氟康唑的药物相互作用,谨慎合用其他药物。     

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

目的寻找广谱、高效、低毒的新一代三唑类抗真菌药物。方法根据靶酶活性位点的空腔大小、各种力场和关键残基分布，设计并合成了19个1-(1,2,4-三唑-1H -1-基)-2-(2,4-二氟苯基)-3-(4-取代苄基-1-哌嗪基)-2-丙醇类化合物，测定了体外抗真菌活性。结果所有化合物对8种致病真菌均有较强的抗真菌活性，对深部真菌的活性明显优于浅部真菌。结论绝大部分化合物的抗真菌活性明显高于氟康唑和特比萘芬，其中化合物VIII-1，10，12，17具有广谱、高活性的优点，值得进一步深入研究。     

3种抗真菌药治疗甲癣真菌病的成本-效果分析

目的:探讨3种抗真菌药治疗甲癣真菌患者的成本和效果.方法:将96例甲癣真菌患者随机分为3组,分别给予国产氟康唑、酮康唑、伊曲康唑治疗.运用药物经济学的成本-效果分析方法进行评价.结果:治疗指甲癣真菌病所需药品成本分别是:国产氟康唑252元、酮康唑398元、伊曲康唑741元;治疗趾指甲癣真菌病所需药品成本分别是:国产氟康唑378元、酮康唑598元、伊曲康唑1111元.远期治疗指甲癣真菌病的有效率分别是91%,75%,97%;趾指甲癣真菌病分别是91%,78%,94%.结论:根据药物经济学成本-效果分析,国产氟康唑是治疗甲真菌病的较佳药物.     

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。     

抗真菌药

三唑类抗真菌药包括氟康唑、伊曲康唑、伏立康唑、特康唑和泊沙康唑。前三者国内已有供应,但伊曲康唑和伏立康唑的价格十分昂贵,其应用受到一定的限制。它们的抗真菌作用机制相同,抗真菌谱和和抗真菌作的比较见附表。下面仅叙述各药的药动学、应用、不良反应等。氟康唑(Fluconazole)【药动学】口服氟康唑后几乎完全从胃肠吸收。无论药物口服或静注,其血浓度基本相同,食物或胃液酸度不影响其生物利用度。重复给药剂量为100mg时,其峰浓度为4～8mg/ml。药物与血浆蛋白结合率约11%～12%。氟康唑易于扩散入体液,包括痰液和唾液;脑脊液浓度为同期…     

新型三唑类化合物的合成及抗真菌活性

目的寻找广谱、高效、低毒的新一代三唑类抗真菌药物。方法根据靶酶活性位点的空间特征、各种力场和关键残基分布，设计并合成了21个1-(1,2,4-三唑-1H -1-基)-2-(2,4-二氟苯基)-3-(4-取代-1-哌嗪基)-2-丙醇类化合物，并测定了体外抑菌活性。结果体外抑菌测试结果表明，所有化合物对8种致病真菌均有一定程度的抗真菌活性，对深部真菌的活性明显优于浅部真菌。在哌嗪的各种取代基中，苯基和杂环取代的抗真菌活性明显优于苯甲酰基取代。结论有多个化合物的体外抗真菌活性明显高于氟康唑和特比萘芬，其中化合物VIII-1,4,5和IX-3具有广谱、高活性的优点，值得进一步研究。     

Identification of Y118 amino acid residue in Candida albicans sterol 14alpha-demethylase associated with the enzyme activity and selective antifungal activity of azole analogues

Our previous publication established a model to predict that the phenyl group of the C-3 side chain of azole antifungal compounds interacts with the phenol group of Tyr118 through the formation of pi-pi face-to-edge interaction. To verify this prediction, wild type and three site-directed mutants of the Y118 residue of Candida albicans sterol 14alpha-demethylase P450 (CACYP51) were constructed and heterologously expressed in Saccharomyces cerevisiae with deletion of the CYP51 gene. With the strains obtained and microsome enzymes separated, cell susceptibility and CACYP51 activity were examined with the 5 novel azole compounds based on the molecular modeling in comparison with fluconazole. After alteration of Y118 with Y118A, Y118F, and Y118T by a single base substitution, the expression levels of CACYP51 protein were not affected. However, these mutations markedly decreased its catalytic activity respectively; the mutation changes also decreased azole susceptibility, indicating the structural importance of the Y118 residue in maintaining CACYP51 activity and in determining azole susceptibility. In addition, our synthetic compounds with the phenyl group side chain attached to C3 produced higher susceptibility against S. cerevisiae with expression of CACYP51 and exhibited more potent inhibitory effects on CACYP51 activity in comparison with fluconazole, suggesting that the phenyl group of C3 side chain substitutes is also important for selective binding to target enzymes.     

