Supervisors info:
Ιωάννα Ανδρεάδου, καθηγήτρια, φαρμακευτικής, ΕΚΠΑ
Summary:
Introduction: F1Fo ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyse ATP leading to cellular energetic deficit, contracture and lethal cardiomyocyte injury. Therefore, inhibition of ATP hydrolase is of major significance and the discovery of selective hydrolase inhibitors could be of particular interest in terms of cardioprotection.
Purpose: In the present study we aimed to: 1) identify and evaluate novel specific inhibitors of the hydrolytic activity of ATP synthase and investigate the infarct size limitation properties of the best candidates in vivo in a mouse model of ischemia reperfusion injury.
Methods: Initially, inhibitors of the hydrolytic activity of ATP synthase were identified using virtual screening methods. Docking-scoring calculations were performed targeting the three major inhibition sites of a holistic model of ATP synthase. In silico ligand-based virtual screening was carried out using known binders such as BMS199264. The workflow was implemented on University of Athens (UOA) in-house library of 2000 compounds, Pharmalab, plus the compounds of the National Cancer Institute (NCI) database. The best candidates were evaluated in vitro on isolated murine heart mitochondria to verify their inhibitory effect on ATP synthase hydrolytic activity. Moreover, their action was confirmed at a cellular level. H9C2 cells were treated with the three best candidates in the presence of rotenone and their ability to maintain membrane potential was monitored. The experiment was repeated in absence of rotenone in order to detect toxic or non-specific effects (n=5 independent experiments). For the in vitro experiments, oligomycin, a non-selective ATPase inhibitor and BTB06584 a selective hydrolase inhibitor were used as positive controls. Finally, 2 from 3 best candidates and the molecules BTB and oligomycin were evaluated in vivo. More precisely, mice were treated iv bolus with the molecules 5’ before ischemia. After that, 30’ minutes of ischemia and 2 hours of reperfusion followed, and the heart tissue was measured.
Results: Different steps of filtering through the virtual screening provided 38 molecules from Pharmalab and 15 candidates form NCI database. From the 53 candidates in total, five compounds displayed in vitro inhibitory activity at 200μΜ on isolated mitochondria and their IC50 values were determined. Among them, three synthetic derivatives possessing a central pyrazolopyridine core displayed the best IC50 values (81.7 ±1.3 μΜ, 99.8 ±1.2 μΜ, 144.8 ±1.3μΜ) and were evaluated on the H9C2 cells (molecule codes:1117,1119 and 1124). They exhibited significant inhibitory activity at 50μΜ (p<0.01 compared to vehicle) while the BTB06584 inhibitor was inactive at the same concentration and two of them were selective. Finally, the 2 candidates which were evaluated in vivo (1117 and 1119) and the molecule oligomycin proved that they can significantly decrease the infract size (33,4% ± 4,8, 34,8% ± 3,7 and 28,3% ± 2,8 respectively), in compare with the control values (45,4% ± 3,8 for the Tween control group (compare with 1117 and 1119) and 44,4% ± 3,5 for the DMSO control group (compare with oligomycin)). On the other hand, the molecule BTB failed to prove a significant decrease (39,6% ± 3,9, compared to the DMSO control group).
Conclusion: 2 novel scaffolds and 2 novel agents were discovered that act as selective inhibitors of ATP hydrolase and decrease in vivo the infract size against myocardial ischemia.
Keywords:
FoF1ATP synthase, myocardial ischemia, inhibitors of ATP hydrolysis, decrease of infract size
