MODIFICATION OF THE STEROL COMPOSITION OF TRYPANOSOMA (SCHIZOTRYPANUM) CRUZI EPIMASTIGOTES BY DELTA(24(25))-STEROL METHYL TRANSFERASE INHIBITORS AND THEIR COMBINATIONS WITH KETOCONAZOLE

We report a detailed analysis of the sterol composition of Trypanosoma cruzi epimastigotes grown in the absence of presence of two sterol analogs previously reported as inhibitors of Delta(24(25)) sterol methyltransferase (24(25)-SMT, E.C.2.1.1.43) in yeast and fungi, a cholestanol analog with a 6-membered aza ring as side chain (22,26-azasterol) and 24-(R,S),25-epiminolanosterol, as well as combinations of these compounds with the C14 demethylase inhibitor ketoconazole. Both sterol analogs produced a dose-dependent reduction in the incorporation of radioactivity from [methyl-C-14]methionine with IC50 values of 640 nM and 70 nM for 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, respectively, indicating a specific inhibition of 24(25)-SMT. Correspondingly, it was found that the sterols present in control cells (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta-ol and precursors) were almost completely replaced by zymosterol (cholesta-8,24-dien-3 beta-ol) or a mixture of zymosterol, cholesta-7,24-dien-3 beta-ol and cholesta-5,7,24-trien-3 beta-ol when the parasites were exposed to the minimal growth inhibitory concentrations of 22,26-azasterol and 24-(R,S),25-epiminolanosterol, respectively. At sub-optimal concentrations of the inhibitors a complete disappearance of the 24-ethyl sterols was observed and a concomitant increase in the proportion of 24-methyl sterols, particularly Delta(24(24')) sterols. This showed that in T. cruzi the second methenylation step (catalyzed by Delta(24(24')) sterol methyl transferase) was significantly more sensitive to these inhibitors than the first and that the sterol analogs were also powerful inhibitors of the Delta(24(24')) sterol reductase. In growth-arrested epimastigotes resulting farm their treatment with low (1-3 mu M) concentrations of either sterol analog combined with sub optimal (100-300 nM) levels of ketoconazole the main sterol was lanosterol with no evidence 24-methylenedihydrolanosterol, the main sterol found in cells treated with growth inhibitory concentrations of the azole alone. Taken together, these results indicated that 24-alkyl sterols are essential growth factors for T. cruzi and that the preferred substrate of the Delta(24(25)) sterol methyl transferase in this organism is zymosterol.