The compound BTB 06584 is an IF 1 ‐dependent selective inhibitor of the mitochondrial F 1 F o‐ ATP ase

Background and Purpose Ischaemia compromises mitochondrial respiration. Consequently, the mitochondrial F 1 F o‐ ATP synthase reverses and acts as a proton‐pumping ATP ase, so maintaining the mitochondrial membrane potential (ΔΨ 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 BMS 199264, which is reported to selectively inhibit F 1 F o‐ ATP ase activity and which is cardioprotective. Results suggested the molecule BTB 06584 (hereafter referred to as BTB ). Fluorescence microscopy was used to study its effects on ΔΨ m and on the rate of ATP consumption following inhibition of respiration in several cell types. The effect of BTB on oxygen ( O 2 ) 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 F 1 subunit ( IF 1 ), the endogenous inhibitor of the F 1 F o‐ ATP ase. Key Results BTB inhibited F 1 F o‐ ATP ase activity with no effect on ΔΨ m or O 2 consumption. ATP consumption was decreased following inhibition of respiration, and ischaemic cell death was reduced. BTB efficiency was increased by IF 1 overexpression and reduced by silencing the protein. In addition, BTB rescued defective haemoglobin synthesis in zebrafish pinotage ( pnt ) mutants in which expression of the A tpif1a gene is lost. Conclusions and Implications BTB may represent a valuable tool to selectively inhibit mitochondrial F 1 F o‐ ATP ase activity without compromising ATP synthesis and to limit ischaemia‐induced injury caused by reversal of the mitochondrial F 1 F o‐ ATP synthase.