Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

Yun, Bogeon; Lee, Hee-Jung; Ghosh, Moumita; Cravatt, Benjamin F.; Hsu, Ku‐Lung; Bonventre, Joseph V.; Ewing, Heather; Gelb, Michael H.; Leslie, Christina C.

doi:10.1074/jbc.m113.497651

Cited by 28 publications

(44 citation statements)

References 68 publications

(69 reference statements)

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“…For imaging cytosolic Ca 2+ , IMLFα were loaded with Fura Red-AM for 15 min as previously described [16]. Imaging of ER and mitochondrial calcium was carried out in IMLFα expressing G-CEPIA er or CEPIA2 mt , respectively.…”

Section: Methodsmentioning

confidence: 99%

“…In a recent study we investigated the role of cPLA 2 α in regulating cell death in lung fibroblasts by using the pyrrolidine derivative pyrrophenone, and by comparing fibroblasts from cPLA 2 α wild type and knockout mice [16]. Cell death was induced in lung fibroblasts with the calcium ionophore A23187, a known inducer of necrotic cell death due to mitochondrial calcium overload and cyclophilin D-dependent opening of the mitochondrial permeability transition pore (MPTP) [16–18].…”

Section: Introductionmentioning

confidence: 99%

“…Cell death was induced in lung fibroblasts with the calcium ionophore A23187, a known inducer of necrotic cell death due to mitochondrial calcium overload and cyclophilin D-dependent opening of the mitochondrial permeability transition pore (MPTP) [16–18]. Cell death was induced to a similar extent in A23187 treated cPLA 2 α +/+ and cPLA 2 α −/− lung fibroblasts indicating no role for cPLA 2 α [16]. However, cell death in response to A23187 was blocked by pyrrophenone in both cPLA 2 α +/+ and cPLA 2 α −/− lung fibroblasts by inhibiting mitochondrial calcium uptake and MPTP [16].…”

Section: Introductionmentioning

confidence: 99%

“…Cell death was induced to a similar extent in A23187 treated cPLA 2 α +/+ and cPLA 2 α −/− lung fibroblasts indicating no role for cPLA 2 α [16]. However, cell death in response to A23187 was blocked by pyrrophenone in both cPLA 2 α +/+ and cPLA 2 α −/− lung fibroblasts by inhibiting mitochondrial calcium uptake and MPTP [16]. The ability of pyrrophenone to block cell death in cells lacking cPLA 2 α represents an off-target effect suggesting that it may target a novel pathway involving a serine hydrolase that regulates mitochondrial calcium uptake.…”

Section: Introductionmentioning

confidence: 99%

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Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum

Yun

Lee

Ewing

et al. 2016

Biochemical and Biophysical Research Communications

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Cytosolic phospholipase A2α (cPLA2α) mediates agonist-induced release of arachidonic acid from membrane phospholipid for production of eicosanoids. The activation of cPLA2α involves increases in intracellular calcium, which binds to the C2 domain and promotes cPLA2α translocation from the cytosol to membrane to access substrate. The cell permeable pyrrolidine-containing cPLA2α inhibitors including pyrrophenone have been useful to understand cPLA2α function. Although this serine hydrolase inhibitor does not inhibit other PLA2s or downstream enzymes that metabolize arachidonic acid, we reported that it blocks increases in mitochondrial calcium and cell death in lung fibroblasts. In this study we used the calcium indicators G-CEPIA1er and CEPIA2mt to compare the effect of pyrrophenone in regulating calcium levels in the endoplasmic reticulum (ER) and mitochondria in response to A23187 and receptor stimulation. Pyrrophenone blocked calcium release from the ER and concomitant increases in mitochondrial calcium in response to stimulation by ATP, serum and A23187. In contrast, ER calcium release induced by the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was not blocked by pyrrophenone suggesting specificity for the calcium release pathway. As a consequence of blocking calcium mobilization, pyrrophenone inhibited serum-stimulated translocation of the cPLA2α C2 domain to Golgi. The ability of pyrrophenone to block ER calcium release is an off-target effect since it occurs in fibroblasts lacking cPLA2α. The results implicate a serine hydrolase in regulating ER calcium release and highlight the importance of careful dose-response studies with pyrrophenone to study cPLA2α function.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum

