Interaction of azole fungicides and related compounds with cytochrome‐P450 isozymes fromPenicillium italicumin in‐vitro assays

摘要

AbstractThe interaction of various sterol demethylation inhibitors (DMls) and experimental compounds with cytochrome‐P450‐dependent sterol 14α‐demethylase (P45014DM) fromPenicillium italicumwas studied by difference spectroscopy using a preparation of microsomal P450 isozymes and assays with cell‐free extracts capable of synthesizing ergosterol from 14Cmevalonate. The EC50values (concentrations of compounds which inhibited radial growth ofP. italicumby 50) of the compounds ranged from 3 × 10−8M to levels higher than 10−3M. All the compounds investigated gave type II difference spectra and interfered with the binding of carbon monoxide (CO) to microsomal P450 isozymes. However, the differences in the IC50values (concentrations of compounds which caused 50 of maximal type II spectral change) between compounds and their inhibition of CO binding did not correlate with the fungicidal activity of the compounds. Hence, neither type II difference spectra nor CO‐displacement tests with microsomal preparations from this fungus can be used for studying the selective fungitoxicity of azoles. Data obtained using the cell‐free sterol 14α‐demethylase assay revealed larger differences between the compounds in their inhibition of sterol 14α‐demethylation. The I50values (concentrations of compounds which inhibited cellfree sterol biosynthesis by 50) varied from 4.3 × 10−9to 4.4 × 10−5M. This assay was able to rank the compounds in order of fungitoxicity, but the I50and EC50values did not correlate quantitatively. Consequently, the observed differential inhibition of P45014DMactivity between the compounds cannot fully explain their selective fungitoxicity. Additional mechanisms must be involved. The present study supports the general opinion that the affinity of azoles for P45014DMdepends on the nature of their N1substituent. However, it was demonstrated that the nature of the azole moiety was also of importance in determining the