Fungal Lanosterol 14α-demethylase Research Articles

Sterol profiling of Leishmania parasites using a new HPLC-tandem mass spectrometry-based method and antifungal azoles as chemical probes reveals a key intermediate sterol that supports a branched ergosterol biosynthetic pathway

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect.

Discovery of 1,2,3-selenadiazole analogues as antifungal agents using a scaffold hopping approach

With the increasing incidence of antifungal resistance, new antifungal agents having novel scaffolds hence are in an urgent need to combat infectious diseases caused by multidrug-resistant (MDR) pathogens. In this study, we reported the design, synthesis, and pharmacological evaluation of novel 1,2,3-selenadiazole analogues by scaffold hopping strategy. Preliminary results of antifungal activity demonstrated that the new class of compounds showed broad-spectrum fungistatic and fungicidal activity. Most importantly, these newly synthesized compounds can eliminate these azole-resistant fungi and inhibit the formation of C. albicans biofilm. In particular, compound S07 showed promising antifungal activity against five azole-resistant strains with MIC values ranging from 4 to 32 μg/mL. Then, further target identification and mechanistic studies indicated that representative compound S07 exert its inhibitory activity by inhibiting fungal lanosterol 14α-demethylase enzyme (CYP51). Interestingly, representative compounds showed low cytotoxicity on mammalian cell lines. In addition, the molecular docking studies elucidated the binding modes of these compounds toward CYP51. Altogether, these results suggest that compound S07 with novel skeleton is a promising CYP51 inhibitor for treatment of fungal infections.

DFT Based Pharmacokinetic, Molecular Docking, and ADMET Studies of Some Glucopyranoside Esters

Monosaccharide esters (MEs) are getting more attention from bioorganic chemists due to their biodegradable and drug-likeness properties. As a consequence, carbohydrate derivatives (sugar-based esters, SEs) are an essential part of medicinal chemistry. In this context, density functional theory (DFT) with B3LYP/ 3-21G has been employed to optimize the methyl 4,6-O-benzylidene-α-D-glucopyranoside (3) of methyl α-D-glucopyranoside (2) and its protected acyl esters 4-6. The prediction of activity spectra for substances (PASS) of these compounds showed better antifungal functionalities than the antibacterial potentiality. Thermodynamic properties and molecular electrostatic potential (MEP) of these MEs indicated their stability and both the electrophilic and nucleophilic attack sites. Due to their better antifungal potentiality, molecular docking was conducted against fungal protein lanosterol 14α-demethylase (3JUS), and SARS-CoV-2 main protease (6LU7) along with absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies. The study indicated a better binding affinity of some esters compared to the standard antifungal and COVID-19 related drug hydroxychloroquine (HCQ).

Azole derivatives with naphthalene showing potent antifungal effects against planktonic and biofilm forms of Candida spp.: an in vitro and in silico study.

Candida infections pose a serious public health threat due to increasing drug resistance. Azoles are first-line antifungal drugs for fungal infections. In this study, we tested an in-house azole collection incorporating naphthalene ring to find hits against planktonic and biofilm forms of resistant Candida spp. In the collection, potent derivatives were identified against the susceptible strains of Candida with minimum inhibitory concentration (MIC) values lower than those of the reference drug, fluconazole. MIC values of 0.125 μg/ml against C. albicans, 0.0625 μg/ml against C. parapsilosis, and 2 μg/ml against C. krusei, an intrinsically azole-resistant non-albicans Candida, were obtained. Some of the derivatives were highly active against fluconazole-resistant clinical isolate of C. tropicalis. Inhibition of C. albicans biofilms was also observed at 4 μg/ml similar as amphotericin B, the reference drug known for its antibiofilm activity. Through molecular docking studies, affinities and key interactions of the compounds with fungal lanosterol 14α-demethylase (CYP51), the target enzyme of azoles, were predicted. The interactions of imidazole with heme cofactor and of the naphthalene with Tyr118 were highlighted in line with the literature data. As a result, this study proves the importance of naphthalene for the antifungal activity of azoles against Candida spp. in both planktonic and biofilm forms.

