Antiproliferative effects and mechanism of action of ICI 195,739, a novel bis-triazole derivative, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi

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Abstract
The in vitro antiproliferative effects of ICI 195,739, a recently developed bis-triazole derivative (T. Boyle, D. J. Gilman, M. B. Gravestock, and J. M. Wardleworth, Ann. N.Y. Acad. Sci. 544:86-100, 1988; J. F. Ryley, S. McGregor, and R. G. Wilson, Ann. N.Y. Acad. Sci. 544:310-328, 1988), on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi and some aspects of its mechanism of action are described. Despite previous claims that triazole compounds act on susceptible organisms by essentially the same mechanism demonstrated for the imidazole compounds, i.e., by interfering with the synthesis of ergosterol at the level of the cytochrome P-450-dependent C-14 demethylation of lanosterol, our results indicate that ICI 195,739 acts on T. cruzi epimastigotes by a dual mechanism which involves blockade of ergosterol byosynthesis and a second, still-unidentified target whose alteration leads to immediate growth arrest. Although ICI 195,739 blocks ergosterol biosynthesis at the level of C-14 lanosterol demethylation, as shown by gas-liquid and thin-layer chromatography, growth arrest in ICI 195,739-treated cells is not related to the depletion of the endogenous ergosterol pool, contrary to what was previously found for ketoconazole, the reference compound among antifungal and antiprotozoal azole derivatives. Consistent with this observation is the fact that the concentration of ICI 195,739 required to inhibit de novo synthesis of ergosterol in epimastigotes by 50% is 60 nM, which is essentially identical to that previously found for ketoconazole under identical conditions, while the minimum concentration required to produce complete growth inhibition is 0.1 microM, which is 300 times lower than that of ketoconazole. With respect to the intracellular amastigote form proliferating inside vertebrate (Vero) cells, 10 nM is sufficient to eradicate the parasite completely in 96 h, with no effects on the host cells; this concentration is identical to that previously found for ketoconazole. Growth inhibition and morphological alterations induced by ketoconazole can be reserved by exogenously added ergosterol but not by cholesterol; for ICI 195, 739, neither sterol is capable of reserving the drug effects. Contrary to what was observed for ketoconazole, the in vitro antiproliferative effects of ICI 195, 739 on both forms of the parasite are not potentiated by the simultaneous presence of terbinafine, an allylamine which blocks ergosterol production by the parasite at a different level of the sterol biosynthetic pathway. These results, together with those of an accompanying study of the ultrastructural alterations induced by the drug, strongly support the notion that ICI 195, 739 acts on T. cruzi by a novel combination of biochemical and cellular effects, which could explain its extraordinary potency in vivo against the parasite.