Hydrolytic activity of mitochondrial F1Fo ATP synthase as a target for myocardial ischemia: discovery and in vitro evaluation of novel inhibitors
Authors: Nikolaou P.E , Bessis-Lazarou P , Efentakis P , Karagiannis D , Lougiakis N , Lambrinidis G , Pouli N , Marakos P , Palmeira C , Mikros E , Davidson S.M , Andreadou I 
Published: November 2020

Abstract

Background/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 cardiac response to ischemia.

Purpose
We performed an in vitro screening in order to identify and evaluate novel specific inhibitors of the hydrolytic activity of ATP synthase.

Methods
Initially, we identified inhibitors of the hydrolytic activity of ATP synthase using virtual screening methods. Docking-scoring calculations were performed targeting the three major inhibition sites of a holistic model of ATP synthase of mus musculus that was modelled for the first time. In silico ligand-based virtual screening was carried out using known binders such as BMS199264. The workflow was implemented on our in-house library of 2000 compounds, Pharmalab, plus 266,151 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, we confirmed their action 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). Finally, the compounds were tested using an assay in which primary adult rat ventricular cardiomyocytes (ARVCs) were challenged with the mitochondrial uncoupler CCCP to induce reverse ATPase activity, and time until contracture was determined. For the in vitro experiments, oligomycin, a non-selective ATPase inhibitor and BTB06584 a selective hydrolase inhibitor were used as positive controls.

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 200μM 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 μM, 99.8±1.2 μM, 144.8±1.3μM) and were evaluated on the H9C2 cells. They exhibited significant inhibitory activity at 50μM (p&lt;0.01 compared to vehicle) while the BTB06584 inhibitor was inactive at the same concentration and two of them were selective. Finally, the final novel inhibitors significantly extended (p&lt;0.001) the time until shortening of the ARVCs.

Conclusion
We have discovered two novel agents that act as selective inhibitors of ATP hydrolase and can be tested against myocardial ischemia.

Funding Acknowledgement
Type of funding source: None