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

Abstract

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.

Bioactivity of Novel Pyrazole-Thiazolines Scaffolds against Trypanosoma cruzi: Computational Approaches and 3D Spheroid Model on Drug Discovery for Chagas Disease

“…Tridimensional Vero cell cultures were obtained as previously described [ 31 ]. Briefly, the isolated cells (2 × 10 4 /well) were cultured in 96-well U-bottom plates previously coated with 1% agarose.…”

“…Thereafter, the cell monolayers were washed with phosphate-buffered saline (PBS) and 200 μL of fresh medium with a serial dilution of the pyrazole-thiazoline derivatives and Bz (1.9–500 µM) added. The cell viability was determined by luminescent assay using the CellTiter-Glo Kit (Promega Corporation, Madison, WI, USA) based on the ATP level [ 31 , 36 ]. After 72 h of treatment, 20 μL of CellTiter-Glo solution was added per well, and cultures were kept for 2 min under agitation.…”

“…Tissue-derived trypomastigotes (1 × 10 6 parasites/well) were incubated with pyrazole-thiazoline derivatives and Bz in different concentrations (0.41–100 µM) for 24 h at 37 °C in a humidified atmosphere and 5% CO 2 . The parasite viability was determined by the addition of luciferin (300 µg/mL), followed by luminescence detection on the GloMax microplate reader (Promega Corporation, Madison, WI, USA) [ 31 , 36 ].…”

“…Vero cell cultures infected with T. cruzi Dm28c-Luc, as previously described, were incubated for 72 h at 37 °C, with the promising derivatives at concentrations higher than IC 90 [ 31 ]. Then, the cultures were washed with PBS, and maintained for another 72 h at 37 °C in a drug-free medium.…”

“…The promising analogues bind by hydrogen bond and hydrophobic interactions to the active site of cruzipain, and significantly inhibit cysteine protease activity. These derivatives have been used by our research group as a hit system to synthesize new compounds through rational optimization, including ring bioisosterism, functional group interconversion, and functional group removal [ 30 , 31 ].…”