Discovery of Novel α,ß-Unsaturated Amide Derivatives as Candidate Antifungals to Overcome Fungal Resistance.

In our previous study, coumarin-containing CYP51 inhibitor A32 demonstrated potent antiresistance activity. However, compound A32 demonstrated unsatisfied metabolic stability, necessitating modifications to overcome these limitations. In this study, α,ß-unsaturated amides were used to replace the unstable coumarin ring, which increased metabolic stability by four times while maintaining antifungal activity, including activity against resistant strains. Subsequently, the sterol composition analysis and morphological observation experiments indicated that the target of these novel compounds is lanosterol 14α-demethylase (CYP51). Meanwhile, biofilm growth was inhibited and resistance genes (ERG11, CDR1, CDR2, and MDR1) expression was downregulated to find out how the antiresistance works. Importantly, compound C07 demonstrated the capacity to stimulate reactive oxygen species, thus displaying potent fungicidal activity. Moreover, C07 exhibited encouraging effectiveness in vivo following intraperitoneal administration. Additionally, the most potent compound C07 showed satisfactory pharmacokinetic properties and low toxicity. These α,ß-unsaturated amide derivatives, particularly C07, are potential candidates for treating azole-resistant candidiasis.

Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1.

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus.

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3ß,6α-diol, the "toxic diol" responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly "toxic diol". We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.

Listen to what the animals say: a systematic review and meta-analysis of sterol 14-demethylase inhibitor efficacy for in vivo models of Trypanosoma cruzi infection.

Sterol 14-demethylase (CYP51) inhibitors, encompassing new chemical entities and repurposed drugs, have emerged as promising candidates for Chagas disease treatment, based on preclinical studies reporting anti-Trypanosoma cruzi activity. Triazoles like ravuconazole (RAV) and posaconazole (POS) progressed to clinical trials. Unexpectedly, their efficacy was transient in chronic Chagas disease patients, and their activity was not superior to benznidazole (BZ) treatment. This paper aims to summarize evidence on the global activity of CYP51 inhibitors against T. cruzi by applying systematic review strategies, risk of bias assessment, and meta-analysis from in vivo studies. PubMed and Embase databases were searched for original articles, obtaining fifty-six relevant papers meeting inclusion criteria. Characteristics of animal models, parasite strain, treatment schemes, and cure rates were extracted. Primary outcomes such as maximum parasitaemia values, survival, and parasitological cure were recorded for meta-analysis, when possible. The risk of bias was uncertain in most studies. Animals treated with itraconazole, RAV, or POS survived significantly longer than the infected non-treated groups (RR = 4.85 [3.62, 6.49], P < 0.00001), and they showed no differences with animals treated with positive control drugs (RR = 1.01 [0.98, 1.04], P = 0.54). Furthermore, the overall analysis showed that RAV or POS was not likely to achieve parasitological cure when compared with BZ or NFX treatment (OD = 0.49 [0.31, 0.77], P = 0.002). This systematic review contributes to understanding why the azoles had failed in clinical trials and, more importantly, how to improve the animal models of T. cruzi infection by filling the gaps between basic, translational, and clinical research.

Anhydroparthenin as a dual-target inhibitor against Sterol C-24 methyltransferase and Sterol 14-α demethylase of Leishmania donovani: A comprehensive in vitro and in silico study.

