Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

Abstract

Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca2+ uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca2+-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca2+-induced membrane depolarisation and Ca2+ retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature.

Mitochondrial permeability transition pore: sensitivity to opening and mechanistic dependence on substrate availability

“…Mitochondria were isolated in accordance with published protocols 62 . Briefly, fresh rat livers were washed and finely minced in ice-cold wash buffer (250 mM sucrose, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 25 mM HEPES, adjusted to pH 7.5 using NaOH).…”

Mitochondrial Permeability Transition: A Molecular Lesion with Multiple Drug Targets

“…Moreover, its inhibitory effect on respiration and toxicity at higher micromolar concentrations indicates that it might target the OXPHOS machinery, and since the effect of oligomycin is not additive to that of Compound 4, it hints to the involvement of the ATP synthase. Finally, N-(2-benzylphenyl)-2-oxo-1H-quinoline-4carboxamide (ER-000444793) was identified from a screen of 50,000 compounds which delayed Ca 2+ -induced mPTP opening, independent of CypD binding and PPIase inhibition (Table 1) [81]. No further information on the potential target of ER-000444793 is known yet, although its effect on ATP synthesis has been ruled out.…”

Inhibition of the mitochondrial permeability transition exerts sex-specific stimulatory effect on fracture repair

“…As more effective and specific CypD inhibitors are being developed, e.g. NIM811 and JW47 (16,17), our findings suggest CypD/MPTP inhibition as a new avenue for bone anabolic strategies in fracture repair.…”

“…This work, along with previous work in our lab, provides support for the role of mitochondria, as key regulators of the osteogenic program and bone formation and repair. We posit that CypD inhibition using pharmacological inhibitors of CypD, such as NIM811, Debio025, and JW47 (16,17), as potential candidates to induce bone formation and promote bone repair especially in aging and other pathologies.…”

“…Its immunosuppressive function stems from its ability to bind and inhibit calcineurin (16). Non-immunosuppressive derivatives of CsA have been developed, such as NIM811, Debio025, and most recently JW47 (16)(17)(18). CypD genetic deletion or pharmacological inhibition are extensively used in studies of the heart and brain pathologies, where it has been shown to protect against injury, e.g.…”