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Abstract

In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca²⁺ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFP^QL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

Keywords

connexin (Cx)drug discovery hemichannel (HC)high-content screening (HCS)central nervous system (CNS)

Introduction

Cell-to-cell communication is mediated by a family of transmembrane proteins, called connexins (Cx). Cx43 is the most abundant and widespread isoform and can form both gap junctions (GJs) and hemichannels (HCs) on the cell membrane. More specifically, Cx43 HCs allow exchange between the intra- and extracellular environments of small molecules and ions.¹ HC opening or closure probabilities are regulated by inflammation, hypoxia, changes in intra- and extracellular osmolarity concentrations, and cytosolic pH.² In physiological situations, HCs are mostly closed.³ However, in pathological situations, HC pores are opened more frequently than usual, leading to so-called “leaky hemichannels”⁴ and resulting in the excessive release of ATP and glutamate, with damage to neighboring cells,⁵ or in the unnecessary influx of ions, leading to ionic imbalance.⁶ Mutations in Cx-encoding genes have also been identified in disorders of the skin, ear, heart, eye lens, and brain.⁴ HCs containing Cx43 are also upregulated in different pain models, for example, neurodegenerative disorders such as Alzheimer’s disease, cerebral ischemia, amyotrophic lateral sclerosis, and cancer.⁷

Modulation of Cx43 HCs has been successfully achieved using pharmacological approaches^8,9 or mimetic peptide application.¹⁰ Meanwhile, experimental approaches on animal models using those peptides or RNA interference using silencing or short-hairpin RNA oligonucleotides,^11,12 and ongoing clinical trials,¹³ have confirmed that HCs represent innovative—yet largely unexplored—therapeutic targets, notably in central nervous system (CNS) disorders.⁷

Identifying new modulators of Cx-based HCs is therefore a necessity in this therapeutic perspective. Classical methods to assess HC function include the quantification of ATP release¹⁴ and uptake or leakage of fluorescent dyes.^14,15 Additional techniques have been further developed in bacteria deficient in K⁺ uptake and genetically modified to express functional human Cx,¹⁶ or using passive uptake of iodide and the quenching of a iodide-sensor protein (iodide-sensitive yellow fluorescent protein [YFP^QL]).¹⁷ Previous high-content (HCS) and high-throughput (HTS) screening methods have been described to monitor the GJ function using microfluidic chip technology¹⁸ or automated fluorescence imaging assay.^19–21 Here, we developed automated versions of two different cell-based assays^14,15 to specifically identify new modulators of HC function in cell culture. Both assays exploit the sensitivity of HCs to calcium to control channel opening. While high external calcium maintains the HCs in a closed conformation, depletion of extracellular Ca²⁺ increases the probability of opening of HCs.²² The first assay is based on the incorporation of Yo-Pro, a fluorescent dye that enters the cells via open HCs. Previous work on HCs using Yo-Pro incorporation correlated the uptake of Yo-Pro and the total amount of Cx43.²³ In the second assay, iodide uptake through open HCs is indirectly followed by measurement of the iodide-dependent quenching of a fluorescent protein, YFP^QL.¹⁷ These assays were validated with known pharmacological inhibitors of HCs and used for the screening of a focused collection of 2242 approved drugs or compounds under preclinical and clinical development. As far as we know, this study is the first systematic screening of HC modulators using chemical libraries dedicated to the identification of HC modulators for CNS disorders.

Materials and Methods

Reagents, Cell Culture, and Chemical Compounds

Opti-MEM culture media, 0.05% trypsin-EDTA, penicillin/streptomycin, Hank’s Balanced Salt Solution (HBSS), fetal bovine serum (FBS), Yo-Pro-1, iodide, and other chemical compounds were purchased from Invitrogen (Saint-Aubin, France), Sigma-Aldrich (St. Louis, MO), or GE Healthcare Life Science (Velizy-Villacoublay, France). IncuCyte Cytotox Red Reagent was purchased from Sartorius (Royston Hertfordshire, UK). Human glioblastoma U251 cells were purchased from Sigma-Aldrich and Ln215-YFP^QL cells were licensed by Yonsei University, Seoul, Korea.²¹

An in-house selection of 2242 Food and Drug Administration (FDA)- or European Medicines Agency (EMA)-approved drugs and compounds under clinical assays was generated from two commercial sources, including 1280 compounds from the Prestwick Chemical Library (Illkirch, France) and 962 additional drugs that were selected in the tebu-bio library (Le Perray-en-Yvelines, France). Compounds were dissolved in 10 mM DMSO.

