Squalene and ergosterol biosynthesis in fungi treated with naftifine, a new antimycotic agent

Naftifine, a new antimycotic drug of the allylamine class, is a potent inhibitor of ergosterol biosynthesis in Candida albicans. Treated cells showed a dose-dependent drop in ergosterol content; the level was reduced by 60% at concentrations of >50 mg/liter, causing total inhibition of growth. This inhibition coincided with a heavy accumulation of the sterol precursor squalene. Radiolabeling experiments showed that the inhibition of sterol synthesis was complete within 10 min of exposure of cells to the compound. Control cells incorporated [14C]acetate into nonsaponifiable lipids composed primarily of ergosterol, whereas naftifinetreated cells accumulated only labeled squalene. When the drug was removed by washing cells thoroughly in 1% Tween 80, the accumulated squalene was further metabolized to ergosterol. A similar pattern of inhibition was observed in sterol biosynthesis from [14C]mevalonate in a cell-free system. At 50 mg/liter, naftifine gave >99% inhibition of sterol biosynthesis both in whole cells and in cell extracts of C. albicans. The primary action of naftifine appears to be the blocking of fungal squalene epoxidation.Naftifine is an allylamine antimycotic drug with activity against a wide range of pathogenic fungi, particularly dermatophytes both in vitro (4) and in vivo (12). In clinical trials, the compound proved very effective as a topical treatment for dermatomycoses (3). At higher concentrations, naftifine also has in vitro activity against Candida albicans (4) and may be clinically useful against this organism. As naftifine represents an entirely new chemical class of antifungal agents, it is important to establish the mode of action in a range of target organisms. Earlier work with the dermatophyte Trichophyton mentagrophytes and the yeast Candida parapsilosis (11) indicated that naftifine interferes with ergosterol biosynthesis by blocking squalene metabolism. In this report, we describe the activity of naftifine against C. albicans, using several experimental models designed to quantify the effects on ergosterol biosynthesis in comparison with the inhibition of cell growth.( Extraction of lipids. Cells were harvested by centrifugation and treated by saponification in 50% ethanol containing 15% KOH and 0.1% pyrogallol for 2 h under reflux. After the addition of 2 volumes of water, the nonsaponifiable lipid (NSL) was extracted three times with petroleum ether (40 to 60°C). The petrol extract was washed with water, dried over anhydrous sodium sulfate, filtered, and brought to dryness in a rotary evaporator. NSL was dissolved in cyclohexane and stored at -20°C in the dark. For quantitative analysis, a known amount of cholesterol was added as an internal standard before the extraction.Gas chromatography. Components of NSL were separated by using a Siemens gas chromatograph (Sichromat 1) with a glass column (110 by 0.6 cm) with 1% OV3 silicone on Chromosorb G (Supelco, Inc., Bellefonte, Pa.) in the stationary phase. The carrier gas was helium (23 ml/min), and the temperature range program was ...

Section: Methodssupporting

confidence: 78%

mentioning

confidence: 99%

Effect of the antimycotic drug naftifine on growth of and sterol biosynthesis in Candida albicans

Ryder¹,

Seidl²,

Troke³

1984

“…As reported previously (19,22,26,27), all five compounds inhibited ergosterol biosynthesis in growing C. albicans cells and caused the accumulation of squalene (Table 1). Inhibition was defined as a decrease in the sterol-to-squalene ratio, which was typically 20 in control cultures.…”

supporting

confidence: 83%

“…Allylamines and the structurally related thiocarbamates are synthetic antifungal agents which inhibit ergosterol biosynthesis at the level of squalene epoxidase, causing the accumulation of squalene (4,13,18,19,22,23,26,27). The compounds are highly fungicidal against dermatophytes and are used clinically in the treatment of dermatomycoses and onychomycoses (10,14).…”

