The spatiotemporal regulation of Ca2+ by mitochondria drives diverse cellular functions ranging from control of oxidative metabolism to induction of cell death1,2,3,4. help understand its molecular nature. Mitochondrial Ca2+ accumulation is critically important for cellular homeostasis. The spatiotemporal regulation of Ca2+ by mitochondria drives diverse cellular functions ranging from control of oxidative metabolism to induction of cell death1,2,3,4. (24S)-24,25-Dihydroxyvitamin D3 Failure in cellular Ca2+ homeostasis and consequent mitochondrial Ca2+ overload is the principal trigger for mitochondrial permeability transition (mPT)5,6,7. mPT defines a sudden increase in mitochondrial inner membrane permeability to low molecular weight solutes of less than 1500 Daltons7. Stress-induced opening of a voltage- and Ca2+- sensitive, Vamp5 high conductance inner membrane channel, the mitochondrial permeability transition pore (mPTP) is associated with matrix swelling, (24S)-24,25-Dihydroxyvitamin D3 dissipation of mitochondrial (24S)-24,25-Dihydroxyvitamin D3 membrane potential, uncoupling of oxidative phosphorylation and cellular metabolic insufficiency6,8,9,10. Growing evidence suggests that persistent mPTP opening is a key pathophysiological event in cellular death underlying a wide variety of human diseases and disorders, notably ischaemia-reperfusion injury of the heart and brain11,12,13,14, neurodegeneration15,16 and muscular dystrophies17,18. The development of mPTP inhibitors is therefore warranted, as new agents could have a wide range of therapeutic applications in the clinic and also have utility in understanding the biomolecular nature of the pore itself. Cyclophilin D (CypD), although not an integral pore component, is a primary positive regulator of mPTP opening19,20. Pharmacological inhibition or genetic ablation of CypD enzymatic activity desensitises the pore, thereby reducing the probability of pore opening and increasing mitochondrial Ca2+ tolerance21,22,23,24,25. Therapeutic targeting of CypD is therefore somewhat limited, as its effects on pore opening are indirect and mitochondria remain ultimately capable of permeability transition21,25. To date cyclosporin A (CsA) is the best characterised inhibitor of the mPTP, exerting its effect by inhibiting CypD22,24,26. However, CsA demonstrates lack of selectivity for inhibiting CypD over other cyclophilins (consisting of 16 family members27) and shows a strong immunosuppressive effect in humans, restricting its therapeutic development potential for treating mitochondrial dysfunction28,29,30. The challenges of selectively targeting CypD are clear and therefore identification of CypD-independent mPTP inhibitors is desirable31. Despite identification of small molecule inhibitors of mPT presenting an obvious therapeutic opportunity, the availability and development of such agents remains limited. A number of groups have identified novel molecules modulating mitochondrial propensity for permeability transition30,32,33,34,35,36,37. However, as yet, reports of positive clinical development are yet to emerge. In order to screen for novel mPTP inhibitors in a rapid and efficient manner, we exploited and finessed a method to isolate mitochondria and preserve function after freeze-thaw using the cryopreservative agent (24S)-24,25-Dihydroxyvitamin D3 trehalose38. A high-throughput screen (HTS) to identify new inhibitors of mPT was then performed using the trehalose-stabilised mitochondria. This HTS yielded a number of compounds of interest. To investigate mechanism of action of the compounds, a panel of assays (mitochondrial swelling, Ca2+-induced mitochondrial membrane depolarisation and Ca2+ retention capacity) was deployed. This enabled identification of compounds specifically modulating mPT and eliminated compounds dissipating membrane potential and therefore inhibiting mitochondrial Ca2+ uptake. Further studies were performed to understand whether the compounds had CypD-dependence. Compounds were also investigated for general effects on mitochondrial and cellular health. As a result of these studies, we now describe the identification, validation and characterisation of N-(2-benzylphenyl)-2-oxo-1H-quinoline-4-carboxamide (ER-000444793), a small molecule, non-toxic mPTP inhibitor with a mechanism (24S)-24,25-Dihydroxyvitamin D3 of action independent of CypD inhibition. Results Validating the functionality of cryopreserved mitochondria by high resolution respirometry and ATP synthesis Trehalose-preserved mitochondria have previously been demonstrated to retain function, albeit with some respiratory compromise after storage at ?80?C38. To assess the utility of this approach, we isolated mitochondria from rat liver using a slightly modified method from that published by Yamaguchi PPIase reaction is extremely rapid, however using appropriate kinetics, both CsA (Fig. 6b) and SfA (Fig. 6c) were observed to dose-dependently inhibit CypD PPIase activity. In contrast, ER-000444793 had no effect on CypD enzymatic activity up to a concentration of 100?M (Fig. 6a). Inhibition values calculated using area-under-the-curve revealed potent inhibition with CsA and SfA (IC50 ?=?106?nM and IC50?=?99?nM respectively) (Fig. 6d). Open in a separate windowpane Number 6 Assessment of CypD practical activity and compound binding. rhCypD protein was incubated with chymotrypsin and compound for 30?minutes. N-Succinyl-Ala-Ala-Pro-Phe-for 10?moments at 4?C; supernatants were.
The spatiotemporal regulation of Ca2+ by mitochondria drives diverse cellular functions ranging from control of oxidative metabolism to induction of cell death1,2,3,4