Candida Albicans CYP51 Research Articles

Effects of Y132H and F145L Substitutions on the Activity, Azole Resistance and Spectral Properties of Candida albicans Sterol 14-Demethylase P450 (CYP51): A Live Example Showing the Selection of Altered P450 through Interaction with Environmental Compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.

Molecular design of two sterol 14α-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole

Sterol 14alpha-demethylase (CYP51) is one of the known major targets for azole antifungals. Therapeutic side effects of these antifungals are based on interactions of the azoles with the human analogue enzyme. This study describes for the first time a comparison of a human CYP51 (HU-CYP51) homology model with a homology model of the fungal CYP51 of Candida albicans (CA-CYP51). Both models are constructed by using the crystal structure of Mycobacterium tuberculosis MT-CYP51 (PDB code: 1EA1). The binding mode of the azole ketoconazole is investigated in molecular dynamics simulations with the GROMACS force field. The usage of special parameters for the iron azole complex binding is necessary to obtain the correct complex geometry in the active site of the enzyme models. Based on the dynamics simulations it is possible to explain the enantioselectivity of the human enzyme and also to predict the binding mode of the isomers of ketoconazole in the active site of the fungal model.

Structure-based de novo design, synthesis, and biological evaluation of non-azole inhibitors specific for lanosterol 14alpha-demethylase of fungi.

The active site of lanosterol 14alpha-demethylase (CYP51) was investigated via MCSS functional group mapping and LUDI calculations. Several non-azole lead molecules were obtained by coupling structure-based de novo design with chemical synthesis and biological evaluation. All of the lead molecules exhibited a strong inhibitory effect on CYP51 of Candida albicans. They occupy the substrate-binding site and interfere with the binding of azole antifungal agents in a competitive manner. The mode of action of the lead molecules was validated by spectrophotomeric analysis and SAR studies. This is the first successful example reported for the inhibitor design of the cytochrome P450 superfamily using the de novo design strategy. Because the affinity of the lead molecules for CYP51 was mainly attributed to their nonbonding interaction with the apoprotein, the studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s.

1,4-Benzothiazine and 1,4-Benzoxazine imidazole derivatives with antifungal activity: A docking study

We have recently described the synthesis and antifungal activity of a series of 1,4-benzothiazine and 1,4-benzoxazine imidazole derivatives that mainly showed in vivo activity against a murine experimental model of candidiasis but that very often lacked in vitro activity. Here, we report a docking study of a representative set of our molecules in a 3D model of CYP51 of Candida albicans (CA-CYP51). The model was constructed on the basis of the sequence homology relationship with the recently reported crystal structure of the CYP51 of Mycobacterium tuberculosis (MT- CYP51).

Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents

The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14α-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC 50 values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0.2 μM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC 50 values of 0.008 and 0.0076 μM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(−) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra.

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b 5/NADH cytochrome b 5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b 5 and NADH cytochrome b 5 reductase, we show here that fungal CYP51 mediated sterol 14α-demethylation can be wholly and efficiently supported by the cytochrome b 5/NADH cytochrome b 5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b 5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.

Differential inhibition of Candida albicans CYP51 with azole antifungal stereoisomers

Azole antifungal compounds are important in agriculture and in the treatment of mycotic infection The target enzyme, sterol 14α-demethylase (CYP51), is inhibited through binding of triazole N-4 to the haem of this P450, as a sixth ligand together with the N-1 substituent groups interacting in some way with the apoprotein. Here we use Saccharomyces cerevisiae expression systems for the target enzyme of Candida albicans to investigate binding of enantiomers of the azole antifungal compounds SCH39304 and tetraconazole. A molecular model produced previously provided qualitative explanations for these differences. Interaction of the azole antifungal aromatic group with Phe-233 or -235 may cause the higher activity for ( R)-tetraconazole while inactivity of the ( SS)-enantiomer of SCH39304 was predicted to result from incompatibility of the hydrophilic sulfonyl moiety when located into the hydrophobic pocket of the active site.