Parthenium hysterophorus plant has a diverse chemical profile and immense bioactive potential. It exhibits excellent pharmacological properties such as anti-cancer, anti-inflammatory, anti-malarial, microbicidal, and anti-trypanosomal. The present study aims to evaluate the anti-leishmanial potential and toxicological safety of anhydroparthenin isolated from P. hysterophorus. Anydroparthenin was extracted from the leaves of P. hysterophorus and characterized through detailed analysis of 1H, 13C NMR, and HRMS. Dye-based in vitro and ex vivo assays confirmed that anhydroparthenin significantly inhibited both promastigote and amastigote forms of the Leishmania donovani parasites. Both the cytotoxicity experiment and hemolytic assay revealed its non-toxic nature and safety index in the range of 10 to 15. Further, various mechanistic assays suggested that anhydroparthenin led to the generation of oxidative stress, intracellular ATP depletion, alterations in morphology and mitochondrial membrane potential, formation of intracellular lipid bodies, and acidic vesicles, ultimately leading to parasite death. As a dual targeting approach, computational studies and sterol quantification assays confirmed that anhydroparthenin inhibits the Sterol C-24 methyl transferase and Sterol 14-α demethylase proteins involved in the ergosterol biosynthesis in Leishmania parasites. These results suggest that anhydroparthenin could be a promising anti-leishmanial molecule and can be developed as a novel therapeutic stratagem against leishmaniasis.

Prevalence and genetic diversity of azole-resistant Malassezia pachydermatis isolates from canine otitis and dermatitis: A 2-year study.

Despite previous reports on the emergence of Malassezia pachydermatis strains with decreased susceptibility to azoles, there is limited information on the actual prevalence and genetic diversity of azole-resistant isolates of this yeast species. We assessed the prevalence of azole resistance in M. pachydermatis isolates from cases of dog otitis or skin disease attended in a veterinary teaching hospital during a 2-year period and analyzed the ERG11 (encoding a lanosterol 14-α demethylase, the primary target of azoles) and whole genome sequence diversity of a group of isolates that displayed reduced azole susceptibility. Susceptibility testing of 89 M. pachydermatis isolates from 54 clinical episodes (1-6 isolates/episode) revealed low minimum inhibitory concentrations (MICs) to most azoles and other antifungals, but 11 isolates from six different episodes (i.e., 12.4% of isolates and 11.1% of episodes) had decreased susceptibility to multiple azoles (fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, and/or voriconazole). ERG11 sequencing of these 11 azole-resistant isolates identified eight DNA sequence profiles, most of which contained amino acid substitutions also found in some azole-susceptible isolates. Analysis of whole genome sequencing (WGS) results revealed that the azole-resistant isolates from the same episode of otitis, or even different episodes affecting the same animal, were more genetically related to each other than to isolates from other dogs. In conclusion, our results confirmed the remarkable ERG11 sequence variability in M. pachydermatis isolates of animal origin observed in previous studies and demonstrated the value of WGS for disentangling the epidemiology of this yeast species.

We analyzed the prevalence and diversity of azole-resistant Malassezia pachydermatis isolates in a veterinary hospital. A low prevalence of multi-azole resistance (c.10% of isolates and cases) was found. Whole genome and ERG11 sequencing of resistant isolates revealed remarkable genetic diversity.

Novel Pyrido[4,3-d]pyrimidine Derivatives as Potential Sterol 14α-Demethylase Inhibitors: Design, Synthesis, Inhibitory Activity, and Molecular Modeling.

Thirty-four new pyrido[4,3-d]pyrimidine analogs were designed, synthesized, and characterized. The crystal structures for compounds 2c and 4f were measured by means of X-ray diffraction of single crystals. The bioassay results showed that most target compounds exhibited good fungicidal activities against Pyricularia oryzae, Rhizoctonia cerealis, Sclerotinia sclerotiorum, Botrytis cinerea, and Penicillium italicum at 16 µg/mL. Compounds 2l, 2m, 4f, and 4g possessed better fungicidal activities than the commercial fungicide epoxiconazole against B. cinerea. Their half maximal effective concentration (EC50) values were 0.191, 0.487, 0.369, 0.586, and 0.670 µg/mL, respectively. Furthermore, the inhibitory activities of the bioactive compounds were determined against sterol 14α-demethylase (CYP51). The results displayed that they had prominent activities. Compounds 2l, 2m, 4f, and 4g also showed better inhibitory activities than epoxiconazole against CYP51. Their half maximal inhibitory concentration (IC50) values were 0.219, 0.602, 0.422, 0.726, and 0.802 µg/mL, respectively. The results of molecular dynamics (MD) simulations exhibited that compounds 2l and 4f possessed a stronger affinity to CYP51 than epoxiconazole.

