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.

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.

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.

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.

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.

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.

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.

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.

Synthesis of obtusifoliol and analogues as CYP51 substrates.

Sterol 14α-demethylases (CYP51s) are a ubiquitous superfamily of cytochrome P450 enzymes that play an essential role in sterol biosynthesis. As fungal CYP51s are the target of azole-based antifungal agents, which are facing the problem of increasing resistance, the substrate specificity of this enzyme subclass has recently garnered significant attention. Herein we report the first chemical synthesis of the final endogenous substrate of this enzyme class, obtusifoliol, in 1.3% yield across ten steps from a commercially available lanosterol mixture. Intermediates along this pathway provide a basis for further derivatisation of the sterol skeleton and future investigation into CYP51 inhibition to overcome pathogens' azole resistance.

The putative obtusifoliol 14α-demethylase OsCYP51H3 affects multiple aspects of rice growth and development.

Some members of the CYP51G subfamily has been shown to be obtusifoliol 14α-demethylase, key enzyme of the sterol and brassinosteroid (BR) biosynthesis, which mediate plant development and response to stresses. However, little is known about the functions of CYP51H subfamily in rice. Here, OsCYP51H3, an ortholog of rice OsCYP51G1 was identified. Compared with wild type, the mutants oscyp51H3 and OsCYP51H3-RNAi showed dwarf phenotype, late flowering, erected leaves, lower seed-setting rate, and smaller and shorter seeds. In contrast, the phenotypic changes of OsCYP51H3-OE plants are not obvious. Metabolomic analysis of oscyp51H3 mutant indicated that OsCYP51H3 may also encode an obtusifoliol 14α-demethylase involved in phytosterol and BR biosynthesis, but possibly not that of triterpenes. The RNA-seq results showed that OsCYP51H3 may affect the expression of a lot of genes related to rice development. These findings showed that OsCYP51H3 codes for a putative obtusifoliol 14α-demethylase involved in phytosterol and BR biosynthesis, and mediates rice development.

In silico Prediction and Pharmacokinetic Studies on Glucosinolates as a Potential Drug and Key Inhibitor Molecule for Lanosterol-14α- demethylase: A Fungal Membrane Biosynthesis Enzyme.

BACKGROUND: Glucosinolates (ß-thioglucoside-N-hydroxysulfates) are a water-soluble organic anion with sulfur- and nitrogen-containing glycosides which are found in abundance in Cruciferous plants. Ergosterol (ERG13) lanosterol-14α-demethylase protein has been targeted for inhibition studies as a key regulator enzyme of fungal membrane biosynthesis. OBJECTIVES: To understand the molecular mechanism of inhibition of Ergosterol (ERG13) lanosterol- 14α-demethylase by various phytochemicals from brassicales, i.e., glucosinolates and their potential role as putative drug molecules. METHODS: In this study, in silico analyses were performed to predict the molecular basis of various glucosinolates as a potential inhibitor of lanosterol-14α-demethylase protein, which is a key regulator of fungal membrane biosynthesis and its pharmacodynamics and toxicity profile. 3d structures of various glucosinolates were retrieved from PubChem, and the target protein, lanosterol-14α-demethylase (Pdb ID- 4lxj), was retrieved from the RCSB protein data bank. Molecular docking and interactions were carried out using the PyRx software using the AutoDOCK toolbar with default parameters. Dru- LiTo, ORISIS web servers were used to predict various drug likeliness predictions and Lipinski's Rule of 5, whereas admetSAR was used for prediction of toxicity, and PASS Program was used to study the antifungal and antimicrobial properties of these compounds. RESULTS: This study shows that among the different compounds screened, gluconasturtiin, Glucotropaeolin, and Indolylmethyl-Glucosinolate showed the highest binding energies of -8.7 kcal/mol, -8.5 kcal/mol, and -8.3 kcal/mol with the lanosterol-14α-demethylase, respectively. Further all the compounds follow the Lipinski's rule as well as they are found to be non-carcinogenic and non-cytotoxic in nature. These compounds also show antifungal properties. CONCLUSION: This study thus reveals that various glucosinolates interact with the ERG13 enzyme at various amino acid positions, which behaves as a catalytic site, thus indicates the probable mechanism of inactivation, and subsequently, these can be used as potential drug molecules. In vitro studies can be taken to further examine the utility of these compounds as antifungal agents.

The evaluation of the overexpression of the ERG-11, MDR-1, CDR-1, and CDR-2 genes in fluconazole-resistant Candida albicans isolated from Ahvazian cancer patients with oral candidiasis.