Point mutation of cytochrome P450 2A6 (a polymorphic variant CYP2A6.25) confers new substrate specificity towards flavonoids

CYP2A6 is a major hepatic member of the cytochrome P450 family in humans. Much variation in CYP2A6 levels and activity can be attributed to genetic polymorphisms of this gene. CYP2A6*25 comprises an amino acid substitution, F118L. To clarify the effect of the leucine substitution at position 118 in CYP2A6.25, this variant, wild type CYP2A6 and three additional variants consisting of artificial mutations at the substrate binding site (position 481) suggested by earlier reports using random mutagenesis studies [CYP2A6.1, CYP2A6.25, CYP2A6.1(F118A), CYP2A6.1(A481G) and CYP2A6.25(A481G)], were co‐expressed with NADPH‐cytochrome P450 reductase in E. coli. The hydroxylase activity of these variants toward 7‐ethoxycoumarin, coumarin, flavone, α‐naphthoflavone, flavanone and hydroxyflavanone were examined. All the mutants had lower activities for coumarin 7‐hydroxylation than the wild type. All the mutants showed higher activities for flavone and α‐naphthoflavone compared with CYP2A6.1. CYP2A6.1 had the highest flavanone 2′‐hydroxylase activity, whereas CYP2A6.25 had the highest 6‐ and 4′‐hydroxylase activities. CYP2A6.1(F118A), CYP2A6.1(A481G) and CYP2A6.25(A481G) had higher flavanone 3′‐hydroxylase activities than CYP2A6.1 and CYP2A6.25. Furthermore, 4′‐hydroxyflavanone was metabolized by CYP2A6.25. These results indicate that the CYP2A6.25 mutation confers new substrate specificity towards flavonoids. Copyright © 2015 John Wiley & Sons, Ltd.     

The novel antifungal agent PLD-118 is neither metabolized by liver microsomes nor inhibits cytochrome P450 in vitro

PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 microM) incubated for 0-60 min with S9 fractions and NADPH was determined by HPLC, using the Waters AccQ.Tag method after derivatization of amino acids to stable, fluorescent derivatives. CYP assays were performed using pooled human liver microsomes with substrates, selective towards human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, incubated at concentrations around the Km. Incubation mixtures were preincubated with PLD-118 (0.1-100 microM) or control inhibitor for 5 min. No metabolism of PLD-118 was detected with rat and dog S9 fractions. A small (8%) decrease in PLD-118 at 100 microM (not detected at 10 microM) with human microsomes was considered to be biologically irrelevant. PLD-118 did not inhibit any of the tested CYPs. PLD-118, at concentrations up to 100 microM, is not metabolized by rat, dog or human liver S9 homogenates and does not inhibit human CYPs in vitro, suggesting little likelihood for interaction of PLD-118 with drugs metabolized by these enzymes.     

CYP3A4 active site volume modification by mutagenesis of leucine 211.

The leucine 211 --> phenylalanine (L211F) and leucine 211 --> tyrosine (L211Y) mutant forms of cytochrome P450 3A4 have been generated by site-directed mutagenesis and expressed functionally in Escherichia coli. Substrate binding affinities (S50 values) for testosterone and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were similar for the mutants and wild-type CYP3A4 (49 and 21 microM for L211F, 35 and 20 microM for L211Y, and 33 and 20 microM for the wild type, respectively). For erythromycin, however, the K(m) values determined for the L211F and L211Y mutants were 2.4- and 10.5-fold higher than for the wild type. Furthermore, IC50 values for the inhibition of testosterone 6 beta-hydroxylation by erythromycin and troleandomycin for L211F were 2.4- and 3.7-fold higher, and those for L211Y were 3.4- and 9.2-fold higher than those measured for the wild type. Conversely, small inhibitors, such as diazepam, exhibited no significant difference in IC50 values between the wild type and the L211F and L211Y mutants. It is proposed that large substrates bound in the catalytic center of CYP3A4 with molecular volumes greater than approximately 600 A(3) were less well accommodated in the altered active sites, resulting in lower association energies and increased IC50 values.     