Design, Synthesis, Bioactive Evaluation, and Molecular Dynamics Simulation of Novel 4H-Pyrano[3,2-c]pyridine Analogues as Potential Sterol 14α-Demethylase (CYP51) Inhibitors.

To discover potential sterol 14α-demethylase (CYP51) inhibitors, thirty-four unreported 4H-pyrano[3,2-c]pyridine derivatives were designed and synthesized. The assay results indicated that most compounds displayed significant fungicidal activity against Sclerotinia sclerotiorum, Colletotrichum lagenarium, Botrytis cinerea, Penicillium digitatum, and Fusarium oxysporum at 16 µg/mL. The half maximal effective concentration (EC50) values of compounds 7a, 7b, and 7f against B. cinerea were 0.326, 0.530, and 0.610, respectively. Namely, they had better antifungal activity than epoxiconazole (EC50 = 0.670 µg/mL). Meanwhile, their half maximal inhibitory concentration (IC50) values against CYP51 were 0.377, 0.611, and 0.748 µg/mL, respectively, representing that they also possessed better inhibitory activities than epoxiconazole (IC50 = 0.802 µg/mL). The fluorescent quenching tests of proteins showed that 7a and 7b had similar quenching patterns to epoxiconazole. The molecular dynamics simulations indicated that the binding free energy of 7a and epoxiconazole to CYP51 was -35.4 and -27.6 kcal/mol, respectively.

Functional Plasticity, Redundancy, and Specificity of Lanosterol 14α-Demethylase in Regulating the Sensitivity to DMIs in Calonectria ilicicola.

Cytochrome P450 sterol 14α-demethylase (CYP51) is a key enzyme involved in the sterol biosynthesis pathway and serves as a target for sterol demethylation inhibitors (DMIs). In this study, the 3D structures of three CPY51 paralogues from Calonectria ilicicola (C. ilicicola) were first modeled by AlphaFold2, and molecular docking results showed that CiCYP51A, CiCYP51B, or CiCYP51C proteins individually possessed two active pockets that interacted with DMIs. Our results showed that the three paralogues play important roles in development, pathogenicity, and sensitivity to DMI fungicides. Specifically, CiCYP51A primarily contributed to cell wall integrity maintenance and tolerance to abiotic stresses, and CiCYP51B was implicated in sexual reproduction and virulence, while CiCYP51C exerted negative regulatory effects on sterol 14α-demethylase activity within the ergosterol biosynthetic pathway, revealing its genus-specific function in C. ilicicola. These findings provide valuable insights into developing rational strategies for controlling soybean red crown rot caused by C. ilicicola.

Promising Antileishmanial Activity of Micromeria nervosa Essential Oil: In Vitro and In Silico Studies.