mentioning

confidence: 99%

Effects of squalene epoxidase inhibitors on Candida albicans

Georgopapadakou

Bertasso

1992

The relationship between sterol biosynthesis inhibition, membrane integrity, and cell growth inhibition in Candida albicans was examined for five squalene epoxidase inhibitors. The compounds were the thiocarbamates tolnaftate and tolciclate and the allylamines naftifine, terbinafine, and SDZ 87-469. All compounds inhibited sterol biosynthesis, with the concentrations that caused a 50% decrease in the total sterol-to-squalene ratio ranging from less than or equal to 0.01 microM for terbinafine and SDZ 87-469 to 500 microM for tolnaftate. At 100 microM, the compounds also caused up to a 30% release of intracellular [14C]aminoisobutyric acid. With terbinafine and SD2 87-469, aminoisobutyric acid release further increased in cells grown at concentrations that inhibited ergosterol biosynthesis. It is suggested that inhibition of ergosterol synthesis may render the C. albicans membrane susceptible to further damage, including direct damage from squalene epoxidase inhibitors.

“…The antifungal activities of these compounds are based on the inhibition of fungal ergosterol biosynthesis at the point of squalene epoxidation (5,(8)(9)(10).…”

mentioning

confidence: 99%

Antifungal activity of the allylamine derivative terbinafine in vitro

Petrányi

Meingassner

Mieth

1987

231

Terbinafine, an allylamine derivative, represents the most effective of this new chemical class of antimycotic compounds. Under in vitro conditions, terbinafine proved to be highly active against dermatophytes (MIC range, 0.001 to 0.01 ,ug/ml), aspergilli (MIC range, 0.05 to 1.56 tig/ml), and Sporothrix schenckii (MIC range, 0.1 to 0.4 ,ug/ml) and also exerted good activity against yeasts (MIC range, 0.1 to >100 ,ig/ml). The growth of Malasseziafurfur was inhibited also (MIC range, 0.2 to 0.8 ,ig/ml). Terbinafine displays a primary fungicidal action against dermatophytes, other filamentous fungi, and S. schenckii. The type of action against yeasts is species dependent and can be primarily fungicidal (Candida parapsilosis) or fungistatic (Candida albicans). The in vitro activity of terbinafine is pH dependent and rises with increasing pH value.The discovery of the antifungal activity of naftifine (Exoderil), which has been shown in vitro and in vivo against a variety of medically important species of fungi (3, 6), led to intensive chemical and biological investigations of the structure-activity relationships of allylamine derivatives at the Sandoz Forschungsinstitut, Vienna, Austria (12,13). The antifungal activities of these compounds are based on the inhibition of fungal ergosterol biosynthesis at the point of squalene epoxidation (5,(8)(9)(10).The most active derivative of this new chemical class of antimycotics found so far is terbinafine (7, 14; G. Petranyi, J. G. Meingassner, and H. Mieth, 9th Int. Congr. Int. Soc. Hum. Anim. Mycol. (ISHAM), abstr. no. P1-27, 1985). In this report we describe the antifungal activity of this compound in vitro.(These data were presented in part at the 13th Interna- 5%, which were then stored as 1.5-ml portions in liquid nitrogen (2). The CFU of the stock cultures were determined after freezing. Appropriate dilutions were made with saline or broth before use.Malassezia strains were cultivated at 37°C in broth media by the method of Weary and Graham (15) by serial transfers.Assessment of antifungal activity. Serial dilution tests were used to determine the MICs of the test compounds for dermatophytes, molds, biphasic fungi, and yeasts. The MIC for Malassezia strains was evaluated by an agar dilution test. Activity against dermatophytes was assessed by an agar diffusion test also. Since it was found in radiolabeling studies that terbinafine tends to stick to plasticware, the assays were performed in glass.Serial dilution test. The MIC for filamentous fungi was determined in Sabouraud dextrose 2% broth (initial pH, 6.5). Malt extract broth (initial pH, 6.5; E. Merck) was used for yeasts.Glass tubes (16 by 160 mm) were filled with 0