Structure-Aided Computational Design of Triazole-Based Targeted Covalent Inhibitors of Cruzipain.

Cruzipain (CZP), the major cysteine protease present in T. cruzi, the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti-T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti-T. cruzi inhibition when studied at 50 µM; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi. Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents.

Design and synthesis of new 1,2,3-triazoles derived from eugenol and analogues with in vitro and in vivo activity against Trypanosoma cruzi.

Chagas disease (CD) is a neglected tropical disease endemic in 21 countries and affects about 8 million people around the world. The pharmacotherapy for this disease is limited to two drugs (Benznidazole and Nifurtimox) and both are associated with important limitations, as low cure rate in the chronic phase of the disease, high toxicity and increasing resistance by Trypanosoma cruzi. Recently, we reported a bioactive 1,2,3-triazole (compound 35) active in vitro (IC50 42.8 µM) and in vivo (100 mg/kg) against T. cruzi Y strains and preliminary in silico studies suggested the cysteine protease cruzain as a possible target. Considering these initial findings, we describe here the design and synthesis of new 1,2,3-triazoles derivatives of our hit compound (35). The triazoles were initially evaluated against healthy cells derived from neonatal rat cardiomyoblasts (H9c2 cells) to determine their cytotoxicity and against epimastigotes forms of T. cruzi Y strain. The most active triazoles were compounds 26 (IC50 19.7 µM) and 27 (IC50 7.3 µM), while benznidazole was active at 21.6 µM. Derivative 27 showed an interesting selectivity index considering healthy H9c2 cells (>77). Promising activities against trypomastigotes forms of the parasite were also observed for triazoles 26 (IC50 20.74 µM) and 27 (IC50 8.41 µM), mainly 27 which showed activity once again higher than that observed for benznidazole (IC50 12.72 µM). While docking results suggested cruzain as a potential target for these compounds, no significant enzyme inhibition was observed in vitro, indicating that their trypanocidal activity is related to another mode of action. Considering the promising in vitro results of triazoles 26 and 27, the in vivo toxicity was initially verified based on the evaluation of behavioral and physiological parameters, mortality, effect in body weight gain, and through the measurement of AST/ALT enzymes, which are markers of liver toxicity. All these evaluations pointed to a good tolerability of the animals, especially considering triazole 27. A reduction in parasitemia was observed among animals treated with triazole 27, but not among those treated with derivative 26. Regarding the dosage, derivative 27 (100 mg/kg) was the most active sample against T. cruzi infection, showing a 99.4% reduction in parasitemia peak. Triazole 27 at a dosage of 100 mg/kg influenced the humoral immune response and reduced myocarditis in the animals, bringing antibody levels closer to those observed among healthy mice. Altogether, our results indicate compound 27 as a new lead for the development of drug candidates to treat Chagas disease.

Screening the Pathogen Box to Discover and Characterize New Cruzain and TbrCatL Inhibitors.

Chagas disease and Human African Trypanosomiasis, caused by Trypanosoma cruzi and T. brucei, respectively, pose relevant health challenges throughout the world, placing 65 to 70 million people at risk each. Given the limited efficacy and severe side effects associated with current chemotherapy, new drugs are urgently needed for both diseases. Here, we report the screening of the Pathogen Box collection against cruzain and TbrCatL, validated targets for Chagas disease and Human African Trypanosomiasis, respectively. Enzymatic assays were applied to screen 400 compounds, validate hits, determine IC50 values and, when possible, mechanisms of inhibition. In this case, 12 initial hits were obtained and ten were prioritized for follow-up. IC50 values were obtained for six of them (hit rate = 1.5%) and ranged from 0.46 ± 0.03 to 27 ± 3 µM. MMV687246 was found to be a mixed inhibitor of cruzain (Ki = 57 ± 6 µM) while MMV688179 was found to be a competitive inhibitor of cruzain with a nanomolar potency (Ki = 165 ± 63 nM). A putative binding mode for MMV688179 was obtained by docking. The six hits discovered against cruzain and TbrCatL are of great interest for further optimization by the medicinal chemistry community.