Design, synthesis, and antifungal activities of novel 1H-triazole derivatives based on the structure of the active site of fungal lanosterol 14 alpha-demethylase (CYP51)

A series of fluconazole (1) analogues, compounds 3a-k, were prepared as potential antifungal agents. They were designed by computational docking experiments to the active site of the cytochrome P450 14alpha-sterol demethylase (CYP51), whose crystal structure is known. Preliminary biological tests showed that most of the target compounds exhibit significant activities against the eight most-common pathogenic fungi. Thereby, the most potent congener, 1-[(4-tert-butylbenzyl)(cyclopropyl)amino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (3j), was found to exhibit a broad antifungal spectrum, being more active against Candida albicans, Candida tropicalis, Cryptococcus neoformans, Microsporum canis, and Trichophyton rubrum (MIC80 < 0.125 microg/ml) than the standard clinical drug itraconazole (2). The observed affinities of the lead molecules towards CYP51 indicate that a cyclopropyl residue enhances binding to the target enzyme. Our results may provide some guidance for the development of novel triazole-based antifungal lead structures.     

Design,synthesis and biological evaluation of amide-pyridine derivatives as novel dual-target (SE,CYP51) antifungal inhibitors

Based on the analysis of the squalene cyclooxygenase (SE) and 14α-demethylase (CYP51) inhibitors pharmacophore feature and the dual-target active sites, a series of compounds with amide-pyridine scaffolds have been designed and synthesized to treat the increasing incidence of drug-resistant fungal infections. In vitro evaluation showed that these compounds have a certain degree of antifungal activity. The most potent compounds 11a, 11b with MIC values in the range of 0.125–2 μg/ml had a broad-spectrum antifungal activity and exhibited excellent inhibitory activity against drug-resistant pathogenic fungi. Preliminary mechanism studies revealed that the compound 11b might play an antifungal role by inhibiting the activity of SE and CYP51. Notably compounds did not show the genotoxicity through plasmid binding assay. Finally, this study of molecular docking, ADME/T prediction and the construction of 3D QSAR model were performed. These results can point out the direction for further optimization of the lead compound.     

New triazole derivatives as antifungal agents: synthesis via click reaction, in vitro evaluation and molecular docking studies

A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propanols (5a-5y) which are analogues of fluconazole, have been designed and synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition on the basis of computational docking experiments to the active site of the cytochrome P450 14α-demethylase (CYP51). The in vitro antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi. Compound 5l showed the best antifungal activities.     

Synthesis,Antifungal Activity,and Molecular Simulation Study of L–Carvone-Derived 1,3,4-Oxadiazole-Thioether Compounds

To discover potent antifungal molecules with new and distinctive structures, 20 novel L-carvone-derived 1,3,4-oxadiazole-thioether compounds 5 a – 5 t were synthesized through multi-step reaction of L-carvone, and their structures were confirmed by FT-IR, ¹H-NMR, ¹³C-NMR, and HR-MS. The antifungal activities of compounds 5 a – 5 t were preliminarily tested by in vitro method, and the results indicated that all of the title compounds displayed certain antifungal activities against the eight tested plant fungi, especially for P. piricola. Among them, compound 5 i (R=p-F) with the most significant antifungal activity deserved further study for discovering and developing novel natural product-based antifungal agents. Moreover, two molecular simulation technologies were employed for the investigation of their structure–activity relationships (SARs). Firstly, a reasonable and effective 3D-QSAR model was established by the comparative molecular field (CoMFA) method, and the relationship of the substituents linked with the benzene rings and the inhibitory activities of the title compounds against P. piricola was elucidated. Then, the binding mode of compound 5 i (R=p-F) and its potential biological target (CYP51) was simulated by molecular docking, and it was found that compound 5 i could readily bind with CYP51 in the active site, and the ligand-receptor interactions involved three hydrogen bonds and several hydrophobic effects.     