Cx43 Silencing Procedure

As presented in Picoli et al.,²⁴ silencing of Cx43 in U251 cells was efficiently achieved by stable transfection with shRNA-Cx43 TRCN0000344843 (shCx43-843) (targets 30-UTR region) from the Mission Sigma shRNA Library, according to the manufacturer’s instructions. Cells stably transfected with nonrelevant shRNA (indicated as shNE) were used as controls.

Cytotoxicity Assay

U251 and Ln215-YFP^QL cells were seeded at 10,000 and 24,000 cells/well, respectively, in 100 µL of fresh culture medium in 96-well tissue culture microplates. Cellular viability was assessed using the IncuCyte Cytotox Red reagent according to the manufacturer’s protocol, in the presence of Yo-Pro (5 µM) for U251 cells or iodide (140 mM) for Ln215-YFP^QL cells, in the presence or absence of Ca²⁺. Four images per well (covering 27% of the well surface) were automatically acquired using IncuCyte Zoom (Essen BioScience, Ann Arbor, MI) every 5 min for 1 h in phase contrast and in the red fluorescence channel with a 10× objective. A threshold based on the fluorescence intensity of Cytotox Red staining was applied for the selection of the dead cells.

Monitoring of Yo-Pro Incorporation in U251 Cells by Real-Time Imaging

A Yo-Pro incorporation assay has been adapted from Orellana et al.²⁵ in 96-well plates. Briefly, U251 cells were seeded at 10,000 cells/well in 96-well tissue culture microplates (Greiner, Les Ulis, France, no. 655090) in Opti-MEM medium supplemented with 10% FBS and allowed to grow for 24 h. Cells were then washed with HBSS buffer with or without Ca²⁺ and treated simultaneously with 5 µM Yo-Pro and chemical compounds. The microplates were transferred into an automated imaging system (IncuCyte Zoom). Four images per well were acquired in phase contrast and in the green fluorescence channel with a 4× objective, 10, 15, and 20 min after compound addition. A threshold based on the fluorescence intensity of Yo-Pro green staining was applied for the selection of the Yo-Pro-positive (Yo-Pro⁺) cells. Yo-Pro uptake activity was subsequently calculated and expressed as a percentage of maximal uptake obtained in the absence of calcium, according to the following formula:

Y o - P r o u p t a k e = \frac{V a l u e_{c o m p o u n d} - m e a n_{C o n t r o l + C a 2 +}}{m e a n_{C o n t r o l - C a 2 +} - m e a n_{C o n t r o l + C a 2 +}}

where $m e a n_{C o n t r o l + C a 2 +}$ and $m e a n_{C o n t r o l - C a 2 +}$ correspond to the mean number of Yo-Pro fluorescent cells for the bioactive and bioinactive controls, respectively, and $V a l u e_{c o m p o u n d}$ to the number of Yo-Pro⁺ cells after compound treatment.

Automated Monitoring of YFP^QL Quenching by Iodide Incorporation in Ln215 Cells

The YFP^QL quenching assay has been here adapted from Lee et al. to a 96-well plate format.²¹ Briefly, YFP^QL-expressing Ln215 cells were seeded at 24,000 cells/well in 96-well tissue culture microplates (Greiner, no. 655090) in DMEM supplemented with 10% FBS and allowed to grow for 24 h. Cells were then washed with HBSS buffer with or without Ca²⁺ and treated with chemical compounds for 10 min at 37 °C. Immediately after the subsequent addition of 140 mM sodium iodide, the YFP^QL fluorescent signal was monitored using an Infinite M1000 multimode reader (Tecan, Switzerland) with acquisitions every 80 s for 560 s. Results were expressed in percentage of quenching activity according to the following formula:

Q u e n c h i n g a c t i v i t y = \frac{V a l u e_{c o m p o u n d} - m e a n_{C o n t r o l + C a 2 +}}{m e a n_{C o n t r o l - C a 2 +} - m e a n_{C o n t r o l + C a 2 +}}

Results

Setup of a Real-Time Microscopy-Based Assay to Quantify HC Function by Yo-Pro Uptake