The present study aimed to evaluate the leishmanicidal potential of the essential oil (EO) of Micromeria (M.) nervosa and to investigate its molecular mechanism of action by qPCR. Furthermore, in silicointeraction study of the major M. nervosa EO compounds with the enzyme cytochrome P450 sterol 14α-demethylase (CYP51) was also performed. M. nervosa EO was analyzed by gas chromatography-mass spectrometry (GC-MS). Results showed that α-pinene (26.44%), t-cadinol (26.27%), caryophyllene Oxide (7.73 ± 1.04%), and α-Cadinene (3.79 ± 0.12%) are the major compounds of M. nervosa EO. However, limited antioxidant activity was observed, as this EO was ineffective in neutralizing DPPH free radicals and in inhibiting ß-carotene bleaching. Interestingly, it displayed effective leishmanicidal potential against promastigote (IC50 of 6.79 and 5.25 µg/mL) and amastigote (IC50 of 8.04 and 7.32 µg/mL) forms of leishmania (L.) infantum and L. major, respectively. Molecular mechanism investigation showed that M. nervosa EO displayed potent inhibition on the thiol regulatory pathway. Furthermore, a docking study of the main components of the EO with cytochrome P450 sterol 14α-demethylase (CYP51) enzyme revealed that t-cadinol exhibited the best binding energy values (-7.5 kcal/mol), followed by α-cadinene (-7.3 kcal/mol) and caryophyllene oxide (-7 kcal/mol). These values were notably higher than that of the conventional drug fluconazole showing weaker binding energy (-6.9 kcal/mol). These results suggest that M. nervosa EO could serve as a potent and promising candidate for the development of alternative antileishmanial agent in the treatment of leishmaniasis.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

An Insight into the Repurposing of Phytoconstituents obtained from Delhi's Aravalli Biodiversity Park as Antifungal Agents.

The global prevalence of fungal infections is alarming in both the pre- and post- COVID period. Due to a limited number of antifungal drugs, there are hurdles in treatment strategies for fungal infections due to toxic potential, drug interactions, and the development of fungal resistance. All the antifungal targets (existing and newer) and pipeline molecules showing promise against these targets are reviewed. The objective was to predict or repurpose phyto-based antifungal compounds based on a dual target inhibition approach (Sterol-14-α- demethylase and HSP-90) using a case study. In pursuit of repurposing the phytochemicals as antifungal agents, a team of researchers visited Aravalli Biodiversity Park (ABP), Delhi, India, to collect information on available medicinal plants. From 45 plants, a total of 1149 ligands were collected, and virtual screening was performed using Schrodinger Suite 2016 software to get 83 hits against both the target proteins: Sterol-14-α-demethylase and HSP-90. After analysis of docking results, ligands were selected based on their interaction against both the target proteins and comparison with respective standard ligands (fluconazole and ganetespib). We have selected Isocarthamidin, Quercetin and Boeravinone B based on their docking score and binding interaction against the HSP-90 (Docking Score -9.65, -9.22 and -9.21, respectively) and 14-α-demethylase (Docking Score -9.19, -10.76 and -9.74 respectively). The docking protocol was validated and MM/GBSA studies depicted better stability of selected three ligands (Isocarthamidin, Quercetin, Boeravinone B) complex as compared to standard complex. Further, MD simulation studies were performed using the Desmond (67) software package version 2018-4. All the findings are presented as a case study for the prediction of dual targets for the repurposing of certain phytochemicals as antifungal agents.

Reduced Susceptibility to Azoles in Cryptococcus gattii Correlates with the Substitution R258L in a Substrate Recognition Site of the Lanosterol 14-α-Demethylase.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a life-threatening fungal infection affecting mostly immunocompromised patients. In fact, cryptococcal meningitis accounts for about 19% of AIDS-related deaths in the world. Because of long-term azole therapies to treat this mycosis, resistance to fluconazole leading to treatment failure and poor prognosis has long been reported for both fungal species. Among the mechanisms implicated in resistance to azoles, mutations in the ERG11 gene, encoding the azole target enzyme lanosterol 14-α-demethylase, have been described. This study aimed to establish the amino acid composition of ERG11 of Colombian clinical isolates of C. neoformans and C. gattii and to correlate any possible substitution with the in vitro susceptibility profile of the isolates to fluconazole, voriconazole, and itraconazole. Antifungal susceptibility testing results showed that C. gattii isolates are less susceptible to azoles than C. neoformans isolates, which could correlate with differences in the amino acid composition and structure of ERG11 of each species. In addition, in a C. gattii isolate with high MICs for fluconazole (64 µg/mL) and voriconazole (1 µg/mL), a G973T mutation resulting in the substitution R258L, located in substrate recognition site 3 of ERG11, was identified. This finding suggests the association of the newly reported substitution with the azole resistance phenotype in C. gattii. Further investigations are needed to determine the exact role that R258L plays in the decreased susceptibility to fluconazole and voriconazole, as well as to determine the participation of additional mechanisms of resistance to azole drugs. IMPORTANCE The fungal species Cryptococcus neoformans and C. gattii are human pathogens for which drug resistance or other treatment and management challenges exist. Here, we report differential susceptibility to azoles among both species, with some isolates displaying resistant phenotypes. Azoles are among the most commonly used drugs to treat cryptococcal infections. Our findings underscore the necessity of testing antifungal susceptibility in the clinical setting in order to assist patient management and beneficial outcomes. In addition, we report an amino acid change in the sequence of the target protein of azoles, which suggests that this change might be implicated in resistance to these drugs. Identifying and understanding possible mechanisms that affect drug affinity will eventually aid the design of new drugs that overcome the global growing concern of antifungal resistance.

