A class of sterol 14-demethylase inhibitors as anti-Trypanosoma cruzi agents

Abstract

Chronic infection with the protozoan parasite Trypanosoma cruzi is a major cause of morbidity and mortality in Latin America. Drug treatments for the associated illness, Chagas disease, are toxic and frequently unsuccessful. In a screening effort against the drug target protein farnesyltransferase, we identified a series of disubstituted imidazoles with highly potent anti-T. cruzi activity that apparently acted through a mechanism independent of protein farnesylation. Metabolic labeling studies of T. cruzi suggested that sterol biosynthesis was inhibited. Combined GC/MS analysis confirmed depletion of cellular sterols and suggested that the site of action was sterol 14-demethylase, a cytochrome P450 enzyme. Spectral studies with recombinant T. cruzi sterol 14-demethylase demonstrated that the compounds bind directly to this enzyme. Two of the compounds were well absorbed when given orally to mice, gave sustained plasma levels, and were well tolerated. The compounds were administered orally to mice with acute T. cruzi infection and caused dramatic decrease in parasitemia and led to 100% survival. These disubstituted imidazole compounds can be prepared by a relatively short synthetic route and represent a structural class with potent anti-T. cruzi activity.

Fig. 1.

Peptidomimetic compounds and derivatives. (a) Methionine containing FTI-2220 patterned after the tetrapeptide CVIM. (b) FTI-2220 with CO₂CH₃ group replacing the methionyl unit (compound 1). (c) Disubstituted imidazole compounds 2-5 (R₁ and R₂ substituents are defined in Table 1).

Fig. 2.

Disubstituted imidazole compounds block the synthesis of sterols in T. cruzi cells and bind to recombinant T. cruzi sterol 14α-demethylase. (a) TLC analysis of ³H-sterols from T. cruzi epimastigotes grown with [³H]mevalonactone and no drug (lane 1), 25 μM ketoconazole (lane 2), or 25 μM compound 2 (lane 3). Sterol standards are shown in lane 4. (b) Difference spectra of recombinant T. cruzi sterol 14-demethylase (ferric state) mixed with increasing amounts of compound 1. The flat line represents no added compound, and the subsequent plots that show serially increasing difference spectra represent additions of 0.05, 0.10, 0.15, and 0.20 nmol of compound 1.

Fig. 3.

Reduced parasitemia and 100% survival in mice treated with compounds 4 or 5. BALB/c mice (six per group) were infected with T. cruzi on day 0 then dosed with vehicle or compounds from days 1 to 14. Mice received compounds at 50 mg/kg (≈100 μmol/kg) twice per day by oral gavage. (Upper) Parasitemia. (Lower) Survival. ▪, vehicle; ▴, compound 4; ▾, compound 5. (The experiment was also performed on a smaller scale with three mice per group, and similar results were observed.)