Yo-Pro incorporation by U251 cells was analyzed in a 96-well format from 10 to 20 min after calcium depletion ( Fig. 1A ). The seeding density was optimized for image analysis and levels of Yo-Pro incorporation in the bioactive and bioinactive controls, that is, in the presence and absence of Ca²⁺ respectively, by monitoring the Z′ factor (see below) in different cell density conditions (from 6000 to 12,000 cells/well). As expected, in the presence of Ca²⁺, only a few cells incorporated the fluorescent dye due to the closed conformation of the HCs. In contrast, removal of calcium triggered a massive entry of Yo-Pro ( Fig. 1B ). Over the time course of the assay, the number of dead cells in the absence of Ca²⁺ is similar to the number of dead cells in the presence of Ca²⁺, with around 10 dead cells ( Suppl. Fig. S1 ), that is, a negligible value compared with the number of seeded cells (10,000 cells/well). This result indicates that the Yo-Pro uptake in the absence of Ca²⁺ is not attributable to cell death. The Z′ factor was calculated according to Zhang et al.²⁶ for each time point in the presence or absence of Ca²⁺ used as bioactive and bioinactive controls, respectively (n = 24 for each condition). The Z′ factors were 0.46, 0.46, and 0.51 and signal-to-noise (S/N) ratios reached 5.66, 6.15, and 8.01 at 10, 15, and 20 min, respectively, indicating robust differences between bioactive and bioinactive controls (S/N ratio > 5) and sufficient robustness for automated HCS at 20 min (Z′ > 0.50) ( Fig. 1C ). As anticipated, calcium depletion resulted in a dose-dependent activation of the HC activity ( Fig. 1D ). Importantly, in the range of concentration used in the assay, DMSO had no effect on the entry of Yo-Pro ( Fig. 1E ). Since U251 glioblastoma cells express high levels of Cx43, Cx43 was assumed to be the major channel for Yo-Pro incorporation. The contribution of Cx43 to Yo-Pro entry has been validated in silencing experiments using a previously validated shRNA.²⁴ In Cx43 knockdown cells, Yo-Pro incorporation showed a ~60 % inhibition of uptake compared with the control cells at 20 min. This result clearly indicates that Yo-Pro incorporation mainly depends on Cx43 expression ( Suppl. Fig. S2 ). Using this assay, the previously described HC modulator meclofenamic acid inhibited Yo-Pro entry up to 55% at 25 and 50 µM ( Fig. 1F ), whereas carbenoxolone, another known inhibitor, had no effect at any concentration tested in the specific conditions of the assay ( Fig. 1G ).

Figure 1.

Cell-based assay setup to follow the incorporation of the Yo-Pro fluorescent marker in U251 cells by real-time imaging. (A) Scheme of the procedure. (B) Example of image acquisition and image analysis processing of cells treated with 0.25% DMSO with or without Ca²⁺ at 20 min. A threshold was applied on the green channel, allowing the detection of Yo-Pro⁺ cells (pink mask); scale = 200 µm. (C) Scatterplots representing the number of Yo-Pro⁺ cells at 10, 15, and 20 min in the absence or presence of Ca²⁺. Z′ factors and S/N ratios are indicated for each time point (n = 24 for each control). (D,E) Dose effect of Ca²⁺ (2.5–1260 µM) and DMSO (0.01%–0.4%) on the incorporation of Yo-Pro at 20 min. Values are mean ± SD (n = 4). (F,G) Dose-dependent effect of meclofenamic acid (F) and carbenoxolone (G) tested from 6.3 to 40 µM and 1.5 to 40 µM, respectively, on the incorporation of Yo-Pro at 20 min in the absence of Ca²⁺. Values are mean ± SD (n = 5 for F or n = 3 for G).

Setup of an Automated Assay to Quantify HC Function through the Rapid YFP^QL Fluorescence Quenching after Iodide Incorporation