Yun

Lee

Ewing

et al. 2016

Biochemical and Biophysical Research Communications

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“…It is generally accepted that MCU responds acutely to mitochondrial Ca 2+ levels, with very high uptake rates in the presence of Ca 2+ levels that exceed the threshold value [10]. It is also well established that MCU has a high capacity for Ca 2+ uptake into mitochondria and can thus induce mitochondrial Ca 2+ overload [11, 12]. In addition, unregulated MCU is insensitive to matrix Ca 2+ concentrations, and the ensuing excessive Ca 2+ uptake promotes the opening of permeability transition pores (PTP) and a series of detrimental consequences that are associated with disorders of Ca 2+ homeostasis and ATP depletion [13].…”

Section: Introductionmentioning

confidence: 99%

Astragaloside-IV Protects Against Heat-induced Apoptosis by Inhibiting Excessive Activation of Mitochondrial Ca2+ Uniporter

Dong

Zhang

Chen

et al. 2017

Cell Physiol Biochem

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Background: Heat causes airway damage during inhalation injury because of bronchial epithelial cell damage. Accumulating evidence shows that mitochondrial uniporter (MCU) is involved in cell damage. We investigated the MCU activity after heat treatment and assessed whether Astragaloside-IV (AS-IV) suppresses heat-induced apoptosis in bronchial epithelial cells by inhibiting the activation of the mitochondrial Ca²⁺ uniporter (MCU), mitochondrial depolarisation and reactive oxygen species (ROS) production. Methods: The bronchial epithelial cell line 16HBE14o- was heat treated, and cell apoptosis was induced in vitro and in vivo. AS-IV was inorganically administered to Wistar rats twice a day after thermal inhalation injury, and 16HBE140- cells were treated with AS-IV after incubation at 47°C for 5 min. Protein expression was determined using Western blotting and commercial kits, apoptosis with TUNEL staining, mitochondrial channel activity by patch clamp, reactive oxygen species by MitoSOX^TM fluorescence, ATP levels and enzyme activities by commercial kits as well as mitochondrial respiration and calcium by fluorescence. Results: AS-IV markedly inhibited heat-induced apoptosis, as indicated by the increased expression of the pro-apoptotic genes Bak, Bik and Bmf and increased expression of the apoptosis markers Bax, cleaved parp, cleaved caspase3 and cytochrome C. We found that MCU activation promoted mitochondrial Ca²⁺ overload, ATP depletion, mitochondrial ROS production and cytochrome c release and rapidly induced apoptosis. However, AS-IV treatment reduced excessive MCU activation and led to resistance against mitochondrial Ca²⁺ overload and excessive cytochrome C release; these effects were blocked by the MCU activator spermine. AS-IV treatment elevated ATP production and decreased ROS activity. Conclusions: MCU plays crucial roles in heat-induced mitochondrial apoptosis in 16HBE140- cells, suggesting a potential target for AS-IV treatment.

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Discovery, Synthesis, and Optimization of Diarylisoxazole‐3‐carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

et al. 2015

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The mitochondrial permeability transition pore (mtPTP) is a Ca2+-requiring mega-channel which, under pathological conditions, leads to the deregulated release of Ca2+ and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5-(3-hydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)isoxazole-3-carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50 < 0.39 µm) and showed no interference on the inner mitochondrial membrane potential (rhodamine 123 uptake EC50 > 100 µm). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N-(3-chloro-2-methylphenyl)-5-(4-fluoro-3-hydroxyphenyl)isoxazole-3-carboxamide (60), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies.

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Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

Cited by 28 publications

References 68 publications

Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum

Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum

Astragaloside-IV Protects Against Heat-induced Apoptosis by Inhibiting Excessive Activation of Mitochondrial Ca2+ Uniporter

Discovery, Synthesis, and Optimization of Diarylisoxazole‐3‐carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

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