Repurposing Carvedilol as a Novel Inhibitor of the Trypanosoma cruzi Autophagy Flux That Affects Parasite Replication and Survival

Abstract

T. cruzi, the causal agent of Chagas disease, is a parasite able to infect different types of host cells and to persist chronically in the tissues of human and animal hosts. These qualities and the lack of an effective treatment for the chronic stage of the disease have contributed to the durability and the spread of the disease around the world. There is an urgent necessity to find new therapies for Chagas disease. Drug repurposing is a promising and cost-saving strategy for finding new drugs for different illnesses. In this work we describe the effect of carvedilol on T. cruzi. This compound, selected by virtual screening, increased the accumulation of immature autophagosomes characterized by lower acidity and hydrolytic properties. As a consequence of this action, the survival of trypomastigotes and the replication of epimastigotes and amastigotes were impaired, resulting in a significant reduction of infection and parasite load. Furthermore, carvedilol reduced the whole-body parasite burden peak in infected mice. In summary, in this work we present a repurposed drug with a significant in vitro and in vivo activity against T. cruzi. These data in addition to other pharmacological properties make carvedilol an attractive lead for Chagas disease treatment.

Keywords: Chagas disease; Trypanosoma cruzi; autophagy flux inhibitor; drug repurposing; mice infection; trypanocidal drugs.

Figure 1

Cruzipain competitive ligands. Chemical structures of K777 (A), carvedilol (B) and B95 (C) generated by using Chemdraw V19.0 (Perkin Elmer Informatics). Cruzipain bound to K777 (PDB entry 2OZ2) was used as a model for our virtual screening, where carvedilol (B) was identified as a hit. It is evident the resemblance between carvedilol and B95, which has been crystallized bound to the active site of cruzipain (PDB entry 3KKU).

Figure 2

Phenotypic alterations suffered by T. cruzi epimastigotes under carvedilol treatment. (A) TEM images of control parasites (Ctr) showing the typical elongated body, with terminal flagellum (f) emerging from the flagellar pocket (fp) and normal morphology of reservosomes (r), acidocalcisomes (ac), nucleus (n) and kinetoplast (k); carvedilol treated parasites (Carve) displayed abnormal vacuolization (v) and accumulation of multivesicular structures at 24 and 48 h and after 10 days of treatment. Bars: 2 μm. Insets Insets show the magnifications (5x) delimited in the original photo. Number (B) and diameter (C) of vacuoles quantified from the TEM images obtained from epimastigotes incubated with DMSO (Ctr) or 10 μM carvedilol (Carve) at the indicated times. Data are shown as mean +/- standard error of 3 independent experiments. **p < 0.01, ***p < 0.001 (Tukey test). A total of 50 cells per group was counted.

Figure 3

Carvedilol is an autophagy flux inhibitor on T. cruzi. T. cruzi epimastigotes (Y-GFP strain) were incubated under control (Ctr) or starvation medium (Stv) in the absence (DMSO) or the presence of 10 μM carvedilol for 24 h and then processed for confocal microscopy. (A) Detection of the TcAtg8.1 protein by IIF using a specific antibody. Confocal images depict autophagosomes labeled in red (TcAtg8.1) and the phase contrast (PC) for each condition. Scale bar: 10 μm. Insets show the magnifications delimited in the original photo. (B) Percentage of parasites with more than two Atg8.1 positive vesicles under each condition. Data shown represent the mean +/- SE from 3 independent experiments. **p < 0.01, ***p < 0.001 (Tukey test). A total of 100 parasites per group was counted. Detection of hydrolytic (C) and acidic (D) compartments were performed by incubation with DQ-BSA and Lysotracker probes respectively and analyzed by in vivo confocal studies. Graphics represent the percentage of parasites positive for DQ-BSA or Lysotracker staining in each condition. Data shown represent the mean +/- SE from 3 independent experiments. ***p < 0.001 (Tukey test). A total of 100 parasites per group was counted.

Figure 4

Antiparasitic effect of carvedilol in epimastigotes and amastigotes of T. cruzi. (A) Epimastigotes (10x10⁶ parasites/ml) of T. cruzi Y-GFP strain were incubated in the absence (DMSO, Ctr) or the presence of 10 μM carvedilol (Carve) at the indicated times. Data shown represent the mean +/- SE from 3 independent experiments. ***p < 0.001 (t-test). H9C2 cells were infected with trypomastigotes of T. cruzi Y-GFP strain (MOI=10) for 24 h followed by a chase of 48 h in the presence (Carve) or the absence (Ctr) of carvedilol at different concentrations (2.5, 5 and 10 μM). After fixation, cells were prepared for microscopic studies. (B) Confocal images of GFP-expressing amastigotes multiplying within the host cells 48 h after treatment with 10 μM carvedilol versus control (DMSO). H9C2 cells actin myofibrils were stained with rhodamine-phalloidine probe (red). Percentage of infected cells (C) and number of amastigotes/cell (D) at the different concentrations of carvedilol versus control. Data are shown as mean +/- standard error of 3 independent experiments. *p < 0.05, **p < 0.01 (Tukey test). A total of 100 cells per group was counted.

Figure 5

Opera Phenix system and qPCR analysis confirmed action of carvedilol in vitro. HG39 cells were infected with trypomastigotes of T. cruzi Tulahuen strain (MOI=10) for 24 hours and treated with 10 μM carvedilol for 24, 48 and 72 h (Carve) or DMSO (Ctr). (A) confocal images of each condition taken by the system. (B) Number of amastigotes/cell in each group at the indicated times. Data are shown as mean +/- standard error of 3 independent experiments. *p < 0.05, ***p < 0.001 (Tukey test). A total of 100 cells per group was counted. (C) Relative concentration of T. cruzi DNA in each condition detected by dual-labeled qPCR. Data are shown as mean +/- standard error of 3 independent experiments. ***p < 0.001 (Tukey test). (D) Parasite load of HG39 cells infected for 24 h with trypomastigotes of T. cruzi Tulahuen strain (MOI=10) previously treated with 10 μM carvedilol for 24 h were compared to cells infected in control conditions (DMSO) or cells pre-treated with 10 μM carvedilol before infection with T. cruzi Tulahuen strain (MOI=10). Data are shown as mean +/- standard error of 3 independent experiments. ***p < 0.001 (Tukey test).

Figure 6

Carvedilol has a trypanocidal effect on T. cruzi trypomastigotes. Trypomastigotes of T. cruzi Tulahuen strain were incubated in the absence (DMSO, Ctr) or the presence of 10 μM carvedilol for 24 h followed by Tunel assay for flow cytometry. Other samples of parasites were treated with DNAse or heated to 90°C for 5 minutes and used as negative and positive controls respectively. (A) Graphs depict the 2D plot of each condition. (B) Percentage of apoptosis-like process calculated from the data obtained in (A) Data are shown as mean +/- standard error of 2 independent experiments.

Figure 7

Live imaging of bioluminescent parasites shows action of carvedilol in vivo. 2 groups of 5 mice were infected with 5000 trypomastigotes Tulahuen strain with the Luc-mNeonGreen incorporated. At 7 DPI, 2 animals from each group were treated with carvedilol 25 mg/kg/day for a period of 14 days and periodically measure their body luminescence (see materials and methods). (A) Images show the level of parasitism detected by the live imaging luminescence representative of the 2 groups at different days after infection. Right panel indicates the luminescence scale. (B) The scatter curve graph shows the means ± SD of radiance measurements obtained in each group at the different days after infection. One-way ANOVA statistical analysis was performed with Tukey multiple comparison test. Significance levels established by p-values ​​*≤0.05.