Structural Optimization and Biological Activity of Pyrazole Derivatives: Virtual Computational Analysis, Recovery Assay and 3D Culture Model as Potential Predictive Tools of Effectiveness against Trypanosoma cruzi.

Chagas disease, a chronic and silent disease caused by Trypanosoma cruzi, is currently a global public health problem. The treatment of this neglected disease relies on benznidazole and nifurtimox, two nitroheterocyclic drugs that show limited efficacy and severe side effects. The failure of potential drug candidates in Chagas disease clinical trials highlighted the urgent need to identify new effective chemical entities and more predictive tools to improve translational success in the drug development pipeline. In this study, we designed a small library of pyrazole derivatives (44 analogs) based on a hit compound, previously identified as a T. cruzi cysteine protease inhibitor. The in vitro phenotypic screening revealed compounds 3g, 3j, and 3m as promising candidates, with IC50 values of 6.09 ± 0.52, 2.75 ± 0.62, and 3.58 ± 0.25 µM, respectively, against intracellular amastigotes. All pyrazole derivatives have good oral bioavailability prediction. The structure-activity relationship (SAR) analysis revealed increased potency of 1-aryl-1H-pyrazole-imidazoline derivatives with the Br, Cl, and methyl substituents in the para-position. The 3m compound stands out for its trypanocidal efficacy in 3D microtissue, which mimics tissue microarchitecture and physiology, and abolishment of parasite recrudescence in vitro. Our findings encourage the progression of the promising candidate for preclinical in vivo studies.

Role of FAK signaling in chagasic cardiac hypertrophy

Abstract Cardiac hypertrophy and dysfunction are a significant complication of chronic Chagas disease, with heart failure, stroke, and sudden death related to disease progression. Thus, understanding the signaling pathways involved in the chagasic cardiac hypertrophy may provide potential targets for pharmacological therapy. Herein, we investigated the implication of focal adhesion kinase (FAK) signaling pathway in triggering hypertrophic phenotype during acute and chronic T. cruzi infection. C57BL/6 mice infected with T. cruzi (Brazil strain) were evaluated for electrocardiographic (ECG) changes, plasma levels of endothelin-1 (ET-1) and activation of signaling pathways involved in cardiac hypertrophy, including FAK and ERK1/2, as well as expression of hypertrophy marker and components of the extracellular matrix in the different stages of T. cruzi infection (60-210 dpi). Heart dysfunction, evidenced by prolonged PR interval and decrease in heart rates in ECG tracing, was associated with high plasma ET-1 level, extracellular matrix remodeling and FAK signaling activation. Upregulation of both FAK tyrosine 397 (FAK-Y397) and serine 910 (FAK-S910) residues phosphorylation as well as ERK1/2 activation, lead to an enhancement of atrial natriuretic peptide gene expression in chronic infection. Our findings highlight FAK-ERK1/2 signaling as a regulator of cardiac hypertrophy in Trypanosoma cruzi infection. Both mechanical stress, induced by cardiac extracellular matrix (ECM) augment and cardiac overload, and ET-1 stimuli orchestrated FAK signaling activation with subsequent activation of the fetal cardiac gene program in the chronic phase of infection, highlighting FAK as an attractive target for Chagas disease therapy.

Role of FAK signaling in chagasic cardiac hypertrophy.

Cardiac hypertrophy and dysfunction are a significant complication of chronic Chagas disease, with heart failure, stroke, and sudden death related to disease progression. Thus, understanding the signaling pathways involved in the chagasic cardiac hypertrophy may provide potential targets for pharmacological therapy. Herein, we investigated the implication of focal adhesion kinase (FAK) signaling pathway in triggering hypertrophic phenotype during acute and chronic T. cruzi infection. C57BL/6 mice infected with T. cruzi (Brazil strain) were evaluated for electrocardiographic (ECG) changes, plasma levels of endothelin-1 (ET-1) and activation of signaling pathways involved in cardiac hypertrophy, including FAK and ERK1/2, as well as expression of hypertrophy marker and components of the extracellular matrix in the different stages of T. cruzi infection (60-210 dpi). Heart dysfunction, evidenced by prolonged PR interval and decrease in heart rates in ECG tracing, was associated with high plasma ET-1 level, extracellular matrix remodeling and FAK signaling activation. Upregulation of both FAK tyrosine 397 (FAK-Y397) and serine 910 (FAK-S910) residues phosphorylation as well as ERK1/2 activation, lead to an enhancement of atrial natriuretic peptide gene expression in chronic infection. Our findings highlight FAK-ERK1/2 signaling as a regulator of cardiac hypertrophy in Trypanosoma cruzi infection. Both mechanical stress, induced by cardiac extracellular matrix (ECM) augment and cardiac overload, and ET-1 stimuli orchestrated FAK signaling activation with subsequent activation of the fetal cardiac gene program in the chronic phase of infection, highlighting FAK as an attractive target for Chagas disease therapy.

