The LRRC8 volume‐regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel

Afzal, Aqeela; Figueroa, Eric E.; Kharade, Sujay V.; Bittman, Kevin; Matlock, Brittany K.; Flaherty, David K.; Denton, Jerod S.

doi:10.14814/phy2.14303

Cited by 20 publications

(15 citation statements)

References 47 publications

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“…The most potent, efficacious, and selective VRAC inhibitor identified to date is 4-(2-butyl-6,7-dichlor-2cyclopentylindan-1-on-5-yl) oxybutyric acid or DCPIB. However, even DCPIB has significant off-target activity toward the H-K-ATPase, inward rectifier and two-pore domain potassium channels, connexin hemichannels, glutamate transporters, and mitochondrial respiration (12)(13)(14)(15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism

Figueroa

Denton

2021

American Journal of Physiology-Cell Physiology

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LRRC8 volume-regulated anion channels (VRACs) play important roles in diverse cell types and may represent therapeutic targets for diseases. To date, however, the pharmacological tools for evaluating the druggability of VRAC have been limited to inhibitors, as no activators of the channel have been reported. We performed a fluorescence-based high-throughput screen (HTS) of 1,184 FDA-approved drugs for compounds that increase VRAC activity. The most potent VRAC potentiator identified was zinc pyrithione (ZPT), which is used commercially for treating dandruff and other skin disorders. In intracellular YFP(F46L/H148Q/I152L)-quenching assays, ZPT potentiates the rate and extent of swelling-induced iodide influx dose-dependently with a half-maximal effective concentration (EC50) of 5.7 µM. Whole-cell voltage-clamp experiments revealed that co-application of hypotonic solution and 30 µM ZPT to HEK293 or HCT116 cells increases the rate of swelling-induced VRAC activation by approximately 10-fold. ZPT potentiates swelling-induced VRAC currents after currents have reached a steady state and activates currents in the absence of cell swelling. Neither ZnCl2 nor free pyrithione activated VRAC, however, treating cells with a mixture of ZnCl2 and pyrithione led to robust channel activation. Finally, the effects of ZPT on VRAC were inhibited by reactive oxygen species (ROS) scavenger NAC and NAD(P)H oxidase inhibitor DPI, suggesting the mechanism of action involves ROS generation. The discovery of ZPT as a potentiator/activator of VRAC demonstrates the utility of HTS for identifying small-molecule modulators of VRAC and adds to a growing repertoire of pharmacological tool compounds for probing the molecular physiology and regulation of this important channel.

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Section: Introductionmentioning

confidence: 99%

Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism

Figueroa

Denton

2021

American Journal of Physiology-Cell Physiology

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“…It is important to consider that DCPIB might have significant off-target effects. A recent study demonstrated that DCPIB suppresses mitochondrial respiration independently of VSOR Cl − channel activity (Afzal et al, 2019). We suggest that the exposure to 10 µM DCPIB or Tamoxifen for several hours might similarly impair the viability of chondrocytes and promote cell death by exerting cytotoxic effects.…”

Section: Discussionmentioning

confidence: 66%

Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes

Kittl

Winklmayr

Preishuber-Pflügl

et al. 2022

Front. Cell Dev. Biol.

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In a variety of physiological and pathophysiological conditions, cells are exposed to acidic environments. Severe synovial fluid acidification also occurs in a progressive state of osteoarthritis (OA) affecting articular chondrocytes. In prior studies extracellular acidification has been shown to protect cells from apoptosis but the underlying mechanisms remain elusive. In the present study, we demonstrate that the inhibition of Cl− currents plays a significant role in the antiapoptotic effect of acidification in human articular chondrocytes. Drug-induced apoptosis was analyzed after exposure to staurosporine by caspase 3/7 activity and by annexin-V/7-actinomycin D (7-AAD) staining, followed by flow cytometry. Cell viability was assessed by resazurin, CellTiter-Glo and CellTiter-Fluor assays. Cl− currents and the mean cell volume were determined using the whole cell patch clamp technique and the Coulter method, respectively. The results reveal that in C28/I2 cells extracellular acidification decreases caspase 3/7 activity, enhances cell viability following staurosporine treatment and gradually deactivates the volume-sensitive outwardly rectifying (VSOR) Cl− current. Furthermore, the regulatory volume decrease (RVD) as well as the apoptotic volume decrease (ADV), which represents an early event during apoptosis, were absent under acidic conditions after hypotonicity-induced cell swelling and staurosporine-induced apoptosis, respectively. Like acidosis, the VSOR Cl− current inhibitor DIDS rescued chondrocytes from apoptotic cell death and suppressed AVD after induction of apoptosis with staurosporine. Similar to acidosis and DIDS, the VSOR channel blockers NPPB, niflumic acid (NFA) and DCPIB attenuated the staurosporine-induced AVD. NPPB and NFA also suppressed staurosporine-induced caspase 3/7 activation, while DCPIB and Tamoxifen showed cytotoxic effects per se. From these data, we conclude that the deactivation of VSOR Cl− currents impairs cell volume regulation under acidic conditions, which is likely to play an important role in the survivability of human articular chondrocytes.

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“…Moreover, SN-401 mediated rescue of islet insulin secretion under glucolipotoxic conditions in vitro also requires SWELL1. Finally, it is important to note that the studies demonstrating promiscuity of SN-401/ DCPIB with other ion channel targets all applied DCPIB at ~10-200 µM [68][69][70][71][72][73] . This is 100-200-fold higher than the concentrations required to potentiate SWELL1-dependent signaling in vitro (Figs.…”

Section: Discussionmentioning

confidence: 99%

Small molecule SWELL1 complex induction improves glycemic control and nonalcoholic fatty liver disease in murine Type 2 diabetes

et al. 2022

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Type 2 diabetes is associated with insulin resistance, impaired pancreatic β-cell insulin secretion, and nonalcoholic fatty liver disease. Tissue-specific SWELL1 ablation impairs insulin signaling in adipose, skeletal muscle, and endothelium, and impairs β-cell insulin secretion and glycemic control. Here, we show that ICl,SWELL and SWELL1 protein are reduced in adipose and β-cells in murine and human diabetes. Combining cryo-electron microscopy, molecular docking, medicinal chemistry, and functional studies, we define a structure activity relationship to rationally-design active derivatives of a SWELL1 channel inhibitor (DCPIB/SN-401), that bind the SWELL1 hexameric complex, restore SWELL1 protein, plasma membrane trafficking, signaling, glycemic control and islet insulin secretion via SWELL1-dependent mechanisms. In vivo, SN-401 restores glycemic control, reduces hepatic steatosis/injury, improves insulin-sensitivity and insulin secretion in murine diabetes. These findings demonstrate that SWELL1 channel modulators improve SWELL1-dependent systemic metabolism in Type 2 diabetes, representing a first-in-class therapeutic approach for diabetes and nonalcoholic fatty liver disease.

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The LRRC8 volume‐regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel

Cited by 20 publications

References 47 publications

Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism

Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism

Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes

Small molecule SWELL1 complex induction improves glycemic control and nonalcoholic fatty liver disease in murine Type 2 diabetes

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