Atomic-Level Structures Reveal How a Repurposed 3-Aminopyrazine-2-carboxamide Scaffold Inhibits Mycobacterium tuberculosis Prolyl-tRNA Synthetase

Abstract Tuberculosis (TB) is again the world’s leading cause of death from a single infectious agent. Aminoacyl-tRNA synthetases are essential for protein synthesis and are promising drug targets because bacterial and human enzymes differ. We repurposed a human prolyl-tRNA synthetase (ProRS) inhibitor scaffold, 3-aminopyrazine-2-carboxamide, to inhibit the Mycobacterium tuberculosis enzyme (MtbProRS). These derivatives show strong activity against multidrug-resistant Mtb strains and are not cytotoxic to HepG2 cells. We determined high-resolution crystal structures of MtbProRS bound to six derivatives to define their binding modes. The compounds bind in the ATP site and trigger local conformational changes that disrupt the proline pocket, supporting a dual-site mechanism of inhibition. Comparison with human cytosolic and mitochondrial ProRS reveals differences in active-site residues, electrostatics, and dynamics that explain species selectivity. Together, these structures provide a framework for designing next-generation, Mtb-selective ProRS inhibitors as new antitubercular candidates.