Abstact

Trypanosoma cruzi, the etiological agent of Chagas disease, depends on glycolysis for ATP production, rendering its glycolytic enzymes attractive targets for therapeutic development. Here, we report the high-resolution crystal structures of two essential glycolytic enzymes, glucose-6-phosphate isomerase (Tc PGI, 1.8 A) and enolase (Tc enolase, 2.4 A) and provide structural and computational analyses to support structure-based drug design. Tc PGI adopts a dimeric alphabetaalpha sandwich fold and features a parasite-specific 53-residue N-terminal extension and a unique C-terminal hook region which both distinguish it from its human ortholog. Tc enolase exhibits the conserved (alpha/beta) 8 TIM barrel fold but harbors minor distinct structural deviations, including an extended alpha17 helix and a structured alpha1 region, which differentiate it from human isoforms. Both enzymes exhibited high thermal stability, consistent with adaptation to the parasite's complex life cycle. Structure-based virtual screening using a scaffold with known multi-target potential identified distinct high-affinity inhibitors for each enzyme. Molecular dynamics simulations further confirmed stable enzyme-inhibitor interactions and favorable binding energetics. Collectively, these findings reveal structural signatures unique to T. cruzi glycolytic enzymes and lay the groundwork for the development of antiparasitic therapeutics.