As described previously, HC function can be monitored through the HC-dependent passive uptake of iodide revealed by the quenching of the YFP^QL, stably expressed in Ln215 cells.²¹ We set up the conditions to monitor the quenching of YFP^QL fluorescence following Ca²⁺ depletion in 96-well microplates ( Fig. 2A ). The adequate number of cells was determined using different concentrations of cells per well (from 10,000 to 24,000 cells) in the presence or absence of Ca²⁺, with an optimal condition of robustness and reproducibility obtained with 24,000 cells. Gain of the multimode reader was adjusted to avoid saturation of the fluorescent signal (65,000 AU of fluorescence) and finally set at less than 50% of the maximum (value maximum of <25,000 in Fig. 2B ). Furthermore, in comparison to the number of cells seeded initially (24,000/well), the number of dead cells in the absence of Ca²⁺ is comparable to the number of dead cells in the presence of Ca²⁺ and negligible when there are fewer than 20 dead cells over the time course of the assay ( Suppl. Fig. S3 ). Using this setup, calculated Z′ factors (n = 20 for each control) ranged from 0.3 to 0.5. Interestingly, S/N ratios were all strictly above 2, ranging from 2.8 to 3.1, indicating at least twofold differences between bioactive and bioinactive controls ( Fig. 2B ). Furthermore, as described above with the Yo-Pro incorporation assay, a dose-dependent effect of Ca²⁺ was observed on the YFP^QL fluorescence quenching ( Fig. 2C ), whereas DMSO had no significant effect ( Fig. 2D ). In this assay, meclofenamic acid had a light inhibitory effect on the quenching of YFP^QL fluorescence with a maximal inhibitory effect of 55% observed at 15.6 and 25 µM, while carbenoxolone inhibited the quenching in a dose-dependent manner with a graphically estimated IC₅₀ of 9.1 µM ( Fig. 2E,F ).

Figure 2.

Cell-based assay for automated monitoring of YFP^QL quenching by iodide incorporation. (A) Scheme of the procedure. (B) Ln215-YFP^QL cells were incubated with or without Ca²⁺ for 10 min; YFP^QL fluorescence was measured every 80 s immediately after iodide injection for 600 s. Values are mean ± SD (n = 20). #Calculated Z′ factors above 0.5. (C,D) Evaluation of the effect of a concentration range of Ca²⁺ (2.5–1260 µM) and DMSO (0.01%–0.40%) on the quenching of YFP^QL fluorescent protein at 160 s after iodide addition. Values are mean ± SD (n = 4). (E,F) Effect of meclofenamic acid and carbenoxolone tested from 1.5 to 40 µM on the quenching of YFP^QL fluorescence at 160 s in the absence of Ca²⁺. Values are mean ± SD (n = 3).

Screening of an In-House Collection of FDA-Approved Drugs

To identify putative modulators of HCs, an in-house collection of 2242 compounds was screened using the Yo-Pro incorporation cell-based assay by real-time imaging ( Fig. 3A ). A subset of 148 compounds reducing Yo-Pro uptake to or below 35% of bioinactive controls (fixed at 100%) were selected as primary hits ( Fig. 3B ). A set of 23 cytotoxic compounds leading to a decrease of cell confluence was discarded. Visual inspection of green channel images was then performed to eliminate an additional set of 38 false-positive hits, mainly due to fluorescence interference or compound precipitation. Finally, 50 compounds were considered as nonrelevant for the field of CNS drug discovery. This final selection led to a set of 37 FDA-approved drugs, thus representing a final hit rate of 1.65% ( Fig. 3A ).

Figure 3.

Screening workflow and results. (A) Scheme of the screening and selection process. (B) Scatterplot representing the percentage of Yo-Pro incorporation compared with controls (DMSO) at 20 min for the 2242 compounds tested at a final concentration of 25 µM (n = 2 per condition; each dot corresponds to the mean of two replicates). Compounds presenting less than 35% incorporation activity were selected for further validation. (C) Scatterplot representing the lowest value (i.e., the highest inhibitory activity) of iodide-induced YFP^QL quenching activity for the 37 compounds selected after the primary screen out of the 10 concentrations tested (1.04–40.0 µM) at a 160 s time point. The 11 compounds that also inhibited the quenching of YFP^QL by iodide incorporation are highlighted in red (B,C). (D,E) Two compounds (circles in C) were tested in dose response from 1.5 to 40 µM on either the Yo-Pro incorporation assay (dark circle) or the iodide-induced YFP^QL quenching assay (empty circle). Yo-Pro uptake or quenching activity percentages are relative to the controls. Values are mean ± SD (n = 3).