Recent advances in antifungal drug development targeting lanosterol 14α-demethylase (CYP51): A comprehensive review with structural and molecular insights

Fungal infections are posing serious threat to healthcare system due to emerging resistance among available antifungal agents. Among available antifungal agents in clinical practice, azoles (diazole, 1,2,4-triazole and tetrazole) remained most effective and widely prescribed antifungal agents. Now their associated side effects and emerging resistance pattern raised a need of new and potent antifungal agents. Lanosterol 14α-demethylase (CYP51) is responsible for the oxidative removal of 14α-methyl group of sterol precursors lanosterol and 24(28)-methylene-24,25-dihydrolanosterol in ergosterol biosynthesis hence an essential component of fungal life cycle and prominent target for antifungal drug development. This review will shed light on various azole- as well as non-azoles-based derivatives as potential antifungal agents that target fungal CYP51. Review will provide deep insight about structure activity relationship, pharmacological outcomes, and interactions of derivatives with CYP51 at molecular level. It will help medicinal chemists working on antifungal development in designing more rational, potent, and safer antifungal agents by targeting fungal CYP51 for tackling emerging antifungal drug resistance.     

The interaction of methylphenidate and benztropine with the dopamine transporter is different than other substrates and ligands

A substantial body of evidence suggests that the dopamine transporter (DAT) is the principal site for cocaine-induced reward and euphoria. Interactions between the DAT and its substrates and ligands may therefore be of clinical relevance. The pharmacological characteristics of DAT compounds were compared in wild type (WT) and mutant DATs. The DAT mutants chosen for study were those with reduced binding and uptake activities (aspartic acid 79 mutated to alanine, termed D79A), reduced binding but normal uptake (tyrosine 251 mutated to alanine, termed Y251A; tyrosine 273 mutated to alanine, termed, Y273A), and normal binding but reduced uptake (a double mutation: serines 356 and 359 mutated to alanine, termed S356,359A). The WT and mutant DATs were transfected into COS-7 cells, and their pharmacological activities were examined 3 days later. Different patterns of pharmacological activity emerged. GBR 12909, cocaine, and mazindol each showed reduced affinity for the Y251A and the Y273A mutants, but their affinity for the S356,359A mutant was similar to that of the WT DAT. d-Amphetamine, MPP+, and dopamine each showed reduced affinity for the S356,359A mutant. Benztropine and methylphenidate had a different effect. Relative to the WT DAT, they both showed reduced affinity for the S356,359A mutant when displacing radioactive carboxyfluorotropane (CFT) binding, but similar affinity when inhibiting radioactive dopamine uptake. These results indicate that methylphenidate and benztropine may interact with the DAT in a different fashion then other substrates and ligands.     

Characterization of the biochemical and structural phenotypes of four CYP1B1 mutations observed in individuals with primary congenital glaucoma

OBJECTIVE: The objective of this study was to examine the biochemical and physical properties of cytochrome P450 1B1 (CYP1B1) mutants, test our hypothesis that primary congenital glaucoma (PCG)-causing mutants have altered metabolic activity, and correlate these to structural changes in the molecule. METHODS: CYP1B1.1 cDNA was mutated to four forms found in individuals with the PCG phenotype, Y81N, E229K, A330F, and R368H. Expression and stability of the mutant hemoproteins and their ability to metabolize beta-estradiol, arachidonic acid, and retinoids, were determined. Alterations in mutant properties were related to structural changes by in silico examination, on the basis of the CYP1A2 crystal structure. RESULTS: CYP1B1 mutations strongly affected the stability, ease of heterologous expression, and enzymatic properties of the protein. These were related to the location of the amino acid substitutions in the CYP1B1 structure. Three of the mutations involve residues located on the surface of CYP1B1, Y81N, and E229K near the distal surface, and R368H near the proximal surface. The former two substitutions, Y81N and E229K, caused greatly reduced stability at 4 degrees C. Y81N severely inhibited all substrate turnover, but E229K only inhibited arachidonate turnover and exhibited minimal effect on efficiency of retinoid metabolism and estradiol metabolism. The R368H mutation is relatively conservative, affecting charge-pairing with the deeper-located D374, but it severely inhibited metabolism of all substrates tested, and, like Y81N, expression of the enzyme is less facile than CYP1B1wt. The A330F mutation replaces a small alanine by a bulky phenylalanine in the enzyme active site and had major impact on substrate binding, turnover, uncoupling, and metabolite pattern. CONCLUSION: Consistent with the hypothesis, these PCG-related mutations cause identifiable structural changes negatively impacting CYP1B1 biochemistry and stability.     

