CHARACTERIZATION OF SQUALENE EPOXIDASE ACTIVITY FROM THE DERMATOPHYTETRICHOPHYTON-RUBRUM AND ITS INHIBITION BY TERBINAFINE AND OTHER ANTIMYCOTIC AGENTS

Squalene epoxidase (SE) is the primary target of the allylamine antimy cotic agents terbinafine and naftifine and also of the thiocarbamates. Although all of these drugs are employed primarily in dermatological therapy, SE from dermatophyte fungi has not been previously investigat ed. We report here the biochemical characterization of SE activity fro m Trichophyton rubrum and the effects of terbinafine and other inhibit ors. Microsomal SE activity from T. rubrum was not dependent on solubl e cytoplasmic factors but had an absolute requirement for NADPH or NAD H and was stimulated by flavin adenine dinucleotide. Kinetic analyses revealed that under optimal conditions the K-m for squalene was 13 mu M and its V-max was 0.71 nmol/h/mg of protein. Terbinafine was the mos t potent inhibitor tested, with a 50% inhibitory concentration (IC50) of 15.8 nM. This inhibition was noncompetitive with regard to the subs trate squalene. A structure-activity relationship study with some anal ogs of terbinafine indicated that the tertiary amino structure of terb inafine was crucial for its high potency, as well as the tert-alkyl si de chain. Naftifine had a lower potency (IC50, 114.6 nM) than terbinaf ine. Inhibition was also demonstrated by the thiocarbamates tolciclate (IC50, 28.0 nM) and tolnaftate (IC50, 51.5 nM). Interestingly, the mo rpholine amorolfine also displayed a weak but significant effect (IC50 30 mu M). T rubrum SE was only slightly more sensitive (approximately twofold) to terbinafine inhibition than was the Candida albicans enzy me. Therefore, this difference cannot fully explain the much higher su sceptibility (greater than or equal to 100-fold) of dermatophytes than of yeasts to this drug. The sensitivity to terbinafine of ergosterol biosynthesis in whole cells of T. rubrum (IC50, 1.5 nM) is 10-fold hig her than that of SE activity, suggesting that the drug accumulates in the fungus.