Specific inhibition of fungal sterol biosynthesis by SF 86-327, a new allylamine antimycotic agent

Abstract

SF 86-327 is a new antimycotic agent of the ailylamine type. Its primary action appears to be the inhibition of ergosterol biosynthesis at the point of squalene epoxidation, as was previously found with the related compound naftifine. Biosynthesis was measured by incorporation of [14C]acetate into sterols in cells of Candida albicans, Candida parapsilosis, Torulopsis glabrata, and the dermatophyte Trichophyton mentagrophytes. There was a positive correlation between the SF 86-327 concentrations needed for inhibition of growth and of sterol synthesis in these four fungi. The greater antifungal efficacy of SF 86-327 in comparison with naftifine was also reflected in the relative activities of the two compounds as sterol synthesis inhibitors. Inhibition was maximal at neutral pH. A similar degree of inhibition was found in cell-free extracts when [14C]mevalonate was used as substrate. In all cases, inhibition of sterol synthesis was accompanied by a parallel accumulation of labeled squalene. SF 86-327 and naftifine had no significant effect on initial enzymes of the ergosterol pathway, measured by incorporation of [14C]acetyl coenzyme A, or on steps distal to squalene epoxidation, measured by conversion of labeled squalene 2,3-epoxide or lanosterol. Both allylamines were highly selective for fungal, as opposed to mammalian, sterol biosynthesis. SF 86-327 caused slight inhibition of squalene epoxidation in a rat liver cell-free system, but at concentrations three to four orders of magnitude greater than those required for inhibition of the fungal pathway.

Effect of Allylamine Antimycotic Agents on Fungal Sterol Biosynthesis Measured by Sterol Side-chain Methylation

“…In C. parupsilosis, no evidence could be found for inhibition of steps distal to squalene epoxidase (Ryder, 1985). In addition, SF 86-327 and naftifine up to 10 mg 1-1 had no effect on the cell-free sterol side-chain methylation assay in this fungus.…”

“…(1 mg 1-l) caused full inhibition of squalene epoxidation (acetate incorporation) in cells of all three fungi (Ryder, 1985). However, residual side-chain methylation was reproducibly low in C. parapsilosis and virtually absent in T. mentagrophytes (Table 4).…”

“…This was found to be due to a rapid acidification of the medium to pH 2.5 as a result of NH,+ uptake into the cells. The pH sensitivity of the inhibitory action of SF 86-327 has previously been shown in Candida (Ryder, 1985), by measuring acetate incorporation into sterols. The sidechain methylation technique was used to demonstrate the pH sensitivity of inhibition in T. mentagrophytes at low concentrations of SF 86-327 (Fig.…”

“…Inhibition was maximal at pH 6-5 in C. albicans cells, measured either by acetate incorporation (Ryder, 1985) or side-chain methylation as described here. Maximal rates of incorporation of radioactivity from [Me-*4C]methionine into sterols in control cells occurred at pH 6.5 in T.…”

“…Parallel incubations were done using as substrate [U-'Tlacetate (Amersham), final specific activity 0.4 Ci (14.8 GBq) mol-I . The standard incubation period was 3 h. Subsequent procedures for harvesting cells, extracting non-saponifiable lipids, isolating sterols by thin layer chromatography and counting radioactivity were as previously described Ryder, 1985).…”