Cytochrome P450 Sterol 14alpha-demethylase Research Articles

In silico Discovery of a New Potent Inhibitor for Sterol 14-alpha Demethylase as a Promising Antifungal Drug against Aspergillus fumigatus Infection

Aspergillus fumigatus is a dangerous opportunistic pathogen that causes severe consequences for human beings when its conidia are inhaled. Several inhibitory drugs have recently been suggested to eradicate these fungi by inhibiting the cytochrome P450 sterol 14-alpha demethylase B (CYP51B). These drugs are designed to exhibit high specificity to the heme that is incorporated in the active site of this enzyme. Though effective binding with heme can be achieved, administration of these drugs can be accompanied by variable risks to the user’s health. Series of in silico screenings were conducted to find out more eligible drug-like compounds to inhibit CYP51B-heme with fewer side effects on patients. Using stringent ZINCPharmer restrictions, seventeen compounds were found to have efficient binding to the heme group of CYP51B. Their effectiveness against CYP51B was tested using molecular docking, drug-likeness prediction, and molecular dynamics (MD) simulation. One compound (ZINC000015774018 or molecule-8) was found to inhibit the heme group with better drug-likeness than that found in the other sixteen drug-like compounds. MD simulations showed that this ligand introduced stabilized interactions with the targeted protein upon interacting with its heme and amino acid residues. Thus it may be used as a potent antifungal inhibitor against A. fumigatus.

Molecular Docking Study of Active Diazenyl Scaffolds as Inhibitors of Essential Targets Towards Antimicrobial Drug Discovery.

The diazenyl compounds (-N=N- linkage) have been reported to have antimicrobial activity. In modern drug discovery, the drug-receptor interactions are generally explored by the molecular docking studies. Three categories of diazenyl scaffolds were screened for the docking studies to explore the binding mechanism of interaction with various microbial targets. The diazenyl Schiff bases (SBN-20, SBN-21, SBN-25, SBN-33, SBN-39, SBN-40 and SBN-42), naphthol pharmacophore based diazenyl Schiff bases (NS-2, NS-8, NS-12, NS-15, NS-21, and NS-23), morpholine based diazenyl chalcones (MD-6, MD-9, MD-14, MD-16, MD-20, and MD-21) were docked against various bacterial and fungal proteins in comparison with different standard drugs. Further, the drug likeliness and ADME properties of these molecules were predicted by QikProp module of the Schrodinger software. Most of the derivatives had shown less docking scores and binding energies towards bacterial proteins, such as dihydropteroate synthase (PDB:2VEG), glucosamine-6-phosphate synthase (PDB:2VF5), dihydrofolate reductase (PDB:3SRW) in comparison with the standard drugs. The naphthol based diazenyl Schiff bases NS-21 and NS-23 were predicted to act on the cytochrome P450 sterol 14-alpha-demethylase (CYP51) (PDB:5FSA) involved in sterol biosynthesis, an essential target for antifungal drugs. The derivative MD-6, NS-2, NS-21, and NS-23 had shown high docking scores against bacterial DNA topoisomerase (PDB:3TTZ) in comparison with the standard drug ciprofloxacin. Further, most of the synthesized derivatives had shown drug like characters. Hence, these compounds can be developed as novel antibacterial agents as potent DNA topoisomerase inhibitors and antifungal agents as CYP51 inhibitors.

Cloning and Characterization of the Lanosterol 14α-Demethylase Gene from Antrodia cinnamomea

Sterol 14alpha-demethylase (CYP51) is one of the key enzymes for sterol biosynthesis in fungi; it is widely distributed in all members of the cytochrome P450 superfamily. In this study, AcCyp51, encoding a cytochrome P450 sterol 14alpha-demethylase, was obtained from the sequences of EST libraries of Antrodia cinnamomea by using 5' RACE and genome walking methods. The open reading frame of AcCyp51 is 1635 bp and encodes 544 amino acids. The recombinant protein of AcCYP51 fused with glutathione-S-transferase from Escherichia coli revealed the demethylating activity by using lanosterol as substrate and GC-MS analysis. Gene expression levels of AcCYP51 were higher in natural basidiomes than in other cell types. Transcription of AcCYP51 increased in various culture conditions including adding squalene, lanosterol, itroconazole, and oleic acid as inducers. These reveal the important functions of AcCYP51 in basidiomatal formation and suggest that it might participate in other biological processes.

Multiple mechanisms account for variation in base-line sensitivity to azole fungicides in field isolates of Mycosphaerella graminicola.

Molecular mechanisms that account for variation in base-line sensitivity to azole fungicides were examined in a collection of twenty field isolates, collected in France and Germany, of the wheat pathogen Mycosphaerella graminicola (Fuckel) Schroeter. The isolates tested represent the wide baseline sensitivity to the azole fungicide tebuconazole described previously. The isolates were cross-sensitive to other azoles tested, such as cyproconazole and ketoconazole, but not to unrelated chemicals like cycloheximide, kresoxim-methyl or rhodamine 6G. Progenies from a genetic cross between an isolate with an intermediate and a high sensitivity to azoles displayed a continuous range of phenotypes with respect to cyproconazole sensitivity, indicating that variation in azole sensitivity in this haploid organism is polygenic. The basal level of expression of the ATP-binding cassette transporter genes MgAtr1-MgAtr5 from Mgraminicola significantly varied amongst the isolates tested, but no clear increase in the transcript level of a particular MgAtr gene was found in the less sensitive isolates. Cyproconazole strongly induced expression of MgAtr4, but no correlation between expression levels of this gene and azole sensitivity was observed. One isolate with intermediate sensitivity to azoles over-expressed CYP51, encoding cytochrome P450 sterol 14alpha-demethylase from M graminicola. Isolates with a low or high sensitivity to azoles were tested for accumulation of cyproconazole, but no clear correlation between reduced accumulation of the fungicide in mycelium and sensitivity to azoles was observed. Therefore, differences in accumulation cannot account exclusively for the variation in base-line sensitivity of the isolates to azoles. The results indicate that multiple mechanisms account for differences in base-line sensitivity to azoles in field isolates of M graminicola.

Aspergillus fumigatus CYP51 sequence: potential basis for fluconazole resistance.

We have cloned and sequenced the Aspergillus fumigatus CYP51 gene which encodes the target of azole antifungal agents, namely cytochrome P450 sterol 14alpha-demethylase. Since A. fumigatus is intrinsically resistant to the widely used azole fluconazole, we compared its predicted CYP51 sequence to the CYP51 sequences from fluconazole-susceptible and resistant Candida albicans. This analysis generated specific hypotheses regarding the basis for A. fumigatus fluconazole resistance; in particular, A. fumigatus residue Ile301 corresponds to C. albicans residue Thr315 which is mutated to Ala in resistant strains and is proposed to hydrogen bond with the sterol substrate.