Comparison of the Activity of Primary Hits on Yo-Pro and Iodide Incorporation Assays

The selected 37 primary hits were tested at 10 concentrations in simplicate, together with meclofenamic acid and carbenoxolone, used here as controls on both the Yo-Pro incorporation assay and the iodide-induced YFP^QL quenching assay. The ability to inhibit Yo-Pro incorporation was confirmed for 25 compounds, with at least one efficient concentration (not shown). In this assay, meclofenamic acid displayed an IC₅₀ above 5 µM (not shown), a value consistent with our previous results obtained during the setup of the assay (see Fig. 1F ). Interestingly, among the 25 previously validated hits, 11 compounds also inhibited the incorporation of iodide ( Fig. 3C ). Two molecules of interest displaying different profiles of activity that are representative of those obtained with the 37 primary hits were chosen and replicated. These two molecules were hit-picked in the original library and tested in triplicates at eight doses (from 1.5 to 40 µM). Molecule 1 induced a dose–response inhibitory effect on Yo-Pro incorporation (IC₅₀ = 8.6 µM) but not on iodide entry; molecule 2 repeatedly inhibited both Yo-Pro incorporation and iodide entry (IC₅₀ = 30 and 25 µM, respectively) ( Fig. 3D,E ).

Discussion

Cx, and notably Cx43, are dysregulated in numerous indications⁷ and have been considered as therapeutic targets²⁷ in sleep disorders,^28–30 neuropathic pain,⁹ skin and corneal wounds, or cardiac ischemic injury.³¹ Marketed drugs routinely used in patients, such as antidepressants,³² have been revealed to act on Cx43-based HCs. Conclusively, investigating a library of FDA- and EMA-approved drugs using HC-targeting assays may therefore identify compounds with so far unsuspected HC activity-modulating properties and prove particularly fruitful.

Cx43 GJ function has been previously monitored in cell culture through the quantification of cell–cell transfer of fluorescent dyes,³³ calcium,³⁴ or iodide.¹⁷ Assays have been adapted to HTS, using microfluidic chips³⁵ and automated fluorescence imaging assays.³⁶ However, those assays are valid to measure GJ but not HC function, as it is largely admitted that under regular culture conditions, the contribution of those HCs is minor^37,38—hence the need to use nonphysiological conditions to specifically monitor their activity. In physiological conditions, HCs are actually mostly in their closed conformation, due to extracellular Ca²⁺ and Mg²⁺ in the millimolar range, negative membrane potentials, or posttranslational modifications.³ Depletion of extracellular Ca²⁺ is hence classically used in cell culture to assess HC function.^14,15,17

We present here the adaptation to automated screening of two previously described cell-based HC assays.¹⁵ The two cell lines have been selected due to endogenous expression of Cx43 in U251¹⁷ and the conclusive use of Ln215 in GJ and HC assays.^17,21,39 Supporting previous reports, extracellular calcium depletion increased HC-mediated uptake of the probes in a dose-dependent manner, and DMSO, the solvent used to solubilize compounds, was revealed to be inactive. Finally, meclofenamic acid, a broad Cx blocker,^30,40 confirmed the sensitivity of the assays. Surprisingly, carbenoxolone displayed inconsistent effects in the two assays, potentially correlating with its previously described relative potency on Cx43.^41,42 The two assays have been described as dependent upon Cx43 HC function;^17,43 this has been further demonstrated in the present study using RNA silencing in U251, and elsewhere in Ln215 using CRISPR-Cas9 KO.³⁹ In addition, the Yo-Pro incorporation assay notably depends on the transfer of Yo-Pro from the cytoplasm to the nucleus after uptake, while the YFP quenching assay notably requires an active quenching of YFP by iodide after iodide uptake. We chose to focus on the search for compounds active on both assays to be considered as properly active against HCs.

These two assays were used for the screening of a chemical collection and further hit characterization. An in-house chemical library composed of 2242 approved drugs was screened on the Yo-Pro incorporation assay (Z′ > 0.5). A threshold was fixed at 35% activity relative to the control, as it allowed the clear discrimination between bioactive and bioinactive controls. A total of 148 primary hits were identified with Yo-Pro uptake under this threshold. Nonrelevant (poor ability to cross the blood-brain barrier, low oral bioavailability, adverse effect profile, etc.) and toxic compounds were eliminated from this first set, leading to a subset of 25 confirmed hits. Among them, 11 also inhibited the quenching of YFP^QL by iodide incorporation after calcium depletion, representing a discovery rate of 0.49%.

Besides, a subset of 1280 compounds of this collection have already been screened on Cx43 GJ function using an automated assay monitoring dye transfer;²⁰ 3 out of 11 compounds efficient on both HC assays also inhibited GJ function. Interestingly, five compounds were active on HCs but not on GJs, and additional studies should confirm their specificity toward HCs and not GJs and evaluate their effects using a newly developed in vivo imaging technique based on manganese-enhanced magnetic resonance.⁴⁴

To our knowledge, this study represents the first large screening on HC activity and suggests a new use for already approved drugs in CNS disorders with HC impairments.

