Azole Affinity of Sterol 14α-Demethylase (CYP51) Enzymes from Candida albicans and Homo sapiens

Candida albicans CYP51 (CaCYP51) (Erg11), full-lengthHomo sapiens CYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed inEscherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (Ks , 14 to 18 μM) and catalyzed the 14α-demethylation of lanosterol usingHomo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [Kd s], 42 to 131 nM) but bound fluconazole (Kd , ∼30,500 nM) and voriconazole (Kd , ∼2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (Kd s, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (Kd s, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (Kd , ∼40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC50 ) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC50 , ∼150 μM) and did not significantly inhibit Δ60HsCYP51.