Trypanosoma cruzi down-regulates mechanosensitive proteins in cardiomyocytes.

BACKGROUND: Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES: Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS: We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS: Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in ß-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION: The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.

Trypanosoma cruzi down-regulates mechanosensitive proteins in cardiomyocytes

BACKGROUND Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in β-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.

Effect of Toxoplasma gondii infection on the junctional complex of retinal pigment epithelial cells.

Ocular toxoplasmosis is the most frequent cause of uveitis, leading to partial or total loss of vision, with the retina the main affected structure. The cells of the retinal pigment epithelium (RPE) play an important role in the physiology of the retina and formation of the blood-retinal barrier. Several pathogens induce barrier dysfunction by altering tight junction (TJ) integrity. Here, we analysed the effect of infection by Toxoplasma gondii on TJ integrity in ARPE-19 cells. Loss of TJ integrity was demonstrated in T. gondii-infected ARPE-19 cells, causing increase in paracellular permeability and disturbance of the barrier function of the RPE. Confocal microscopy also revealed alteration in the TJ protein occludin induced by T. gondii infection. Disruption of junctional complex was also evidenced by scanning and transmission electron microscopy. Cell-cell contact loss was noticed in the early stages of infection by T. gondii with the visualization of small to moderate intercellular spaces. Large gaps were mostly observed with the progression of the infection. Thus, our data suggest that the alterations induced by T. gondii in the structural organization of the RPE may contribute to retinal injury evidenced by ocular toxoplasmosis.

Trypanosoma cruzi alters adherens junctions in cardiomyocytes.

We analyzed the distribution and expression of cadherin and beta-catenin during Trypanosoma cruzi-cardiomyocyte interaction. Confocal microscopy revealed cadherin associated with beta-catenin at the cell-cell contacts. After 24h of infection, the spatial distribution and expression of both adherens junction (AJ) proteins remained unaltered. In contrast, loss of N-cadherin-catenin complex was visualized in highly infected cardiomyocytes. Immunoblotting assays corroborated the spatial disorder, showing a 46% reduction in both N-cadherin and beta-catenin expression at later infection (72h of infection). Our data demonstrate that T. cruzi infection disturbs AJs, which can result in loss of cardiac tension and may contribute to the cardiac dysfunctions present in T. cruzi infection.

Cytoskeleton reassembly in cardiomyocytes infected by Trypanosoma cruzi is triggered by treatment with ergosterol biosynthesis inhibitors.

We have previously shown that Trypanosoma cruzi-infected cardiomyocytes present alterations in cytoskeletal organisation in vitro. The remarkable change in the host cell cytoskeleton opened up the question of whether treatment of infected cells with antitrypanosomal compounds, such as ergosterol biosynthesis inhibitors (EBIs), allows the reconstruction of myofibrils and the microtubule network, restoring the cell biological function. Therefore, 48-h-old T. cruzi-infected cardiomyocyte cultures were treated with 10 nM ketoconazole or posaconazole and cytoskeletal remodelling of the host cells was analysed by indirect immunofluorescence assay. Both compounds displayed a potent antiparasitic effect and dramatically reduced the infection ratio. After 120 h of treatment, actin polygonal configuration was frequently visualised in the host cell cytoplasm, suggesting the initial stage of actin framework restoration. Rearrangement of myofibrils and the microtubule network was achieved 168 h after the start of drug treatment. Our data demonstrate that the trypanocidal effect of EBIs lead to reconstruction of the cytoskeleton of T. cruzi-infected cardiomyocytes in vitro.

Heparan sulfate proteoglycans mediate the invasion of cardiomyocytes by Trypanosoma cruzi.

Cytoadherence is an important step for the invasion of a mammalian host cell by Trypanosoma cruzi. Cell surface macromolecules are implicated in the T. cruzi-cardiomyocyte recognition process. Therefore, we investigated the role of cell surface proteoglycans during this invasion process and analyzed their expression after the parasite infected the target cells. Treatment of trypomastigote forms of T. cruzi with soluble heparan sulfate resulted in a significant inhibition in successful invasion, while chondroitin sulfate had no effect. Removal of sulfated glycoconjugates from the cardiomyocyte surface using glycosaminoglycan (GAG) lyases demonstrated the specific binding of the parasites to heparan sulfate proteoglycans. Infection levels were reduced by 42% whenthe host cells were previously treated with heparitinase II. No changes were detected in the expression of GAGs infected cardiomyocytes even after 96 h of infection. Our data demonstrate that heparan sulfate proteoglycans, but not chondroitin sulfate, mediate both attachment and invasion of cardiomyocytes by T. cruzi.

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75 per cent inhibition in the T. cruzi-cardiomyocytes invasion.