Hydrolytic Activity of Mitochondrial F 1 F O -ATP Synthase as a Target for Myocardial Ischemia-Reperfusion Injury: Discovery and In Vitro and In Vivo Evaluation of Novel Inhibitors

F F -ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, it reverses its activity, hydrolyzing ATP and leading to energetic deficit and cardiac injury. We aimed to discover novel inhibitors of ATP hydrolysis, accessing the druggability of the target within ischemia(I)/reperfusion(R) injury. New molecular scaffolds were revealed using ligand-based virtual screening methods. Fifty-five compounds were tested on isolated murine heart mitochondria and H9c2 cells for their inhibitory activity. A pyrazolo[3,4- ]pyridine hit structure was identified and optimized in a hit-to-lead process synthesizing nine novel derivatives. Three derivatives significantly inhibited ATP hydrolysis , while , they reduced myocardial infarct size (IS). The novel compound was the most effective in reducing IS, validating that inhibition of F F -ATP hydrolytic activity can serve as a target for cardioprotection during ischemia. Further examination of signaling pathways revealed that the cardioprotection mechanism is related to the increased ATP content in the ischemic myocardium and increased phosphorylation of PKA and phospholamban, leading to the reduction of apoptosis.