Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes

Leishmania mexicana mexicana promastigotes grown with cholesterol, supplied in natural products as the free sterol and as cholesteryl esters, were exposed to [2- 14C]mevalonate and to the antimycotic drug ketoconazole. Growth was inhibited and cholesterol and 14α-methyl sterols accumulated in free and esterified forms ( cholesterol >>> 4α,14α-dimethylcholesta-8,24-dien-3β-ol >> 14α-methylcholesta-8,24-dien-3β-ol ⋍ 14α-methylergosta-8,24(28)-dien-3β-ol >> 4α,14α-dimethylergosta-8,24(28)-dien-3β-ol ; identified by capillary gas chromatography/mass spectrometry, and by 1H and 13C nuclear magnetic resonance spectrometry). The 14α-methyl sterols were preferentially labelled with 14C. The cholesterol was unlabelled and substituted for a substantial fraction of the major product of sterol biosynthesis, ergosta-5,7,24(28)-trien-3β-ol (5-dehydroepisterol), but did not replace it and did not offer remarkable protection against either growth inhibition or alteration of sterol biosynthesis. Promastigotes grown with [6- 2H]cholesterol or [4- 14C]cholesterol did not contain labelled forms of Leishmania sterols, or other sterols. The chromatographic and spectrometric sterol analyses and the isotopic tracer findings suggested that ketoconazole impaired the cytochrome P-450 dependent 14α-demethylation of lanosterol, that cholesterol was neither biosynthesized nor metabolized, and that the physiological functions of 5-dehydroepisterol had sterol structural requirements not entirely met by cholesterol. In all these studies, L. mexicana mexicana demonstrated a sterol biochemistry remarkably similar to that of fungi. This recommends an increase in interest in antimycotic drugs as chemotherapeutic agents for leishmanial infections.