Supplemental Material

Figure_S1_v4 – Supplemental material for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

Supplemental material, Figure_S1_v4 for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function by Emmanuelle Soleilhac, Marjorie Comte, Anaelle da Costa, Caroline Barette, Christèle Picoli, Magda Mortier, Laurence Aubry, Franck Mouthon, Marie-Odile Fauvarque and Mathieu Charvériat in SLAS Discovery

Supplemental Material

Figure_S2_v3 – Supplemental material for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

Supplemental material, Figure_S2_v3 for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function by Emmanuelle Soleilhac, Marjorie Comte, Anaelle da Costa, Caroline Barette, Christèle Picoli, Magda Mortier, Laurence Aubry, Franck Mouthon, Marie-Odile Fauvarque and Mathieu Charvériat in SLAS Discovery

Supplemental Material

Figure_S3_v3 – Supplemental material for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

Supplemental material, Figure_S3_v3 for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function by Emmanuelle Soleilhac, Marjorie Comte, Anaelle da Costa, Caroline Barette, Christèle Picoli, Magda Mortier, Laurence Aubry, Franck Mouthon, Marie-Odile Fauvarque and Mathieu Charvériat in SLAS Discovery

Footnotes

Acknowledgements

This work has notably been performed at the CMBA screening platform (http://irig.cea.fr/drf/irig/english/Pages/Platform/CMBA.aspx) supported by the GRAL LabEX GRAL, ANR-10-LABX-49-01, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche), CBH-EUR-GS (ANR-17-EURE-0003). The CMBA is a member of the Chembiofrance research infrastructure (www.chembiofrance.fr) and the GAP2D consortium supported by the GIS-IBiSA (). Ln215 YFPQL cells were produced by Dr. Jinu Lee and licensed by Yonsei University (Seoul, Korea).

Supplemental material is available online with this article.

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.D., C.P., F.M., and M.C. are full-time employees of Theranexus Company.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the CEA, Theranexus, and a research grant from the Auvergne-Rhone-Alpes district (Pack Ambition 2017, no. 17 011104 01-8748).

ORCID iD

Mathieu Charvériat

References

Burra

Jiang

J. X.

Regulation of Cellular Function by Connexin Hemichannels. Int. J. Biochem. Mol. Biol. 2011, 2, 119–128.

Tarzemany

Jiang

J. X.

; et al. Connexin 43 Hemichannels Regulate the Expression of Wound Healing-Associated Genes in Human Gingival Fibroblasts. Sci. Rep. 2017, 7, 14157.

Contreras

J. E.

Saez

J. C.

Bukauskas

F. F.

; et al. Gating and Regulation of Connexin 43 (Cx43) Hemichannels. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11388–11393.

Retamal

M. A.

Reyes

E. P.

Garcia

I. E.

; et al. Diseases Associated with Leaky Hemichannels. Front. Cell. Neurosci. 2015, 9, 267.

Orellana

J. A.

Shoji

K. F.

Abudara

; et al. Amyloid Beta-Induced Death in Neurons Involves Glial and Neuronal Hemichannels. J. Neurosci. 2011, 31, 4962–4977.

Orellana

J. A.

Retamal

M. A.

Moraga-Amaro

; et al. Role of Astroglial Hemichannels and Pannexons in Memory and Neurodegenerative Diseases. Front. Integr. Neurosci. 2016, 10, 26.

Xing

Yang

Cui

; et al. Connexin Hemichannels in Astrocytes: Role in CNS Disorders. Front. Mol. Neurosci. 2019, 12, 23.

Delvaeye

Vandenabeele

Bultynck

; et al. Therapeutic Targeting of Connexin Channels: New Views and Challenges. Trends Mol. Med. 2018, 24, 1036–1053.

Jeanson

Duchêne

Richard

; et al. Potentiation of Amitriptyline Anti-Hyperalgesic-Like Action by Astroglial Connexin 43 Inhibition in Neuropathic Rats. Sci. Rep. 2016, 6, 38766.

10.

Leybaert

Lampe

P. D.

Dhein

; et al. Connexins in Cardiovascular and Neurovascular Health and Disease: Pharmacological Implications. Pharmacol. Rev. 2017, 69, 396–478.

11.

