The compound BTB06584 is an IF1-dependent selective inhibitor of the mitochondrial F1Fo-ATPase

Background and Purpose Ischaemia compromises mitochondrial respiration. Consequently, the mitochondrial F1Fo-ATPsynthase reverses and acts as a proton-pumping ATPase, so maintaining the mitochondrial membrane potential (m), while accelerating ATP depletion and cell death. Here we have looked for a molecule that can selectively inhibit this activity without affecting ATP synthesis, preserve ATP and delay ischaemic cell death. Experimental Approach We developed a chemoinformatic screen based on the structure of BMS199264, which is reported to selectively inhibit F1Fo-ATPase activity and which is cardioprotective. Results suggested the molecule BTB06584 (hereafter referred to as BTB). Fluorescence microscopy was used to study its effects on m and on the rate of ATP consumption following inhibition of respiration in several cell types. The effect of BTB on oxygen (O2) consumption was explored and protective potential determined using ischaemia/reperfusion assays. We also investigated a potential mechanism of action through its interaction with inhibitor protein of F1 subunit (IF1), the endogenous inhibitor of the F1Fo-ATPase. Key Results BTB inhibited F1Fo-ATPase activity with no effect on m or O2 consumption. ATP consumption was decreased following inhibition of respiration, and ischaemic cell death was reduced. BTB efficiency was increased by IF1 overexpression and reduced by silencing the protein. In addition, BTB rescued defective haemoglobin synthesis in zebrafish pinotage (pnt) mutants in which expression of the Atpif1a gene is lost. Conclusions and Implications BTB may represent a valuable tool to selectively inhibit mitochondrial F1Fo-ATPase activity without compromising ATP synthesis and to limit ischaemia-induced injury caused by reversal of the mitochondrial F1Fo-ATPsynthase.