Design,synthesis, and biological evaluation of novel 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols

Based on the results of computational docking to the active site of the cytochrome P450 14α-demethylase (CYP51), a series of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols as analogs of fluconazole were designed, synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against eight human pathogenic fungi in vitro showed that all the title compounds exhibited excellent activities with broad spectrum.     

Homology modeling,molecular docking and spectra assay studies of sterol 14α-demethylase from <Emphasis Type="Italic">Penicillium digitatum</Emphasis>

Sterol 14α-demethylase from Penicillium digitatum (PdCYP51) is a prime target of antifungal drugs for citrus disease in plants. To design novel antifungal compounds, a homology model of PdCYP51 was constructed using the recently reported crystal structure of human CYP51 as the template. Molecular docking was performed to investigate the interaction of four commercial fungicides with the modeled enzyme. The side chain of these compounds interplayed with PdCYP51 mainly through hydrophobic and van der Waals interactions. Biochemical spectra analysis of inhibitors combined with PdCYP51 are also compatible with the docking results. This is the first molecular modeling for PdCYP51 based on the eukaryotic crystal structure of CYP51. The structural information and binding site mapping of PdCYP51 for different inhibitors obtained from this study could aid in screening and designing new antifungal compounds targeting this enzyme.     

New vinyl-1,2,4-triazole derivatives as antimicrobial agents: Synthesis,biological evaluation and molecular docking studies

1,2,4-Triazole is a very important scaffold in medicinal chemistry due to the wide spectrum of biological activities and mainly antifungal activity of 1,2,4-triazole derivatives. The main mechanism of antifungal action of the latter is inhibition of 14-alpha-demethylase enzyme (CYP51). The current study presents synthesis and evaluation of eight triazole derivatives for their antimicrobial activity. Docking studies to elucidate the mechanism of action were also performed. The designed compounds were synthesized using classical methods of organic synthesis. The in vivo evaluation of antimicrobial activity was performed by microdilution method. All tested compounds showed good antibacterial activity with MIC and MBC values ranging from 0.0002 to 0.0069 mM. Compound 2 h appeared to be the most active among all tested with MIC at 0.0002–0.0033 mM and MBC at 0.0004–0.0033 mM followed by compounds 2f and 2g. The most sensitive bacterium appeared to be Xanthomonas campestris while Erwinia amylovora was the most resistant. The evaluation of antifungal activity revealed that all compounds showed good antifungal activity with MIC values ranging from 0.02 mM to 0.52 mM and MFC from 0.03 mM to 0.52 mM better than reference drugs ketoconazole (MIC and MFC values at 0.28–1.88 mM and 0.38 mM to 2.82 mM respectively) and bifonazole (MIC and MFC values at 0.32–0.64 mM and 0.64–0.81 mM). The best antifungal activity is displayed by compound 2 h with MIC at 0.02–0.04 mM and MFC at 0.03–0.06 mM while compound 2a showed the lowest activity. The results showed that these compounds could be lead compounds in search for new potent antimicrobial agents. Docking studies confirmed experimental results.     

Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B′ helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.     

Novel ketoconazole analogues based on the replacement of 2,4-dichlorophenyl group with 1,4-benzothiazine moiety: design, synthesis, and microbiological evaluation

As a part of a program to develop novel antifungal agents, new compounds which incorporate the 1,4-benzothiazine moiety into the structure of ketoconazole (KTZ) were prepared. These compounds were computationally investigated to assess whether the 1,4-benzothiazine moiety was a suitable bioisosteric replacement for the 2,4-dichlorophenyl group of KTZ in order to obtain a more potent inhibition of CYP51 enzyme of Candida albicans. Results of preliminary microbiological studies show that the racemic cis-7 analogue has a good in vivo activity, comparable to that of KTZ, but the best activity was observed in the racemic trans-7 analogue.     

