In Vivo Efficacy and Biochemical Target Engagement of a Chromene-Based Inhibitor of Trypanosoma cruzi Lysyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) have emerged as essential and attractive antiparasitic targets. In Trypanosoma cruzi, in vivo evidence supporting lysyl-tRNA synthetase (LysRS) as a valid target has thus far been restricted to a single chemotype. Here, we report the repositioning of a chromene-based LysRS inhibitor developed for Plasmodium falciparum and Cryptosporidium parvum, providing an independent chemical and structural validation of LysRS in T. cruzi. Compound 5 displayed potent activity against intracellular T. cruzi amastigotes (EC50 = 0.9 μM), low host-cell toxicity, and oral efficacy in an acute murine model of Chagas disease, suppressing parasitemia and reducing tissue parasite burden. Pharmacokinetic (PK) profiling revealed favorable oral bioavailability with delayed absorption, highlighting the importance of PK context in efficacy assessment. Biochemical inhibition and crystallographic structures provide evidence of T. cruzi LysRS target engagement. These findings reinforce T. cruzi LysRS as a tractable target and reveal a new chemical scaffold for Chagas disease drug development.