Zhang

Yang

Zhu

; et al. Relationship of Cx43 Regulation of Vascular Permeability to Osteopontin-Tight Junction Protein Pathway after Sepsis in Rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2018, 314, R1–R11.

12.

Han

X. J.

Chen

Hong

; et al. Lentivirus-Mediated RNAi Knockdown of the Gap Junction Protein, Cx43, Attenuates the Development of Vascular Restenosis Following Balloon Injury. Int. J. Mol. Med. 2015, 35, 885–892.

13.

Montgomery

Ghatnekar

G. S.

Grek

C. L.

; et al. Connexin 43-Based Therapeutics for Dermal Wound Healing. Int. J. Mol. Sci. 2018, 19, 1778.

14.

Lohman

A. W.

Isakson

B. E.

Differentiating Connexin Hemichannels and Pannexin Channels in Cellular ATP Release. FEBS Lett. 2014, 588, 1379–1388.

15.

Johnson

R. G.

H. C.

Evenson

; et al. Connexin Hemichannels: Methods for Dye Uptake and Leakage. J. Membr. Biol. 2016, 249, 713–741.

16.

Krishnan

Fiori

M. C.

Whisenant

T. E.

; et al. An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels. SLAS Discov. 2017, 22, 135–143.

17.

Lee

J. Y.

Yoon

S. M.

Choi

E. J.

; et al. Terbinafine Inhibits Gap Junctional Intercellular Communication. Toxicol. Appl. Pharmacol. 2016, 307, 102–107.

18.

Bathany

Beahm

Felske

J. D.

; et al. High Throughput Assay of Diffusion through Cx43 Gap Junction Channels with a Microfluidic Chip. Anal. Chem. 2011, 83, 933–939.

19.

Picoli

Nouvel

Aubry

; et al. Human Connexin Channel Specificity of Classical and New Gap Junction Inhibitors. J. Biomol. Screen. 2012, 17, 1339–1347.

20.

Picoli

Soleilhac

Journet

; et al. High-Content Screening Identifies New Inhibitors of Connexin 43 Gap Junctions. Assay Drug Dev. Technol. 2019, 17, 240–248.

21.

Lee

J. Y.

Choi

E. J.

Lee

A New High-Throughput Screening-Compatible Gap Junctional Intercellular Communication Assay. BMC Biotechnol. 2015, 15, 90.

22.

Ransom

Giaume

Gap Junctions and Hemichannels. In Neuroglia, 3rd Ed.; Kettenmann, H., Ransom, B., Eds.; Oxford University Press: Oxford, 2012.

23.

Saez

J. C.

Vargas

A. A.

Hernandez

D. E.

; et al. Permeation of Molecules through Astroglial Connexin 43 Hemichannels Is Modulated by Cytokines with Parameters Depending on the Permeant Species. Int. J. Mol. Sci. 2020, 21, 3970.

24.

Picoli

Soleilhac

Journet

; et al. High-Content Screening Identifies New Inhibitors of Connexin 43 Gap Junctions. Assay Drug Dev. Technol. 2019, 17, 240–248.

25.

Orellana

J. A.

Diaz

Schalper

K. A.

; et al. Cation Permeation through Connexin 43 Hemichannels Is Cooperative, Competitive and Saturable with Parameters Depending on the Permeant Species. Biochem. Biophys. Res. Commun. 2011, 409, 603–609.

26.

Zhang

J. H.

Chung

T. D.

Oldenburg

K. R.

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screen. 1999, 4, 67–73.

27.

Charvériat

Naus

C. C.

Leybaert

; et al. Connexin-Dependent Neuroglial Networking as a New Therapeutic Target. Front. Cell. Neurosci. 2017, 11, 174.

28.

Sauvet

Erblang

Gomez-Merino

; et al. Efficacy of THN102 (a Combination of Modafinil and Flecainide) on Vigilance and Cognition during 40-Hour Total Sleep Deprivation in Healthy Subjects: Glial Connexins as a Therapeutic Target. Br. J. Clin. Pharmacol. 2019, 85, 2623–2633.

29.

Vodovar

Duchene

Wimberley

; et al. Cortico-Amygdala-Striatal Activation by Modafinil/Flecainide Combination. Int. J. Neuropsychopharmacol. 2018, 21, 687–696.

30.

Duchêne

Perier

Zhao

; et al. Impact of Astroglial Connexins on Modafinil Pharmacological Properties. Sleep 2016, 39, 1283–1292.

31.