Carbohydrate hitched imidazoles as agents for the disruption of fungal cell membrane

The fungal diseases represent an increasing global health burden and have transformed from a rare curiosity to the leading cause of human mortality. The present manuscript reports the antifungal potential of two novel compounds possessing a carbohydrate and an imidazole moiety. Antifungal susceptibility test determined the growth inhibition potential of the synthesized compounds against Aspergillus niger 9689 and it was observed that compounds D and E gave an antifungal inhibitory index of 66.66 and 56.67% respectively. Further, ultra-structure analysis of the treated fungal mycelia through scanning electron microscope (SEM) and confocal microscopy indicated significant membrane permeability and disintegration of fungal cell membrane, thus highlighting the probable role of the synthesized compounds as inhibitors of fungal lanosterol 14α-demethylase. In silico studies corroborated with the in-vitro results, as the synthesized compounds interacted with the critical amino acids present at the active site of the fungal enzyme (lanosterol 14α-demethylase).

Novel nucleosides as potential inhibitors of fungal lanosterol 14α-demethylase: an in vitro and in silico study.

Aim: The global burden of fungal infections has transitioned from a case-specific observation to a major cause of high human mortality. Therefore, novel compounds with innovative methodologies need to be synthesized and evaluated for their antifungal potential to keep pace with the current clinical demands. Results: An efficient synthetic pathway was developed for the synthesis of 21 synthetic novel nucleosides. Two compounds had significant antifungal effect on Aspergillus fumigatus 3007, which was comparable to fluconazole. The experimental data (confocal microscopy, ultrahigh-performance liquid chromatography and flow cytometry) demonstrated the inhibition of fungal lanosterol 14α-demethylase. Conclusion: Owing to the therapeutic relevance of the synthesized nucleosides and simplicity of the procedure, the method may find its potential application for synthesis of antifungal agents.

Identification of novel antifungal agents: antimicrobial evaluation, SAR, ADME\u2013Tox and molecular docking studies of a series of imidazole derivatives

Thirty-four imidazole-based compounds synthesized by one-pot catalytic method were evaluated for their antifungal and antibacterial activities against several fungal and bacterial strains. None of the compounds had antibacterial activity. Interestingly, compounds 1, 2, 3, 10 and 15 displayed a strong antifungal activity against all the tested fungal species, while compounds 5, 7, 9, 11, 21 and 27 showed a moderate antifungal activity. To better understand the biological activity of the most active compounds ADME–Tox and molecular docking studies were carried out. Interestingly, compounds 1, 2, 3, 7, 10 and 15 showed excellent bioavailability. In addition, compounds 1, 2 and 3, exhibited good toxicity profiles. Docking studies of the two most active compounds 2 (IC50 of 95 ± 7.07 μM) and 10 (IC50 of 235 ± 7.07 μM) suggested that they might act by inhibiting the fungal lanosterol 14α-demethylase. Therefore, these novel antifungal agents merit further characterization for the development of new antifungal therapeutics.

Discovery of new azoles with potent activity against Candida spp. and Candida albicans biofilms through virtual screening

Systemic candidiasis is a rampant bloodstream infection of Candida spp. and C. albicans is the major pathogen isolated from infected humans. Azoles, the most common class of antifungals which suffer from increasing resistance, and especially intrinsically resistant non-albicans Candida (NAC) species, act by inhibiting fungal lanosterol 14α-demethylase (CYP51). In this study we identified a number of azole compounds in 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol/ethanone oxime ester structure through virtual screening using consensus scoring approach, synthesized and tested them for their antifungal properties. We reached several hits with potent activity against azole-susceptible and azole-resistant Candida spp. as well as biofilms of C. albicans. 5i's minimum inhibitor concentration (MIC) was 0.125 μg/ml against C. albicans, 0.5 μg/ml against C. krusei and 1 μg/ml against azole-resistant C. tropicalis isolate. Considering the MIC values of fluconazole against these fungi (0.5, 32 and 512 μg/ml, respectively), 5i emerged as a highly potent derivative. The minimum biofilm inhibitor concentration (MBIC) of 5c, 5j, and 5p were 0.5 μg/ml (and 5i was 2 μg/ml) against C. albicans biofilms, lower than that of amphotericin B (4 μg/ml), a first-line antifungal with antibiofilm activity. In addition, the active compounds showed neglectable toxicity to human monocytic cell line. We further analyzed the docking poses of the active compounds in C. albicans CYP51 (CACYP51) homology model catalytic site and identified molecular interactions in agreement with those of known azoles with fungal CYP51s and mutagenesis studies of CACYP51. We observed the stability of CACYP51 in complex with 5i in molecular dynamics simulations.

