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.

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.

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.

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.

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.

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.

Unravelling the role of transient redox partner complexes in P450 electron transfer mechanics.

The molecular evolution of cytochromes P450 and associated redox-driven oxidative catalysis remains a mystery in biology. It is widely believed that sterol 14α-demethylase (CYP51), an essential enzyme of sterol biosynthesis, is the ancestor of the whole P450 superfamily given its conservation across species in different biological kingdoms. Herein we have utilized X-ray crystallography, molecular dynamics simulations, phylogenetics and electron transfer measurements to interrogate the nature of P450-redox partner binding using the naturally occurring fusion protein, CYP51-ferredoxin found in the sterol-producing bacterium Methylococcus capsulatus. Our data advocates that the electron transfer mechanics in the M. capsulatus CYP51-ferredoxin fusion protein involves an ensemble of ferredoxin molecules in various orientations and the interactions are transient. Close proximity of ferredoxin, however, is required to complete the substrate-induced large-scale structural switch in the P450 domain that enables proton-coupled electron transfer and subsequent oxygen scission and catalysis. These results have fundamental implications regarding the early evolution of electron transfer proteins and for the redox reactions in the early steps of sterol biosynthesis. They also shed new light on redox protein mechanics and the subsequent diversification of the P450 electron transfer machinery in nature.

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.

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.

Design, Synthesis, and Evaluation of Novel 4-Chloropyrazole-Based Pyridines as Potent Fungicide Candidates.

A rational molecular design approach was developed in our laboratory to guide the discovery of novel sterol biosynthesis inhibitors. Based on the application of bioactivities of heterocyclic rings and molecular docking targeting the sterol biosynthesis 14α-demethylase, a series of 4-chloropyrazole-based pyridine derivatives were rationally designed, synthesized, and characterized and their fungicidal activities were also evaluated. Bioassay results showed that 7e, 7f, and 7m exhibited commendable, diverse antifungal actions that are comparable to those of the positive controls imazalil and triadimefon. The active compounds' mode of action was further studied by microscopy observations, Q-PCR, and enzyme inhibition assay and discovered that target compounds affect fungal sterol biosynthesis via disturbing RcCYP51 enzyme system. These findings support that their fungicidal mode of action still targets the cytochrome P450-dependent 14α-demethylase as the molecular design did at first. The above results strongly suggest that our rational molecular design protocol is not only practical but also efficient.

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.

Gene Amplification of CYP51B: a New Mechanism of Resistance to Azole Compounds in Trichophyton indotineae.

Trichophyton indotineae causes dermatophytosis that is resistant to terbinafine and azole compounds. The aim of this study was to determine the mechanisms of resistance to itraconazole (ITC) and voriconazole (VRC) in strains of T. indotineae. Two azole-sensitive strains (ITC MIC < 0.125 µg/mL; VRC MIC < 0.06 µg/mL) and four azole-resistant strains (ITC MIC ≥ 0.5 µg/mL; VRC MIC ≥ 0.5 µg/mL) were used for the investigation. The expression of MDR genes encoding multidrug transporters of the ABC family for which orthologs have been identified in Trichophyton rubrum and those of CYP51A and CYP51B encoding the targets of azole antifungal compounds were compared between susceptible and resistant strains. TinMDR3 and TinCYP51B were overexpressed in T. indotineae resistant strains. Only small differences in susceptibility were observed between TinMDR3 disruptants and parental strains overexpressing TinMDR3. Whole-genome sequencing of resistant strains revealed the creation of a variable number of TinCYP51B tandem repeats at the specific position of their genomes in three resistant strains. Downregulation of TinCYP51B by RNA interference (RNAi) restored the susceptibility of azole-resistant strains. In contrast, overexpression of TinCYP51B cDNA conferred resistance to a susceptible strain of T. indotineae. In conclusion, the reduced sensitivity of T. indotineae strains to azoles is mainly due to the overexpression of TinCYP51B resulting from additional copies of this gene.

A comparison of the bacterial CYP51 cytochrome P450 enzymes from Mycobacterium marinum and Mycobacterium tuberculosis.

Members of the CYP51 family of cytochrome P450 enzymes are classified as sterol demethylases involved in the metabolic formation of cholesterol and related derivatives. The CYP51 enzyme from Mycobacterium marinum was studied and compared to its counterpart from Mycobacterium tuberculosis to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding of the purified CYP51 enzymes from M. marinum and M. tuberculosis were performed. The catalytic oxidation of lanosterol and related steroids was investigated. M. marinum CYP51 was structurally characterized by X-ray crystallography. The CYP51 enzyme of M. marinum is sequentially closely related to CYP51B1 from M. tuberculosis. However, differences in the heme spin state of each enzyme were observed upon the addition of steroids and other ligands. Both enzymes displayed different binding properties to those reported for the CYP51-Fdx fusion protein from the bacterium Methylococcus capsulatus. The enzymes were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol, but no products were detected for the related species dihydrolanosterol and eburicol. The crystal structure of CYP51 from M. marinum in the absence of added substrate but with a Bis-Tris molecule within the active site was resolved. The CYP51 enzyme of M. marinum displays differences in how steroids and other ligands bind compared to the M. tuberculosis enzyme. This was related to structural differences between the two enzymes. Overall, both of these CYP51 enzymes from mycobacterial species displayed significant differences to the CYP51 enzymes of eukaryotic species and the bacterial CYP51-Fdx enzyme of Me. capsulatus.

K143R Amino Acid Substitution in 14-α-Demethylase (Erg11p) Changes Plasma Membrane and Cell Wall Structure of Candida albicans.

The opportunistic pathogen Candida albicans is responsible for life-threating infections in immunocompromised individuals. Azoles and polyenes are two of the most commonly used antifungals and target the ergosterol biosynthesis pathway or ergosterol itself. A limited number of clinically employed antifungals correspond to the development of resistance mechanisms. One resistance mechanism observed in clinical isolates of azole-resistant C. albicans is the introduction of point mutations in the ERG11 gene, which encodes a key enzyme (lanosterol 14-α-demethylase) on the ergosterol biosynthesis pathway. Here, we demonstrate that a point mutation K143R in ERG11 (C. albicans ERG11K143R/K143R) contributes not only to azole resistance, but causes increased gene expression. Overexpression of ERG11 results in increased ergosterol content and a significant reduction in plasma membrane fluidity. Simultaneously, the same point mutation caused cell wall remodeling. This could be facilitated by the unmasking of chitin and ß-glucan on the fungal cell surface, which can lead to recognition of the highly immunogenic ß-glucan, triggering a stronger immunological reaction. For the first time, we report that a frequently occurring azole-resistance strategy makes C. albicans less susceptible to azole treatment while, at the same time, affects its cell wall architecture, potentially leading to exposure of the pathogen to a more effective host immune response.