Laird

D. W.

Lampe

P. D.

Therapeutic Strategies Targeting Connexins. Nat. Rev. Drug Discov. 2018.

32.

Jeanson

Pondaven

Ezan

; et al. Antidepressants Impact Connexin 43 Channel Functions in Astrocytes. Front. Cell. Neurosci. 2015, 9, 495.

33.

Abbaci

Barberi-Heyob

Blondel

; et al. Advantages and Limitations of Commonly Used Methods to Assay the Molecular Permeability of Gap Junctional Intercellular Communication. Biotechniques 2008, 45, 33–52, 56–62.

34.

Haq

Grose

Ward

; et al. A High-Throughput Assay for Connexin 43 (Cx43, GJA1) Gap Junctions Using Codon-Optimized Aequorin. Assay Drug Dev. Technol. 2013, 11, 93–100.

35.

Bathany

Hua

S. Z.

Assay for Molecular Transport across Gap Junction Channels in One-Dimensional Cell Arrays. Lab Chip 2011, 11, 1096–1101.

36.

Dukic

A. R.

McClymont

D. W.

Tasken

A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43-Ezrin Interaction Disruptors Using IncuCyte ZOOM. SLAS Discov. 2017, 22, 77–85.

37.

Verselis

V. K.

Srinivas

Divalent Cations Regulate Connexin Hemichannels by Modulating Intrinsic Voltage-Dependent Gating. J. Gen. Physiol. 2008, 132, 315–327.

38.

Rana

Dringen

Gap Junction Hemichannel-Mediated Release of Glutathione from Cultured Rat Astrocytes. Neurosci. Lett. 2007, 415, 45–48.

39.

Choi

E. J.

Yeo

J. H.

Yoon

S. M.

; et al. Gambogic Acid and Its Analogs Inhibit Gap Junctional Intercellular Communication. Front. Pharmacol. 2018, 9, 814.

40.

Harks

E. G.

de Roos

A. D.

Peters

P. H.

; et al. Fenamates: A Novel Class of Reversible Gap Junction Blockers. J. Pharmacol. Exp. Ther. 2001, 298, 1033–1041.

41.

Chaytor

A. T.

Marsh

W. L.

Hutcheson

I. R.

; et al. Comparison of Glycyrrhetinic Acid Isoforms and Carbenoxolone as Inhibitors of EDHF-Type Relaxations Mediated via Gap Junctions. Endothelium 2000, 7, 265–278.

42.

Burnham

M. P.

Sharpe

P. M.

Garner

; et al. Investigation of Connexin 43 Uncoupling and Prolongation of the Cardiac QRS Complex in Preclinical and Marketed Drugs. Br. J. Pharmacol. 2014, 171, 4808–4819.

43.

Giaume

Theis

Pharmacological and Genetic Approaches to Study Connexin-Mediated Channels in Glial Cells of the Central Nervous System. Brain Res. Rev. 2010, 63, 160–176.

44.

Droguerre

Tsurugizawa

Duchene

; et al. A New Tool for In Vivo Study of Astrocyte Connexin 43 in Brain. Sci. Rep. 2019, 9, 18292.

Supplementary Material

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Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

Abstract

Keywords

Introduction

Materials and Methods

Reagents, Cell Culture, and Chemical Compounds

Cx43 Silencing Procedure

Cytotoxicity Assay

Monitoring of Yo-Pro Incorporation in U251 Cells by Real-Time Imaging

Automated Monitoring of YFPQL Quenching by Iodide Incorporation in Ln215 Cells

Results

Setup of a Real-Time Microscopy-Based Assay to Quantify HC Function by Yo-Pro Uptake

Setup of an Automated Assay to Quantify HC Function through the Rapid YFPQL Fluorescence Quenching after Iodide Incorporation

Screening of an In-House Collection of FDA-Approved Drugs

Comparison of the Activity of Primary Hits on Yo-Pro and Iodide Incorporation Assays

Discussion

Supplemental Material

Figure_S1_v4 – Supplemental material for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

Supplemental Material

Figure_S2_v3 – Supplemental material for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

Supplemental Material

Figure_S3_v3 – Supplemental material for Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

ORCID iD

References

Supplementary Material

Automated Monitoring of YFP^QL Quenching by Iodide Incorporation in Ln215 Cells

Setup of an Automated Assay to Quantify HC Function through the Rapid YFP^QL Fluorescence Quenching after Iodide Incorporation