Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa.
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.
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea, Wales SA2 8PP, United Kingdom.
Andrew G S
AG
Warrilow
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea, Wales SA2 8PP, United Kingdom.
Claire L
CL
Price
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea, Wales SA2 8PP, United Kingdom.
Josie E
JE
Parker
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea, Wales SA2 8PP, United Kingdom.
Nicola J
NJ
Rolley
Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064 USA.
Christopher J
CJ
Smyrniotis
Plant Chemistry Group, School of Chemistry, Bangor University, Bangor, Gwynedd, Wales, LL57 2UW, United Kingdom.
David D
DD
Hughes
Plant Chemistry Group, School of Chemistry, Bangor University, Bangor, Gwynedd, Wales, LL57 2UW, United Kingdom.
Vera
V
Thoss
Center for Chemical Biology, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061.
W David
WD
Nes
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea, Wales SA2 8PP, United Kingdom.
Diane E
DE
Kelly
Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064 USA.
Theodore R
TR
Holman
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea, Wales SA2 8PP, United Kingdom.
Steven L
SL
Kelly
D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology; D002176:Candida albicans / Q000378:metabolism; D003022:Clotrimazole / Q000494:pharmacology; D000072505:Cytochrome P450 Family 51 / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D015725:Fluconazole / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism*; D017964:Itraconazole / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008290:Malassezia / Q000187:drug effects / Q000201:enzymology*; D013053:Spectrophotometry; D058886:Sterol 14-Demethylase / Q000378:metabolism*; D013261:Sterols / Q000737:chemistry; D065819:Voriconazole / Q000494:pharmacology
D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D008055:Lipids; D013261:Sterols; D017964:Itraconazole; D015725:Fluconazole; D000072505:Cytochrome P450 Family 51; D058886:Sterol 14-Demethylase; D003022:Clotrimazole; D065819:Voriconazole; D007654:Ketoconazole