Synthesis and antifungal activities in vitro of novel pyrazino [2,1-a] isoquinolin derivatives

A series of novel pyrazino[2,1-a]isoquinolin compounds were designed and synthesized, and their antifungal activities in vitro were evaluated. The results showed that all of the compounds exhibited antifungal activities. Some of them exhibited stronger antifungal activities than that of lead compounds and among them compound 11b was the most potent one, which showed more potent than that of the active control fluconazole to the four of the five tested fungi. The studies presented here provide a new structural type for the development of novel antifungal agents.     

Design,synthesis and evaluation of benzoheterocycle analogues as potent antifungal agents targeting CYP51

To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compound 1, a series of benzoheterocycle analogues were designed, synthesized and evaluated for their in vitro antifungal activity. The most promising compounds 13s and 14a exhibited excellent antifungal activity against C. albicans, C. neoformans, A. fumigatus and fluconazole-resistant C. albicans strains, that was superior or comparable to those of the reference drugs fluconazole and voriconazole. GC–MS analyses suggested that the novel compound 13s might have a similar mechanism to fluconazole by inhibiting fungal lanosterol 14α-demethylase (CYP51). Furthermore, compounds 13s and 14a exhibited low inhibition profiles for various human cytochrome P450 isoforms as well as excellent blood plasma stability.     

Functional expression and characterization of CYP51 from dandruff-causing Malassezia globosa

Malassezia globosa is one of the most common yeasts to cause various human skin diseases including dandruff and seborrheic dermatitis. Genomic analysis of M. globosa revealed four putative cytochrome P450 (CYP) enzymes. Here, we report the purification and characterization of recombinant CYP51, a putative lanosterol 14α-demethylase, from M. globosa. The M. globosa CYP51 was expressed heterologously in Escherichia coli, followed by purification. Purified CYP51 showed a typical reduced CO-difference spectrum of P450, with a maximum absorption at 447?nm. Purified CYP51 exhibited tight binding to azole antifungal agents such as ketoconazole, econazole, fluconazole, or itraconazole, with K(d) values around 0.26-0.84?μM, which suggests that CYP51 is an orthologous target for antifungal agents in the M. globosa. In addition, three mutations (Y127F, A169S, and K176N) in the amino acid sequence of M. globosa CYP51 were identified in one of the azole-resistant strains. Homology modeling of M. globosa CYP51 suggested that the Y127F mutation may influence the resistance to azoles by blocking substrate access channels. Taken together, functional expression and characterization of the CYP51 enzyme can provide a fundamental basis for a specific antifungal drug design for dandruff caused by M. globosa.     

玉米黑粉菌cyp51基因结构分析与克隆表达

通过生物信息学手段分析cyp51基因结构,并根据GenBank登记的玉米黑粉茼cyp51 DNA序列,设计cyp51引物和两对分别截短不同跨膜区的突变体引物,构建了多种重组表达质粒及突变体重组表达质粒.选用不同宿主菌包括Escherichia coli BL21(DE3).BL21(DE3)pLysS和Rosetta(DE3)诱导表达并优化条件.SDS-PAGE分析结果表明:只有突变体pET32-YH-35能够在E. coli BL21(DE3)中高效表达(30℃,0.5 mmol/L IPRG诱导).通过与戊唑醇等4种商品化杀菌剂农药和14种XF系列农药先导化合物的紫外结合光谱分析表明:重组蛋白具有生物学活性.其中一种XF系列化合物的结合常数接近商品化杀菌剂,有可能开发为新的杀菌剂,为设计开发新型高效抗真菌新药提供了理论依据.     

Synthesis and in vitro antifungal activities of new 3-substituted benzopyrone derivatives

A series of new benzopyrone compounds were designed and synthesized and their antifungal activities in vitro were evaluated. The results showed that the benzopyrone derivatives with short terminal alkyl chain exhibited potent antifungal activity, which represent a novel class of promising leads for the development of novel non-azole antifungal agents. Compound 5j is the most potent one with MIC(80) value 1.5 μg/mL against Trichophyton rubrum. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the compounds. The designed compounds interact with CA-CYP51 through hydrophobic and van der Waals interactions.     

Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents

The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14alpha-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC(50) values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0. 2 microM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC(50) values of 0.008 and 0.0076 microM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(-) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra.     

Design and optimization of highly-selective,broad spectrum fungal CYP51 inhibitors

While the orally-active azoles such as fluconazole and posaconazole are effective antifungal agents, they potently inhibit a broad range of off-target human cytochrome P450 enzymes (CYPs) leading to various safety issues (e.g., drug-drug interactions, liver, and reproductive toxicities). Recently we described the rationally-designed, antifungal agent VT-1161 that is more selective for fungal CYP51 than related human CYP enzymes such as CYP3A4. Herein, we describe the use of a homology model of Aspergillus fumigatus to design and optimize a novel series of highly selective, broad spectrum fungal CYP51 inhibitors. This series includes the oral antifungal VT-1598 that exhibits excellent potency against yeast, dermatophyte, and mold fungal pathogens.     

5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. Design,synthesis, molecular docking and evaluation

In continuation of our efforts to develop new compounds with antimicrobial properties we describe design, synthesis, molecular docking study and evaluation of antimicrobial activity of seventeen novel 2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-thiazolidin-4-ones. All compounds showed antibacterial activity against eight Gram positive and Gram negative bacterial species. Twelve out of seventeen compounds were more potent than streptomycin and all compounds exhibited higher potency than ampicillin. Compounds were also tested against three resistant bacterial strains: MRSA, P. aeruginosa and E. coli. The best antibacterial potential against ATCC and resistant strains was observed for compound 8 (2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(4-nitrobenzylidene)-1,3thiazolidin-4-one). The most sensitive bacterium appeared to be S. typhimirium, followed by B. cereus while L. monocitogenes and M. flavus were the most resistant. Compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited antifungal activity better than the reference drugs bifonazole and ketokonazole (3-115 times). It was found that compound 8 appeared again to be the most potent. Molecular docking studies on E. coli MurB, MurA as well as C. albicans CYP 51 and dihydrofolate reductase were used for the prediction of mechanism of antibacterial and antifungal activities confirming the experimental results.     

Evolutionary trace analysis of CYP51 family: implication for site-directed mutagenesis and novel antifungal drug design

Lanosterol 14α-demethylase (CYP51) is an essential enzyme in the fungal life cycle and also an important target for the antifungal drug development. Based on the multiple sequence alignments of CYP51 family, an evolutionary tree of the CYP51 family was constructed by the evolutionary trace (ET) method. The identified trace residues could provide a reliable and rational guide to the design of CYP51 mutations and give more information about the detailed mechanism of substrate (drug) recognition and binding. The reliability of ET analysis to identify residues of functional importance was validated by the reported site-directed mutagenesis studies of CYP51s. Several residues in the active site were also validated by our mutagenesis studies. Mapping the identified trace residues onto the active site of the modeled structure of Candida albicans CYP51 (CACYP51) may provide useful information for the design of novel antifungal agents.     

Synthesis, in vitro evaluation and molecular docking studies of new triazole derivatives as antifungal agents

On the basis of the active site of lanosterol 14α-demethylase from Candida albicans (CACYP51), a series of 1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(4H)-one derivatives were synthesized as fluconazole analogs. Results of the preliminary antifungal tests against eight human pathogenic fungi in vitro showed that these compounds exhibited activities to some extent, and some displayed excellent antifungal activities against C. albicans than reference drug fluconazole. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the target compounds. The designed compounds interact with CACYP51 through hydrophobic, van der Waals and hydrogen-bonding interactions.     

New azoles with antifungal activity: Design, synthesis, and molecular docking

In order to search for many target compounds with excellent activities, a series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluoro-phenyl)-3-[(4-substituted phenyl)-piperazin-1-yl]-propan-2-ols were designed, synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against eight human pathogenic fungi in vitro showed that all the title compounds exhibited excellent activities with broad spectrum. Moreover, a molecular model for the binding between 5a and the active site of CACYP51 was provided based on the computational docking results.