Novel imidazole derivatives as antifungal agents: Synthesis, biological evaluation, ADME prediction and molecular docking studies

A series of 2-(substituteddithiocarbamoyl)-N-[4-((1H-imidazol-1-yl)methyl)phenyl]acetamide derivatives was designed and synthesized to combat the increasing incidence of drug-resistant fungal infections. All synthesized compounds were characterized by IR, 1H-NMR, 13C-NMR, and HRMS spectra and elemental analyses. Antifungal activity tests were performed against four different fungal strains. Molecular docking studies were performed to investigate the mode of action towards the fungal lanosterol 14α-demethylase, a cytochrome P450-dependent enzyme. ADME studies were carried out and a connection between activities and physicochemical properties of the target compounds was determined. Most of the final compounds exhibited significant activity against Candida albicans and Candida krusei with MIC50 value 12.5 μg/mL. The results of in vitro anti-Candida activity, a docking study and ADME prediction revealed that the newly synthesized compounds have potential anti-Candida activity and evidenced the most active derivative, 5b (2-Pyrrolidinthiocarbonylthio-N-[4-((1H-imidazol-1-yl)methyl)phenyl]acetamide), which can be further optimized as a lead compound.

Azole Resistance Reduces Susceptibility to the Tetrazole Antifungal VT-1161.

Tetrazole antifungals designed to target fungal lanosterol 14α-demethylase (LDM) appear to be effective against a range of fungal pathogens. In addition, a crystal structure of the catalytic domain of Candida albicans LDM in complex with the tetrazole VT-1161 has been obtained. We have addressed concern about artifacts that might arise from crystallizing VT-1161 with truncated recombinant CYP51s and measured the impact on VT-1161 susceptibility of genotypes known to confer azole resistance. A yeast system was used to overexpress recombinant full-length Saccharomyces cerevisiae LDM with a C-terminal hexahistidine tag (ScLDM6×His) for phenotypic analysis and crystallographic studies with VT-1161 or with the widely used triazole drug posaconazole (PCZ). We determined the effect of characterized mutations in LDM on VT-1161 activity and identified drug efflux pumps from fungi, including key fungal pathogens, that efflux VT-1161. The relevance of these yeast-based observations on drug efflux was verified using clinical isolates of C. albicans and Candida glabrata VT-1161 binding elicits a significant conformational difference between the full-length and truncated enzymes not found when posaconazole is bound. Susceptibility to VT-1161 is reduced by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) drug efflux pumps, the overexpression of LDM, and mutations within the drug binding pocket of LDM that affect interaction with the tertiary alcohol of the drug.

3,5-Disubstituted Thiazolidine-2,4-Diones: Design, Microwave-Assisted Synthesis, Antifungal Activity, and ADMET Screening

A series of 12 new thiazolidine-2,4-dione derivatives were obtained by microwave-assisted synthesis. All compounds were physicochemically characterized by quantitative elemental C, H, N, S analysis and spectral data (mass spectrometry [MS], infrared [IR], and nuclear magnetic resonance [NMR]), with the results being in agreement with the expected data. An in vitro screening performed on Candida albicans ATCC 10231 showed their moderate antifungal activity, which was further investigated by determining the minimum inhibitory concentration and minimum fungicidal concentration values for the most active compounds on four strains of Candida. The molecular docking studies, performed against a fungal lanosterol 14α-demethylase, emphasized the importance of different molecular fragments in the compounds’ structures for their antifungal activity. The synthesized compounds were subjected to in silico screening for the prediction of their absorption, distribution, metabolism, excretion, and toxicity (ADMET) and molecular properties. The results of the antifungal activity assays, docking study, and ADMET predictions revealed that the synthesized compounds are potential anti-Candida agents that might act by interacting with the fungal lanosterol 14α-demethylase and could be further optimized and developed as 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.

