Toward targeted covalent inhibition of Cruzipain for the treatment of Chagas disease.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), remains a neglected tropical disease with limited therapeutic options. Cruzipain (CZP), the main cysteine protease of T. cruzi, has been extensively validated as a drug target due to its essential roles in parasite biology and pathogenesis. Targeted covalent inhibitors (TCIs) have emerged as a promising strategy to achieve potent and selective CZP inhibition, driven by advances in covalent warhead chemistry and structure-based drug design. However, their rational development faces significant challenges associated with the complex biological features of T. cruzi. In particular, the structural diversity of CZP subtypes and the frequent lack of translation from enzymatic inhibition to trypanocidal activity represent major bottlenecks in the field. This review provides a comprehensive and critical overview of the chemical space explored for CZP TCIs, including both peptidic and non-peptidic scaffolds, as well as the diverse range of covalent warheads employed. We further discuss best practices for biological evaluation of TCIs, emphasizing the importance of integrating structure-kinetics relationships and selectivity profiling. In addition, we highlight the central role of computational approaches in enabling the rational exploration of this complex chemical landscape. By integrating chemical, biological, and computational perspectives, this review delineates key challenges and emerging opportunities in the development of CZP TCIs, providing a framework to improve bioactivity translation and accelerate the discovery of effective antichagasic agents.