Phenotypic Screening In Vitro of Novel Aromatic Amidines against Trypanosoma cruzi

Abstract

The current treatment of Chagas disease (CD), based on nifurtimox and benznidazole (Bz), is unsatisfactory. In this context, we performed the phenotypic in vitro screening of novel mono- and diamidines and drug interaction assays with selected compounds. Ten novel amidines were tested for their activities against bloodstream trypomastigote (BT) and amastigote forms of Trypanosoma cruzi (Y and Tulahuen strains) and their toxicities for mammalian host cells (L929 cells and cardiac cells). Seven of 10 molecules were more active than Bz against BT, with the most active compound being the diamidine DB2267 (50% effective concentration [EC50] = 0.23 μM; selectivity index = 417), which was 28-fold more active and about 3 times more selective than the standard drug. Five of the six monoamidines were also more active than Bz. The combination of DB2267 and DB2236 in fixed-ratio proportions showed an additive effect (sum of fractional inhibitory concentrations < 4) on BT. Interestingly, when intracellular forms were exposed to DB2267, its activity was dependent on the parasite strain, being effective (EC50 = 0.87 ± 0.05 μM) against a discrete typing unit (DTU) II strain (strain Y) but not against a representative DTU VI strain (strain Tulahuen) even when different vehicles (β-cyclodextrin and dimethyl sulfoxide) were used. The intrinsic fluorescence of several diamidines allowed their uptake to be studied. Testing of the uptake of DB2236 (inactive) and DB2267 (active) by amastigotes of the Y strain showed that the two compounds were localized intracellularly in different compartments: DB2236 in the cytoplasm and DB2267 in the nucleus. Our present data encourage further studies regarding the activities of amidines and provide information which will help with the identification of novel agents for the treatment of CD.

FIG 1

Structures of the diamidines and monoamidines evaluated in this study.

FIG 2

Light microscopy images of untreated (A) and DB2267-treated (B) cardiac cells (1.18 μM for 48 h at 37°C) infected with T. cruzi (Y strain) showing a strong decline (60%) in the number of parasites (arrows) present in the treated cell cultures compared to the number present in the untreated samples.

FIG 3

Fluorescence microscopy images (A and C) and corresponding differential interference contrast images (B and D) of cardiac cells infected with T. cruzi (Y strain) after 2 h of exposure to 10 μg/ml of DB2267 (A and B) and DB2236 (C and D), showing the internalization of both compounds with their intracellular localization in the nuclei (A) and cytoplasm (C) of the mammalian host cells and the parasites (arrows).

FIG 4

Representative isobologram. In vitro interaction of DB2267 and DB2236 combined against bloodstream trypomastigotes of T. cruzi (Y strain). The EC₅₀ of DB2236 is plotted on the abscissa, and the EC₅₀ of DB2267 is plotted on the ordinate. The plotted points are the EC₅₀ of each fixed ratio of 5:0, 4:1, 3:2, 1:4, and 0:5 of DB2267 and DB2236. ΣFICs of <0.5 indicate synergism. ΣFICs of >0.5 and ≤4.0 indicate additivity, and ΣFICs of >4.0 indicate antagonism.