Resistance to antifungals that target CYP51

Abstract

Fungal diseases are an increasing global burden. Fungi are now recognised to kill more people annually than malaria, whilst in agriculture, fungi threaten crop yields and food security. Azole resistance, mediated by several mechanisms including point mutations in the target enzyme (CYP51), is increasing through selection pressure as a result of widespread use of triazole fungicides in agriculture and triazole antifungal drugs in the clinic. Mutations similar to those seen in clinical isolates as long ago as the 1990s in Candida albicans and later in Aspergillus fumigatus have been identified in agriculturally important fungal species and also wider combinations of point mutations. Recently, evidence that mutations originate in the field and now appear in clinical infections has been suggested. This situation is likely to increase in prevalence as triazole fungicide use continues to rise. Here, we review the progress made in understanding azole resistance found amongst clinically and agriculturally important fungal species focussing on resistance mechanisms associated with CYP51. Biochemical characterisation of wild-type and mutant CYP51 enzymes through ligand binding studies and azole IC50 determinations is an important tool for understanding azole susceptibility and can be used in conjunction with microbiological methods (MIC50 values), molecular biological studies (site-directed mutagenesis) and protein modelling studies to inform future antifungal development with increased specificity for the target enzyme over the host homologue.

Keywords: Antifungals; Azole resistance; CYP51; Fungicides; Point mutations; Sterol 14-demethylase.

Fig. 1

Structures of the clinical and agricultural azole compounds discussed in this review

Fig. 2

Azole resistance mechanisms in C. albicans. CYP51 is an essential step in the biosynthesis of ergosterol, which is required for membrane stability and functionality. Azoles inhibit CYP51 causing the accumulation of 14 α-methyl-ergosta-8,24(28)-dien-3β-6α-diol. Resistance to azoles can occur through a an altered CYP51 (point mutations), b overexpression of CYP51, c overexpression of efflux transporters and d null mutation of ERG3 which blocks the synthesis of 14 α-methyl-ergosta-8,24(28)-dien-3β-6α-diol, resulting in the accumulation of 14 α-methyl-fecosterol which is capable of supporting membrane function

Fig. 3

Lanosterol binding studies with 5 μM wild-type, S279F and I471T C. albicans CYP51 proteins. The type I difference spectrum obtained for the wild-type protein is shown a along with the lanosterol saturation plots b for wild-type (black circle), S279F (white circle) and I471T (◉) C. albicans CYP51. The Michaelis–Menten equation was used to fit the data and derive K _s values

Fig. 4

Fluconazole binding studies with wild-type, S279F and I471T C. albicans CYP51 proteins. The type II difference spectrum obtained for the wild-type protein is shown a along with the fluconazole saturation plots b for wild-type (black circle), S279F (white circle) and I471T (◉) C. albicans CYP51. A rearrangement of the Morrison equation [172] was used to fit the data and determine K _d values

Fig. 5

Inhibition of lanosterol binding to C. albicans CYP51 by azole fungicides. Type I difference spectra were measured during the progressive titration of 10 μM CYP51 with lanosterol in the absence and presence of azole fungicides. Lineweaver-Burk plots were constructed from the type I binding spectra obtained in order to compare lanosterol binding in the absence (black circle) and presence (white circle) of 4 μM voriconazole, 4 μM prothioconazole-desthio and 100 μM prothioconazole. One representative example of each experiment is shown, although all experiments were performed in triplicate

Fig. 6

Azole IC₅₀ determinations. a The CYP51 activities of 2.5 μM wild-type (black circle), S279F (white circle) and I471T (◉) C. albicans CYP51 were determined at fluconazole concentrations ranging from 0 to 4 μM. A relative velocity of 1.0 corresponds to the velocity observed with the wild-type enzyme in the absence of fluconazole (0.062 nmol min⁻¹). b The CYP51 activities of 2.5 μM wild-type C. albicans CYP51 were determined at voriconazole (black circle) and prothioconazole-desthio (◉) concentrations ranged from 0 to 4 μM and prothioconazole (white circle) concentrations ranged from 0 to 100 μM. Relative velocities of 1.0 equate to actual velocities of 0.080, 0.098 and 0.087 nmol min⁻¹ for the IC₅₀ determinations with voriconazole, prothioconazole and prothioconazole-desthio, respectively. Mean values from three replicates are shown along with associated standard error bars

Fig. 7

Structural modelling of wild-type and S279F C. albicans CYP51 proteins with and without fluconazole docked. a Wild-type protein, with S279 located peripherally on an external β turn. The S279F mutant protein also shown has F279 incorporated within a short section of α helix preceding the long I helix. b The wild-type protein shows fluconazole in the centre (coloured by element) and the haem group to the right, and residues predicted to be within the range of contact of fluconazole are labelled. The S279F mutant protein is also shown with the S279F substitution leading to conformational changes that result in S507 being removed from interaction with fluconazole but still bordering the haem cavity

Fig. 8

Comparison of azole binding parameters for C. albicans and human CYP51 enzymes. a K _d values were determined for azoles binding to human (black bar) and C. albicans (white bar) CYP51 proteins from ligand saturation curves using a rearrangement of the Morrison equation [172]. b IC₅₀ values for azole antifungal drugs and fungicides. IC₅₀ values were determined for azoles binding to human (black bar) and C. albicans (white bar) CYP51 proteins using the CYP51 reconstitution assay system. c Fold selectivity for the fungal drug target over the human homologue was calculated by dividing the IC₅₀ values (black bar) and K _d values (white bar) for human CYP51 by those obtained for C. albicans CYP51

Fig. 9

Prothioconazole binding with C. albicans and human CYP51 proteins. a Weak type I difference spectra were obtained using 5 μM solutions of the CYP51 proteins during progressive titration with prothioconazole. b Ligand saturation curves were constructed from the change in absorbance against prothioconazole concentration using the Michaelis–Menten equation