Phenotypic Evaluation of Nucleoside Analogues against Trypanosoma cruzi Infection: In Vitro and In Vivo Approaches

Abstract

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is a serious public health problem. Current treatment is restricted to two drugs, benznidazole and nifurtimox, displaying serious efficacy and safety drawbacks. Nucleoside analogues represent a promising alternative as protozoans do not biosynthesize purines and rely on purine salvage from the hosts. Protozoan transporters often present different substrate specificities from mammalian transporters, justifying the exploration of nucleoside analogues as therapeutic agents. Previous reports identified nucleosides with potent trypanocidal activity; therefore, two 7-derivatized tubercidins (FH11706, FH10714) and a 3′-deoxytubercidin (FH8513) were assayed against T. cruzi. They were highly potent and selective, and the uptake of the tubercidin analogues appeared to be mediated by the nucleoside transporter TcrNT2. At 10 μM, the analogues reduced parasitemia >90% in 2D and 3D cardiac cultures. The washout assays showed that FH10714 sterilized the infected cultures. Given orally, the compounds did not induce noticeable mouse toxicity (50 mg/kg), suppressed the parasitemia of T. cruzi-infected Swiss mice (25 mg/kg, 5 days) and presented DNA amplification below the limit of detection. These findings justify further studies with longer treatment regimens, as well as evaluations in combination with nitro drugs, aiming to identify more effective and safer therapies for Chagas disease.

Keywords: Chagas disease; Trypanosoma cruzi; experimental chemotherapy; nucleoside derivatives; thymidine transporter.

Figure 1

Structural formulae of the compounds under study. Internal codes between brackets.

Figure 2

Uptake of 50 nM [³H]-adenosine by LmexNT1.1 expressed in L. mexicana promastigotes from which both original alleles of LmexNT1.1/NT1.2 and of LmexNT2 had been deleted by CRISPR/cas9. Incubation time was 10 s, well within the linear phase of uptake [30]. Symbols represent averages of triplicate determinations, and error bars indicate ± SEM when not shown error bars fall within the symbol. The graph shown is a single experiment in triplicate, representative of three identical, independent repeats.

Figure 3

FH11706, FH10714, FH8513 and Bz activity on male Swiss mice infected with 10⁴ bloodstream trypomastigotes (Y strain). (A) Parasitemia curve. (B) Percentage (%) of mouse weight variation.

Figure 4

Light microscopy quantification of parasites released into the supernatant of L929 infected with T. cruzi (Tulahuen strain) after washout assay. Values are the average of two independent determinations. All compounds were statistically significative (p > 0.05) compared to untreated cultures (control).