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.

Structural modeling of cytochrome P450 51 from a deep-sea fish points to a novel structural feature in other CYP51s.

Cytochromes P450 (CYP), enzymes involved in the metabolism of endogenous and xenobiotic substrates, provide an excellent model system to study how membrane proteins with unique functions have catalytically adapted through evolution. Molecular adaptation of deep-sea proteins to high hydrostatic pressure remains poorly understood. Herein, we have characterized recombinant cytochrome P450 sterol 14α-demethylase (CYP51), an essential enzyme of cholesterol biosynthesis, from an abyssal fish species, Coryphaenoides armatus. C. armatus CYP51 was heterologously expressed in Escherichia coli following N-terminal truncation and purified to homogeneity. Recombinant C. armatus CYP51 bound its sterol substrate lanosterol giving a Type I binding spectra (KD 15 µM) and catalyzed lanosterol 14α-demethylation turnover at 5.8 nmol/min/nmol P450. C. armatus CYP51 also bound the azole antifungals ketoconazole (KD 0.12 µM) and propiconazole (KD 0.54 µM) as determined by Type II absorbance spectra. Comparison of C. armatus CYP51 primary sequence and modeled structures with other CYP51s identified amino acid substitutions that may confer an ability to function under pressures of the deep sea and revealed heretofore undescribed internal cavities in human and other non-deep sea CYP51s. The functional significance of these cavities is not known. PROLOGUE: This paper is dedicated in memory of Michael Waterman and Tsuneo Omura, who as good friends and colleagues enriched our lives. They continue to inspire us.

Oteseconazole: First Approved Orally Bioavailable and Selective CYP51 Inhibitor for the Treatment of Patients with Recurrent Vulvovaginal Candidiasis.

Oteseconazole was approved by the US FDA in April 2022. It is the first approved selective and orally bioavailable CYP51 inhibitor for the treatment of patients with recurrent Vulvovaginal candidiasis. Herein, we describe its dosage, administration, chemical structure, physical properties, synthesis, mechanism of action, and pharmacokinetics.

In silico prediction of Antifungal compounds from Natural sources towards Lanosterol 14-alpha demethylase (CYP51) using Molecular docking and Molecular dynamic simulation.