INTRODUCTION: Resistance to azole drugs has been observed in candidiasis due to their long-term use and poor response to treatment. Resistance to azole drugs in Candida albicans isolates is controlled by several genes including ERG11, CDR1, CDR2, and MDR1. In this study, the expression of the mentioned genes was evaluated in C. albicans isolates susceptible and resistant to fluconazole. METHODS: After identifying the Candida isolates using morphological and molecular methods, the minimum inhibitory concentration (MIC) and drug susceptibility were determined using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. RNA was then extracted and cDNA was synthesized from 24 C. albicans isolates from patients with cancer. Then, the mean expressions of these genes were compared in two groups using real-time polymerase chain reaction (RT-PCR). RESULTS: A total of 74 Candida isolates were obtained from the oral cavity of 61 cancer patients with oral candidiasis. After 24 h, 21.6% of the isolates were fluconazole-resistant, 10.8% were identified as dose-dependent, and the rest of the isolates (67.6%) were fluconazole-sensitive. The mean expressions of the CDR1 and MDR1 genes were significantly higher in the resistant isolates than in the sensitive ones. However, the ERG11 and CDR2 genes were not significantly increased in the resistant isolates. CONCLUSION: The increased mean expressions of the CDR1 and MDR1 genes had a greater effect on fluconazole resistance among the drug-resistant strains of C. albicans in chemotherapy patients. It seemed that the accumulation of chemotherapeutic drugs in this organism stimulated some regulatory factors and increased the expression of these two genes and ultimately helped to further increase their expression and resistance to fluconazole.

Matching mouse models to specific human liver disease states by comparative functional genomics of mouse and human datasets.

Omics has broadened our view of transcriptional and gene regulatory networks of multifactorial diseases, such as metabolism associated liver disease and its advanced stages including hepatocellular carcinoma. Identifying liver disease biomarkers and potential treatment targets makes use of experimental models, e.g. genetically engineered mice, which show molecular features of human pathologies but are experimentally tractable. We compared gene expression profiling data from human to our studies on transgenic mice with hepatocyte deletion of Cyp51 from cholesterol synthesis with the aim of identifying the human liver disease state best matched by the Cyp51 knockout model. Gene Expression Omnibus was used to identify relevant human datasets. We identified enriched and deregulated genes, pathways and transcription factors of mouse and human disease samples. Analysis showed a closer match of the Cyp51 knockout to the female patient samples. Importantly, CYP51 was depleted in both mouse and female human data. Among the enriched genes were the oxysterol-binding protein-related protein 3 (OSBPL3), which was enriched in all datasets, and the collagen gene COL1A2, which was enriched in both the mouse and one human dataset. KEGG and Reactome analyses revealed the most enriched pathway to be ECM-receptor interaction. Numerous transcription factors were differentially expressed in mice of both sexes and in the human female dataset, while depleted HNF4α and RXRα:PPARα-isoform1 were a hallmark in all cases. Our analysis exposed novel potential biomarkers, which may provide new avenues towards more personalized approaches and different targets in females and males. The analysis was only possible because of availability of open data resources and tools and broadly consistent annotation.

Phytochemical profiling of Piper crocatum and its antifungal mechanism action as Lanosterol 14 alpha demethylase CYP51 inhibitor: a review.

Mycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community. The development of resistant strains in Fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays. To control fungal infections, substances and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed. Especially from Piperaceae, which have long been used in India, China, and Korea to treat human ailments in traditional medicine. The purpose of this review is to describe the antifungal mechanism action from Piper crocatum and its phytochemical profiling against lanosterol 14a demethylase CYP51. The methods used to search databases from Google Scholar to find the appropriate databases using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Flow Diagram as a clinical information retrieval method. From 1.150.000 results searched by database, there is 73 final results article to review. The review shows that P. crocatum contains flavonoids, tannins, terpenes, saponins, polyphenols, eugenol, alkaloids, quinones, chavibetol acetate, glycosides, triterpenoids or steroids, hydroxychavikol, phenolics, glucosides, isoprenoids, and non-protein amino acids. Its antifungal mechanisms in fungal cells occur due to ergosterol, especially lanosterol 14a demethylase (CYP51) inhibition, which is one of the main target sites for antifungal activity because it functions to maintain the integrity and function of cell membranes in Candida. P. crocatum has an antifungal activity through its phytochemical profiling against fungal by inhibiting the lanosterol 14a demethylase, make damaging cell membranes, fungal growth inhibition, and fungal cell lysis.

Construction and activity evaluation of novel dual-target (SE/CYP51) anti-fungal agents containing amide naphthyl structure.

