Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors against Trypanosoma cruzi: implications for Chagas disease drug discovery and development

Abstract

Advocacy for better drugs and access to treatment has boosted the interest in drug discovery and development for Chagas disease, a chronic infection caused by the genetically heterogeneous parasite, Trypanosoma cruzi. In this work new in vitro assays were used to gain a better understanding of the antitrypanosomal properties of the most advanced antichagasic lead and clinical compounds, the nitroheterocyclics benznidazole, nifurtimox and fexinidazole sulfone, the oxaborole AN4169, and four ergosterol biosynthesis inhibitors--posaconazole, ravuconazole, EPL-BS967 and EPL-BS1246. Two types of assays were developed: one for evaluation of potency and efficacy in dose-response against a panel of T. cruzi stocks representing all current discrete typing units (DTUs), and a time-kill assay. Although less potent, the nitroheterocyclics and the oxaborole showed broad efficacy against all T. cruzi tested and were rapidly trypanocidal, whilst ergosterol biosynthesis inhibitors showed variable activity that was both compound- and strain-specific, and were unable to eradicate intracellular infection even after 7 days of continuous compound exposure at most efficacious concentrations. These findings contest previous reports of variable responses to nitroderivatives among different T. cruzi strains and further challenge the introduction of ergosterol biosynthesis inhibitors as new single chemotherapeutic agents for the treatment of Chagas disease.

Figure 1. The nitroheterocyclics benznidazole and nifurtimox are the most efficacious compounds against a phylogenetically broad panel of Trypanosoma cruzi.

Tissue cultures in 384-well plates were infected with one of the following T. cruzi: Dm28c (DTU I, purple), Y (DTU II, red), ARMA13 cl1 (DTU III, orange), ERA cl2 (DTU IV, light green), 92-80 cl2 (DTU V, light blue), CL Brener (DTU VI, dark blue), and Tulahuen (DTU VI, dark green). Compounds were 2-fold diluted and were added to infected cultures 24 h after T. cruzi plating and incubated for 72 (Dm28c) or 96 h (all others) prior to assessment of antiparasitic activity by high content analysis. Dose-response curves of compounds antiparasitic activity normalized to infected and non-infected controls are shown. The X-axis shows log of compound molar (M) concentrations and Y-axis shows the normalized activity, based on the measurement of ratio of infected cells. Data refers to mean values of at least two independent experiments.

Figure 2. Benznidazole, nifurtimox, fexinidazole sulfone, and AN4169 are fast trypanocidal compounds that can eliminate intracellular T. cruzi within 96 h of continuous exposure in vitro.

U2OS cells tissue cultures in 384-well plates were infected with the Y strain for 24 hours prior to addition of compounds in serial dilution by the factor of 2-fold, as indicated the by colors according to the compound gradient (right, top). The following concentrations are shown: 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 μM for benznidazole and fexinidazole sulfone; 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.20 μM for nifurtimox; and 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.31, 0.16 and 0.078 μM for AN4169. Antiparasitic activity was analyzed every 24 h, starting right after compound addition (time point 0 h), up to 96 h. Data points are means and standard deviations of at least two independent experiments.

Figure 3. TcCYP51 inhibitors are slow trypanocidal compounds that after prolonged exposure can greatly reduce but not eliminate intracellular infection.

U2OS cells tissue cultures in 384-well plates were infected with the Y strain for 24 hours prior to addition of compounds in serial dilution by the factor of 2-fold and as indicated the by the colors according to compound gradient (right, top). The following concentrations are shown: 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.156 nM for posaconazole and ravuconazole; and 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.81 and 3.91 nM for EPL-BS967 and EPL-BS1246. Antiparasitic activity was analyzed every 24 h, starting right after compound addition (time point 0 h), up to 144 h. Data points are means and standard deviations of three independent experiments.