New azole derivatives showing antimicrobial effects and their mechanism of antifungal activity by molecular modeling studies

Azole antifungals are potent inhibitors of fungal lanosterol 14α demethylase (CYP51) and have been used for eradication of systemic candidiasis clinically. Herein we report the design, synthesis, and biological evaluation of a series of 1-phenyl/1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethanol esters. Many of these derivatives showed fungal growth inhibition at very low concentrations. Minimal inhibition concentration (MIC) value of 15 was 0.125 μg/mL against Candida albicans. Additionally, some of our compounds, such as 19 (MIC: 0.25 μg/mL), were potent against resistant C. glabrata, a fungal strain less susceptible to some first-line antifungal drugs. We confirmed their antifungal efficacy by antibiofilm test and their safety against human monocytes by cytotoxicity assay. To rationalize their mechanism of action, we performed computational analysis utilizing molecular docking and dynamics simulations on the C. albicans and C. glabrata CYP51 (CACYP51 and CGCYP51) homology models we built. Leu130 and T131 emerged as possible key residues for inhibition of CGCYP51 by 19.

Design, Synthesis and Antifungal Activity Evaluation of New Thiazolin-4-ones as Potential Lanosterol 14α-Demethylase Inhibitors.

Twenty-three thiazolin-4-ones were synthesized starting from phenylthioamide or thiourea derivatives by condensation with α-monochloroacetic acid or ethyl α-bromoacetate, followed by substitution in position 5 with various arylidene moieties. All the synthesized compounds were physico-chemically characterized and the IR (infrared spectra), 1H NMR (proton nuclear magnetic resonance), 13C NMR (carbon nuclear magnetic resonance) and MS (mass spectrometry) data were consistent with the assigned structures. The synthesized thiazolin-4-one derivatives were tested for antifungal properties against several strains of Candida and all compounds exhibited efficient anti-Candida activity, two of them (9b and 10) being over 500-fold more active than fluconazole. Furthermore, the compounds’ lipophilicity was assessed and the compounds were subjected to in silico screening for prediction of their ADME-Tox properties (absorbtion, distribution, metabolism, excretion and toxicity). Molecular docking studies were performed to investigate the mode of action towards the fungal lanosterol 14α-demethylase, a cytochrome P450-dependent enzyme. The results of the in vitro antifungal activity screening, docking study and ADME-Tox prediction revealed that the synthesized compounds are potential anti-Candida agents that might act by inhibiting the fungal lanosterol 14α-demethylase and can be further optimized and developed as lead compounds.

Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14α-demethylase.

Emergence of fungal strains showing resistance to triazole drugs can make treatment of fungal disease problematic. Triazole resistance can arise due to single mutations in the drug target lanosterol 14α-demethylase (Erg11p/CYP51). We have determined how commonly occurring single site mutations in pathogenic fungi affect triazole binding using Saccharomyces cerevisiae Erg11p (ScErg11p) as a target surrogate. The mutations Y140F/H were introduced into full-length hexahistidine-tagged ScErg11p. Phenotypes and high-resolution X-ray crystal structures were determined for the mutant enzymes complexed with short-tailed (fluconazole and voriconazole) or long-tailed (itraconazole and posaconazole) triazoles and wild type enzyme complexed with voriconazole. The mutations disrupted a water-mediated hydrogen bond network involved in binding of short-tailed triazoles, which contain a tertiary hydroxyl not present in long-tailed triazoles. This appears to be the mechanism by which resistance to these short chain azoles occurs. Understanding how these mutations affect drug affinity will aid the design of azoles that overcome resistance.

Design, synthesis and evaluation of benzotriazole derivatives as novel antifungal agents.