With the increase of fungal infection and drug resistance, it is becoming an urgent task to discover the highly effective antifungal drugs. In the study, we selected the key ergosterol bio-synthetic enzymes (Squalene epoxidase, SE; 14 α-demethylase, CYP51) as dual-target receptors to guide the construction of novel antifungal compounds, which could achieve the purpose of improving drug efficacy and reducing drug-resistance. Three different series of amide naphthyl compounds were generated through the method of skeleton growth, and their corresponding target products were synthesized. Most of compounds displayed the obvious biological activity against different Candida spp. and Aspergillus fumigatus. Among of them, target compounds 14a-2 and 20b-2 not only possessed the excellent broad-spectrum anti-fungal activity (MIC50, 0.125-2 µg/mL), but also maintained the anti-drug-resistant fungal activity (MIC50, 1-4 µg/mL). Preliminary mechanism study revealed the compounds (14a-2, 20b-2) could block the bio-synthetic pathway of ergosterol by inhibiting the dual-target (SE/CYP51) activity, and this finally caused the cleavage and death of fungal cells. In addition, we also discovered that compounds 14a-2 and 20b-2 with low toxic and side effects could exert the excellent therapeutic effect in mice model of fungal infection, which was worthy for further in-depth study.

Construction and activity evaluation of novel benzodioxane derivatives as dual-target antifungal inhibitors.

Ergosterol exert the important function in maintaining the fluidity and osmotic pressure of fungal cells, and its key biosynthesis enzymes (Squalene epoxidase, SE; 14 α-demethylase, CYP51) displayed the obvious synergistic effects. Therefore, we expected to discover the novel antifungal compounds with dual-target (SE/CYP51) inhibitory activity. In the progress, we screened the different kinds of potent fragments based on the dual-target (CYP51, SE) features, and the method of fragment-based drug discovery (FBDD) was used to guide the construction of three different series of benzodioxane compounds. Subsequently, their chemical structures were synthesized and evaluated. These compounds displayed the obvious biological activity against the pathogenic fungal strains. Notably, target compounds 10a-2 and 22a-2 possessed the excellent broad-spectrum anti-fungal activity (MIC50, 0.125-2.0 µg/mL) and the activity against drug-resistant strains (MIC50, 0.5-2.0 µg/mL). Preliminary mechanism studies have confirmed that these compounds effectively inhibited the dual-target (SE/CYP51) activity, they could cause fungal rupture and death by blocking the bio-synthetic pathway of ergosterol. Further experiments discovered that compounds 10a-2 and 22a-2 also maintained a certain of anti-fungal effect in vivo. In summary, this study not only provided the new dual-target drug design strategy and method, but also discover the potential antifungal compounds.

Relaxed Substrate Requirements of Sterol 14α-Demethylase from Naegleria fowleri Are Accompanied by Resistance to Inhibition.

Naegleria fowleri is the protozoan pathogen that causes primary amoebic meningoencephalitis (PAM), with the death rate exceeding 97%. The amoeba makes sterols and can be targeted by sterol biosynthesis inhibitors. Here, we characterized N. fowleri sterol 14-demethylase, including catalytic properties and inhibition by clinical antifungal drugs and experimental substituted azoles with favorable pharmacokinetics and low toxicity. None of them inhibited the enzyme stoichiometrically. The highest potencies were displayed by posaconazole (IC50 = 0.69 µM) and two of our compounds (IC50 = 1.3 and 0.35 µM). Because both these compounds penetrate the brain with concentrations reaching minimal inhibitory concentration (MIC) values in an N. fowleri cellular assay, we report them as potential drug candidates for PAM. The 2.1 Å crystal structure, in complex with the strongest inhibitor, provides an explanation connecting the enzyme weaker substrate specificity with lower sensitivity to inhibition. It also provides insight into the enzyme/ligand molecular recognition process and suggests directions for the design of more potent inhibitors.

Pyrimidinetrione-imidazoles as a Unique Structural Type of Potential Agents towards Candida Albicans: Design, Synthesis and Biological Evaluation.

Substantial morbidity and mortality of fungal infections have aroused concerns all over the world, and common Candida spp. currently bring about severe systemic infections. A series of pyrimidinetrione-imidazole conjugates as potentially antifungal agents were developed. Bioassays manifested that 4-fluobenzyl pyrimidinetrione imidazole 5 f exerted favorable inhibition towards C. albicans (MIC=0.002 mM), being 6.5 folds more active than clinical antifungal drug fluconazole (MIC=0.013 mM). Preliminary mechanism research indicated that compound 5 f could not only depolarize membrane potential but also permeabilize the membrane of C. albicans. Molecular docking was operated to simulate the interaction mode between molecule 5 f and CYP51. In addition, hybrid 5 f might form 5 f-DNA supramolecular complex via intercalating into DNA. The interference of membrane and DNA might contribute to its fungicidal capacity with no obvious tendency to induce the resistance against C. albicans. Conjugate 5 f endowed good blood compatibility as well as low cytotoxicity towards HeLa and HEK-293T cells.