Inhibition of squalene epoxidase by allylamine antimycotic compounds. A comparative study of the fungal and mammalian enzymes

Abstract

The inhibition of squalene epoxidase by the allylamine antimycotic agents naftifine and compound SF 86-327 was investigated, with particulate enzyme preparations from the pathogenic yeasts Candida albicans and Candida parapsilosis and from rat liver. Both naftifine and compound SF 86-327 were potent inhibitors of the Candida epoxidases and showed apparently non-competitive kinetics with respect to the substrate squalene. The Ki values for naftifine and compound SF 86-327 in the C. albicans system were 1.1 microM and 0.03 microM respectively. The C. parapsilosis enzyme was slightly more sensitive to inhibition. Varying the concentrations of cofactors or the soluble cytoplasmic fraction (S200) had no effect on the inhibition. The epoxidase from rat liver was much less sensitive (Ki for compound SF 86-327 was 77 microM). The inhibition was also qualitatively different from that in Candida, being competitive with respect to squalene and also with respect to the S200 fraction. S200 fraction derived from C. albicans also antagonized the inhibition of the epoxidase from liver, but the liver S200 fraction did not affect inhibition of the Candida enzyme by compound SF 86-327. There was no evidence for an irreversible or mechanism-based inhibition of either the fungal or the mammalian epoxidase. The selective inhibition of squalene epoxidase was sufficient to account for the known antimycotic action of the compounds.

Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

“…The correct sequences were confirmed by DNA sequence analysis. By this method we constructed the erg1 alleles G 25 S, G 27 Drug susceptibility testing. Drug susceptibility determination was performed in liquid YPD medium as described previously (15).…”

“…Leucine 37 is part of an ␣ helix, and the N-terminal end of this helix points toward the biphosphate group of the flavin cofactor. Amino acid residues D 209 and G 210 of the nucleotide binding domain and G 334 and D 335 , which comprise the FADII motif, are also close to the biphosphate and ribitol moieties of the flavin cofactor but are located on the opposite side with respect to G 25 , G 27 , and G 30 . In the case of D 209 and G 210 , both amino acids are located close to the cofactor in the region of its adenosyl moiety (Fig.…”

“…To determine the enzymatic activities of the functional squalene epoxidase variants, crude cell extracts of KLN1 containing recombinant plasmids pNS1 (wild-type ERG1), pG 30 S, pG 27 S, pL 37 P, and pR 269 G were prepared from exponential cultures grown in YPD medium at 30°C. Since the G 25 S Erg1p variant did not complement the KLN1 phenotype, the strain expressing this protein was grown in YNB medium under oxygen-limiting conditions.…”

“…Fungal squalene epoxidases are selectively inhibited by the allylamines terbinafine and naftifine in a noncompetitive manner with regard to the substrate squalene (27). In contrast, mammalian squalene epoxidases are competitively inhibited by NB598, which also belongs to the class of allylamines and contains the same side chain as terbinafine (10).…”

“…Inhibitors of mammalian SE, such as NB598 and derivatives thereof (10,30), have not reached the market. For the treatment of fungal infections, however, the allylamine derivatives naftifine and terbinafine have been applied successfully and were shown to specifically inhibit fungal squalene epoxidases (26,27).…”