An increase in the occurrence of fungal infections throughout the world, as well as the rise of novel fungal strains and antifungal resistance to commercially available drugs, suggests that new therapeutic choices for fungal infections are needed. The purpose of this research was to find new antifungal candidates or leads of secondary metabolites derived from natural sources that could effectively inhibit the enzymatic activity of Candida albicans lanosterol 14-alpha demethylase (CYP51) while also having good pharmacokinetics. In silico prediction of the drug-likeness, chemo-informatics and enzyme inhibition indicate that the 46 compounds derived from fungi, sponges, plants, bacteria and algae sources have a high novelty to meet all five requirements of Lipinski's rules and impede enzymatic function. Among the 15 candidate molecules with strong binding affinity to CYP51 investigated by molecular docking simulation, didymellamide A-E compounds demonstrated the strongest binding energy against the target protein at -11.14, -11.46, -11.98, -11.98, and -11.50 kcal/mol, respectively. Didymellamide molecules bind to comparable active pocket sites of antifungal ketoconazole and itraconazole medicines by hydrogen bonds forming to Tyr132, Ser378, Met508, His377 and Ser507, and hydrophobic interactions with HEM601 molecule. The stability of the CYP51-ligand complexes was further investigated using molecular dynamics simulations that took into account different geometric features and computed binding free energy. Using the pkCSM ADMET descriptors tool, several pharmacokinetic characteristics and the toxicity of candidate compounds were assessed. The findings of this study revealed that didymellamides could be a promising inhibitor against these CYP51 protein. However, there is still a need for further in vivo and in vitro studies to support these findings.

Anti-Candida, docking studies, and in vitro metabolism-mediated cytotoxicity evaluation of Eugenol derivatives.

The high morbidity and mortality rates of Candida infections, especially among immunocompromised patients, are related to the increased resistance rate of these species and the limited therapeutic arsenal. In this context, we evaluated the anti-Candida potential and the cytotoxic profile of eugenol derivatives. Anti-Candida activity was evaluated on C. albicans and C. parapsilosis strains by minimum inhibitory concentration (MIC), scanning electron microscopy (SEM), and molecular docking calculations at the site of the enzyme lanosterol-14-α-demethylase active site, responsible for ergosterol formation. The cytotoxic profile was evaluated in HepG2 cells, in the presence and absence of the metabolizing system (S9 system). The results indicated compounds 1b and 1d as the most active ones. The compounds have anti-Candida activity against both strains with MIC ranging from 50 to 100 µg ml-1 . SEM analyses of 1b and 1d indicated changes in the envelope architecture of both C. albicans and C. parapsilosis like the ones of eugenol and fluconazole, respectively. Docking results of the evaluated compounds indicated a similar binding pattern of fluconazole and posaconazole at the lanosterol-14-α-demethylase binding site. In the presence of the S9 system, compound 1b showed the same cytotoxicity profile as fluconazole (1.08 times) and compound 1d had 1.23 times increase in cytotoxicity. Eugenol and other evaluated compounds showed a significant increase in cytotoxicity. Our results suggest compound 1b as a promising starting point candidate to be used in the design of new anti-Candida agent prototypes.

Prediction of novel and potent inhibitors of lanosterol 14-α demethylase.

Lanosterol 14-α demethylase (LDM) is one of the promising drug targets of azoles antifungal. In this study, we have screened a large number of small molecules from different chemical databases (ZINC, DrugBank, ChEMBL, and ChemDiv) to find out novel and potential inhibitors of LDM. As a result, from more than a hundred thousand molecules, the two best candidates, C1 (ZINC000299817826) and C3 (ZINC000095786149), were selected from the top-scoring compounds and further validated in Molecular Dynamic (MD) simulation. The Glide scores of C1 and C3 were -19.33 kcal/mol and -19.13 kcal/mol, suggesting that these compounds bind with LDM with higher binding affinity than the benchmark compound (itraconazole), which has a Glide score of -6.85 kcal/mol. Docking poses reveal that the compounds C1 and C3 bind to the outermost region of the LDM binding site, which can prevent the lanosterol from getting into the catalytic pocket. Furthermore, MD simulation studies were performed to assess the stability of C1 and C3 in complex with LDM and were found to be stable over the 100 nanosecond simulation time. Binding free energy calculated by the MMPBSA method suggested that the C3 forms a more stable complex with the LDM as close to the benchmark compounds. Among the top selected molecules, C1 and C3 were predicted to be the significant inhibitors of LDM.Communicated by Ramaswamy H. Sarma.