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

Cruzipain (CZP), the major cysteine protease present in <i>T. cruzi</i>, 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-<i>T. cruzi</i> 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-<i>T. cruzi</i> inhibition when studied at 50 μM; and <b>Ald-6</b> excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to <i>T. cruzi</i>. Overall, not only <b>Ald-6</b> 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.