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

Abstract

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 ...

Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans

“…Whole-cell ergosterol biosynthesis was measured by incorporation of [1,2-"%C]acetate into nonsaponifiable lipids as described previously (Ryder, 1985 ;Ryder et al, 1984). Sterol synthesis in cell-free extracts, prepared as in Favre & Ryder (1997b) and Ryder (1987), was assayed in a final volume of 500 µl, consisting of 50 mM sodium\potassium phosphate pH 7n4, 0n5 mM DTT, 1 mM NAD, 1 mM NADP, 0n1 mM FAD, 3 mM glucose 6-phosphate, 5 mM ATP, 3 mM glutathione, 2n7 mM MnCl # , 4 mM MgCl # and 0n5 µCi (1n85i10% Bq) (RS)-[2-"%C]mevalonate, supplemented with 1n25 mg protein from cell-free extracts and the appropriate concentration of antimycotic, added from a 50-fold concentrated stock prepared in DMSO.…”

“…After 2 h incubation at 30 mC in a shaking water bath, reactions were terminated by the addition of 1 ml 15 % KOH in 90 % ethanol. Nonsaponifiable lipids were extracted and analysed by TLC (Ryder, 1985 ;Ryder et al, 1984) ; the "%C-radioactivity present in each fraction was measured with an Instant Imager (Packard). Reproducibility of extraction was regularly checked by adding 15 000 d.p.m.…”

Pyridines and Pyrimidines Mediating Activity against an Efflux-Negative Strain of Candida albicans through Putative Inhibition of Lanosterol Demethylase

“…The method for acetate incorporation and the analysis of sterol intermediates was based on that of Ryder et al (16). Strains of C. albicans were grown overnight in 25 ml of YPD in 125-ml flasks at 30°C with shaking at 130 rpm (ISF-4-W; Kühner), which typically resulted in an OD 600 of 5 to 10.…”

Inhibition of fungal and mammalian sterol biosynthesis by 2‐aza‐2,3‐dihydrosqualene

“…The cyclase was measured by conversion of 14C-labelled 2,3-epoxidosqualene to sterols as described in [8]. Ergosterol biosynthesis in whole cells of C. albicans was assayed by incubation with [14C]acetate, extraction of the non-saponifiable lipids and separation by thin layer chromatography as described [8,9]. Sterol biosynthesis in cell-free extracts of C. albicans was similarly measured by incorporation of [2-14C]mevalonate [9].…”