Considering the need for discovery of new antifungal drugs with greater potency and broader spectrum of activity, a new series of 5-substituted benzotriazole derivatives were designed, through structure based design, as inhibitors of fungal cytochrome P450 lanosterol 14-α demethylase. These were further optimized by a combination of iterative medicinal chemistry principles and molecular docking. Based on the best docking scores, some benzotriazole derivatives were synthesized and characterized by IR, (1)H NMR and MS/MS. The molecules were evaluated for their antifungal action against Candida albicans by cup plate method and ergosterol quantification method by UV spectroscopy. Reasonably good correlation between docking scores and antifungal activity were observed. The computational predictions were in consensus with the experimental results.

Structural Insights into Binding of the Antifungal Drug Fluconazole to Saccharomyces cerevisiae Lanosterol 14α-Demethylase.

Infections by fungal pathogens such as Candida albicans and Aspergillus fumigatus and their resistance to triazole drugs are major concerns. Fungal lanosterol 14α-demethylase belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes. This monospanning bitopic membrane protein is involved in ergosterol biosynthesis and is the primary target of azole antifungal drugs, including fluconazole. The lack of high-resolution structural information for this drug target from fungal pathogens has been a limiting factor for the design of modified triazole drugs that will overcome resistance. Here we report the X-ray structure of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase in complex with fluconazole at a resolution of 2.05 Å. This structure shows the key interactions involved in fluconazole binding and provides insight into resistance mechanisms by revealing a water-mediated hydrogen bonding network between the drug and tyrosine 140, a residue frequently found mutated to histidine or phenylalanine in resistant clinical isolates.

Efinaconazole in the treatment of onychomycosis.

Efinaconazole 10% topical solution is a new triazole recently approved for the treatment of onychomycosis. It inhibits fungal lanosterol 14α-demethylase in the ergosterol biosynthesis pathway, has potent antifungal activity against dermatophytes, as well as activity against Candida spp. and non-dermatophyte molds, and showed promising results in clinical trials. This review summarizes the mechanism of action, in vitro and in vivo data, clinical trials, safety, and quality-of-life data of efinaconazole as it applies to the treatment of onychomycosis.

Design, synthesis and antifungal evaluation of novel benzimidazole tertiary amine type of fluconazole analogues

A novel series of compounds containing tertiary amine moiety, substituted benzimidazole and triazole ring, initial design by molecular docking study of this scaffold at the active site of the fungal enzymes lanosterol 14α-demethylase (homology modeled of C. albicans) was synthesized by microwave irradiation and characterized by Proton Nuclear Magnetic Resonance (1H NMR), Infra Red (IR), and Mass Spectroscopy (MS), and by elemental analysis. The screening of compound for in vitro (turbidimetric method) and in vivo (kidney burden test) antifungal activity against C. albicans revealed activity in many of the compounds as comparable to that of fluconazole.

Preclinical pharmacological profile of Eberconazole: A review and update

Eberconazole is a broad-spectrum imidazole antifungal agent used as a topical preparation in the management of cutaneous mycoses. In vitro studies have shown that eberconazole is effective against dermatophytes, candidiasis, yeasts (including those which are triazole resistant) and Pityriasis versicolor. It inhibits fungal lanosterol 14α-demethylase, thereby inhibiting ergosterol synthesis leading to inhibition of fungal growth. In addition to its antifungal activity, it is also effective against Gram-positive bacteria, a property that is useful clinically. It also possesses anti-inflammatory property thus making it a suitable agent in the clinical management of inflamed cutaneous mycoses. Topical application of eberconazole was well tolerated in preclinical studies without any report of delayed hypersensitivity or photosensitivity reactions. There were no phototoxic effects. There was no significant systemic absorption. Animal toxicity studies have shown that it is safe, and the No Observed Effect Level was 2 ml/kg body weight in tested animals. It was not mutagenic and shared similar cytotoxicity profile with other imidazole antifungal products studied. Penetration studies using synthetic membranes revealed that eberconazole intrasets showed less variation as compared to clotrimazole and terbinafine intrasets. Overall amount of eberconazole released was more compared to comparators. In vitro and preclinical studies have demonstrated better therapeutic efficacy with eberconazole than clotrimazole and ketoconazole.