科研之友
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摘要
Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated Delta 60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K-s, 14 to 18 mu M) and catalyzed the 14 alpha-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K(d)s], 42 to 131 nM) but bound fluconazole (K-d, similar to 30,500 nM) and voriconazole (K-d, similar to 2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K(d)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 Delta 60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Delta 60HsCYP51, producing atypical type I UV-visible difference spectra (K(d)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 Delta 60HsCYP51 (K-d, similar to 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 Delta 60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC50, similar to 150 mu M) and did not significantly inhibit Delta 60HsCYP51.
- 出版日期2013-3