Molecular characterisation of cyp51A and cyp51B genes coding for P450 14alpha-lanosterol demethylases A (CYP51Ap) and B (CYP51Bp) from voriconazole-resistant laboratory isolates of Aspergillus flavus

Aspergillus flavus is the second most common Aspergillus spp. causing invasive infections in immunocompromised patients. Extensive prophylactic use of voriconazole (VCZ) in immunocompromised patients may enhance the selection of VCZ-resistant clinical isolates of A. flavus, compromising the effectiveness of this antifungal drug against A. flavus infection. To study triazole resistance, we selected A. flavus isolates in the laboratory showing reduced in vitro susceptibility to VCZ. The cyp51A and cyp51B genes coding for P450 14alpha-sterol demethylases A (CYP51Ap) and B (CYP51Bp) were characterised to examine possible drug target modification-dependent resistance to VCZ in this fungus. High-molecular-weight DNA was isolated from 10 A. flavus isolates showing in vitro resistance to VCZ (minimum inhibitory concentration (MIC) range 4-32 microg/mL) as well as from the drug-susceptible parent isolate X26728 (MIC = 1 microg/mL). The cyp51A and cyp51B genes were cloned and the nucleotide sequences were determined. A comparison of the deduced amino acid sequences of CYP51Ap from 10 VCZ-resistant isolates with that of the drug-susceptible parent showed no amino acid variation in six of the ten isolates. CYP51Ap from isolates Afl-VCZ6 and Afl-VCZ46 showed a K197N change, CYP51Ap from isolate Afl-VCZ114 showed Y132N and T469S changes, whereas that from isolate Afl-VCZ45 showed K197N, D282E and M288L changes. These results suggest that VCZ-resistant A. flavus isolates can be readily isolated in the laboratory under selection pressure. Multiple mechanisms, including drug target modification, may be responsible for the in vitro resistance of A. flavus to VCZ.     

Y111是维持血管内皮细胞生长抑制因子生物活性的关键氨基酸

目的血管内皮细胞生长抑制因子(Vascu lar endothe-lial cell growth inh ib itor,VEG I)是近年来发现的一类TNF家族新成员,具有抑制内皮细胞增殖与新生血管生成的作用。但目前其结构功能关系研究尚为空白,该文研究VEG I空间结构上重要残基的作用极其对生物活性的影响。方法采用定点突变技术突变了四个关键氨基酸E45R,G47A,Y111F,Y111T,构建了表达载体并在大肠肝菌中表达及纯化,采用人脐静脉内皮细胞增殖和鸡胚尿囊膜血管生成实验研究了四个突变体的活性。结果E45R突变体的活性显著下降,G47A突变体的活性略低于野生型VEG I,Y111F,Y111T突变体的活性比野生型VEGI下降10倍以上。结论VEGI的第111位氨基酸Y是发挥其功能的关键氨基酸,可能与受体形成直接结合,且氨基酸Y上的苯环与酚羟基均对其活性的发挥具重要作用;第45位氨基酸E对其活性的发挥也具重要作用,而第47氨基酸对其活性的发挥可能不具重要作用。     

Inhibition of human CYP19 by azoles used as antifungal agents and aromatase inhibitors, using a new LC-MS/MS method for the analysis of estradiol product formation

Azoles are used as fungicides in agriculture or antifungal drugs in medicine. Their therapeutic activity is based on the inhibition of fungal lanosterol-14alpha-demethylase (CYP51). Azoles are also used for the treatment of estrogen-dependent diseases, e.g. in breast cancer therapy. Inhibition of CYP19 (aromatase) is the working principle for tumor therapy, but is an unwanted side effect of azoles used as fungicides or antifungal drugs. The inhibition of recombinant human CYP19 by 21 azoles in use for the three different purposes was investigated using the natural substrate testosterone. Estradiol product formation was measured by a newly developed and fully validated analytical method based on liquid chromatography-tandem mass spectrometry utilizing photospray ionization (APPI). Potency of enzyme inhibition was expressed in terms of IC50 concentrations. The two cytostatic drugs fadrozole and letrozole were the most potent inhibitors. However, azoles used as fungicides, e.g. prochloraz, or as antifungal drugs, e.g. bifonazole, were almost as potent inhibitors of aromatase as the drugs used in tumor therapy. Comparison of plasma concentrations that may be reached in antifungal therapy do not allow for large safety factors for bifonazole and miconazole. The IC50 values were compared to data obtained with other substrates, such as the pseudo-substrate dibenzylfluorescein (DBF). A high correlation was found, indicating that the fluorescence assay with DBF can well be used for potency ranking and screening of chemicals for aromatase inhibition. The data for antifungal drugs show that side effects on steroid hormone synthesis in humans due to inhibition of aromatase should be considered.