Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa.

Andrew G S Warrilow,Nicola J Rolley,Josie E Parker,Vera Thoss,Christopher J Smyrniotis,Theodore R Holman,Diane E Kelly,David D Hughes,Steven L Kelly,W David Nes,Claire L Price

doi:10.1038/srep27690

Abstract

Malassezia globosa cytochromes P450 CYP51 and CYP5218 are sterol 14α-demethylase (the target of azole antifungals) and a putative fatty acid metabolism protein (and a potential azole drug target), respectively. Lanosterol, eburicol and obtusifoliol bound to CYP51 with Kd values of 32, 23 and 28 μM, respectively, catalyzing sterol 14α-demethylation with respective turnover numbers of 1.7 min−1, 5.6 min−1 and 3.4 min−1. CYP5218 bound a range of fatty acids with linoleic acid binding strongest (Kd 36 μM), although no metabolism could be detected in reconstitution assays or role in growth on lipids. Clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole and ketaminazole bound tightly to CYP51 (Kd ≤ 2 to 11 nM). In contrast, fluconazole did not bind to CYP5218, voriconazole and ketaminazole bound weakly (Kd ~107 and ~12 μM), whereas ketoconazole, clotrimazole and itraconazole bound strongest to CYP5218 (Kd ~1.6, 0.5 and 0.4 μM) indicating CYP5218 to be only a secondary target of azole antifungals. IC50 determinations confirmed M. globosa CYP51 was strongly inhibited by azole antifungals (0.15 to 0.35 μM). MIC100 studies showed itraconazole should be considered as an alternative to ketoconazole given the potency and safety profiles and the CYP51 assay system can be used in structure-activity studies in drug development.

Highlights

(Kd 36 μM), no metabolism could be detected in reconstitution assays or role in growth on lipids
CYP52 proteins are known to be involved in growth on alkanes and fatty acids in yeasts and a homolog has been described in Malassezia[25] that might represent a drug target to inhibit growth on these carbon sources
Purification by Ni2+-NTA agarose chromatography resulted in 61% and 66% recoveries of native M. globosa CYP51 and CYP5218 proteins

Summary

Introduction

(Kd 36 μM), no metabolism could be detected in reconstitution assays or role in growth on lipids. Fluconazole did not bind to CYP5218, voriconazole and ketaminazole bound weakly (Kd ~107 and ~12 μM), whereas ketoconazole, clotrimazole and itraconazole bound strongest to CYP5218 (Kd ~1.6, 0.5 and 0.4 μM) indicating CYP5218 to be only a secondary target of azole antifungals. Azole antifungal agents selectively target fungal CYP51 enzymes over the human homolog[23] through the direct coordination of the triazole ring N-4 nitrogen (fluconazole, itraconazole and voriconazole) or the imadazole ring N-3 nitrogen (clotrimazole and ketoconazole) with the heme iron as the sixth axial ligand[24]. CYP52 proteins are known to be involved in growth on alkanes and fatty acids in yeasts and a homolog has been described in Malassezia[25] that might represent a drug target to inhibit growth on these carbon sources. The expression, purification and characterization of two M. globosa cytochrome P450 monooxygenase proteins (CYP51 and CYP5218) are described and the effectiveness of azole antifungal agents against M. globosa CYP51 and CYP512 including inhibition of M. globosa growth are determined

Methods

Results

Conclusion