CRAC channel

At 3 μM, GSK-7975A 50% lowers the release of histamine, leukotriene C4, and cytokines (TNFα, IL-5/-8/-13, and TNFα) while also reducing the FcεRI-consuming Ca2+ influx [1]. -7975A increases the full release of inflammatory cytokines from T cells and inhibits the release of media from mast cells in a variety of animal species. Calcium influx through CRAC channels is inhibited by GSK-7975A. This led to the inhibition of mast cells from different concentration preparations. GSK-7975A did not prevent the release of cytokines from mouse and guinea pig mast cells, but it totally stopped the release of cytokines from T cells in mouse preparations [2]. inhibits, in human pancreatic alveolar cells, toxin-induced ORAI1 activation subsequent to Ca2+ release and/or Ca2+ current activation as a concentration suspension (>90% inhibition level seen in control cells). In mice, GSK-7975A scissors stop necrosis from starting in human pancreatic alveolar cells [3].

In TLCS-AP, CER-AP, and FAEE-AP, GSK-7975A suppresses the systemic and local aspects of acute pancreatitis in a dose- and time-coupled manner. Only at low doses did GSK-7975A considerably reduce lung MPO; at elevated levels, it dramatically lowered pancreatic MPO levels, IL-6, and serum enzymes starch. The pancreatic histopathology of TLCS-AP, CER-AP, and FAEE-AP is considerably reduced by GSK-7975A [3].

Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules. Together these observations provide a critical forward step in the identification of CRAC blockers suitable for clinical development in the treatment of inflammatory disorders.[2]

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Patch-clamp electrophysiology[1]
The whole-cell variant of the patch-clamp technique was used as described previously. Currents in some experiments were also evoked by using a ramp protocol consisting of a continuous voltage ramp from −120 to +120 mV. Further details are provided in this article’s Online Repository at www.jacionline.org.
The CRACM-channel blockers GSK-7975A and Synta-6627, Gd3+, and La3+ were added directly to the recording chamber as required. GSK-7975A is compound 36 from patent WO 2010/1222089.

Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.[3]

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The intraperitoneal injection of GSK-7975A also delays the development of retinal vasculature assessed at postnatal day 6 in mice, since it reduces vessel length and the number of junctions, while it increases lacunarity. Moreover, we find that SARAF and Orai1 are involved in VEGF-mediated [Ca2+]i increase, and their knockdown using siRNA impairs HUVEC tube formation, proliferation, and migration. Finally, immunostaining and in situ proximity ligation assays indicate that SARAF likely interacts with Orai1 in HUVECs. Therefore, these findings show for the first time a functional interaction between SARAF and Orai1 in ECs and highlight their essential role in different steps of the angiogenesis process.[4]

[1]. Ashmole I, et al. CRACM/Orai ion channel expression and function in human lung mast cells. J Allergy Clin Immunol. 2012 Jun;129(6):1628-35.e2.
[2]. Rice LV, et al. Characterization of selective Calcium-Release Activated Calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations. Eur J Pharmacol. 2013 Mar 15;704(1-3):49-57.
[3]. Wen L, et al. Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models. Gastroenterology. 2015 Aug;149(2):481-92.e7.
[4]. SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis. Front Cell Dev Biol . 2021 Mar 4:9:639952.

Background: Extracellular Ca²⁺ influx into human lung mast cells (HLMCs) is crucial for the FcεRI-dependent release of pre-formed granule-derived mediators and newly synthesized autoactive substances and cytokines. However, the ion channels mediating this Ca²⁺ influx remain unclear. Recently discovered members of the CRACM/Orai ion channel family, carrying Ca²⁺ release-activated Ca²⁺ currents, are potential candidates. [1]
Objective: To investigate the expression and function of CRACM channels in HLMCs. [1]
Methods: CRACM mRNA, protein, and functional expression were detected in purified HLMCs and isolated human bronchi. [1]
Results: CRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detected in HLMCs. CRACM-like currents were detected in HLMCs after FcεRI-dependent activation and in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca²⁺-selective currents obtained under both conditions could be blocked by 10 μM La³⁺ and Gd³⁺ (known CRACM channel blockers) and two different specific CRACM channel blockers, GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca²⁺ influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotrienes C₄, and cytokines (IL-5/IL-8/IL-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in isolated tissues. [1]
Conclusion: The presence of CRACM channels, CRACM-like currents, and the functional inhibition of HLMC Ca(2+) influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM channel blockers support the role of CRACM channels in FcεRI-dependent HLMC secretion. Therefore, CRACM channels are potential targets for the treatment of asthma and related allergic diseases. [1]

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Background and Objective: Sustained activation of cytoplasmic calcium concentration leads to pancreatic acinar cell damage and necrosis. ORAI1, a calcium regulator activated by calcium release, is the most abundant Ca(2+) influx channel in pancreatic acinar cells; it maintains calcium overload in mice exposed to induced pancreatitis toxins. We investigated the role of ORAI1 in the development and progression of pancreatic acinar cell damage and acute pancreatitis in mice. [3]
Methods: Mouse and human acinar cells, as well as HEK 293 cells transfected with human ORAI1 and human matrix interaction molecule 1, were overstimulated or incubated with human bile acids, carotenoids, or cyclopyrazolium acid to induce calcium influx. GSK-7975A or CM_128 was added to a subset of cells and analyzed by confocal microscopy, video microscopy, and patch-clamp recording. Acute pancreatitis was induced in C57BL/6J mice by intraductal injection of taurine 3-sulfate or intravenous injection of cilulin or ethanol and palmitoleic acid. Subsequently, a subset of mice were treated with the ORAI1 inhibitor GSK-7975A or CM_128 at different time points to evaluate their local and systemic effects. [3]
Results: Both GSK-7975A and CM_128 inhibited, in a concentration-dependent manner, ORAI1 activation induced by toxins and/or activation of Ca²⁺ currents following Ca²⁺ release in mouse and human pancreatic acinar cells (inhibition rates exceeding 90% in control cells). ORAI1 inhibitors also prevented activation of necrotic cell death pathways in mouse and human pancreatic acinar cells. Both GSK-7975A and CM_128 inhibited all local and systemic features in all three acute pancreatitis models in a dose- and time-dependent manner. Administration 1 hour after induction of pancreatitis showed significantly higher efficacy across multiple parameters compared to administration 6 hours after induction. [3]
Conclusion: Cytoplasmic calcium overload (mediated by ORAI1) is part of the pathogenesis of acute pancreatitis. ORAI1 inhibitors hold promise for the treatment of pancreatitis patients. [3]

C18H12F5N3O2

397.298801422119

397.084

C, 54.42; H, 3.04; F, 23.91; N, 10.58; O, 8.05

1253186-56-9

59547990

White to off-white solid powder

1.5±0.1 g/cm3

462.2±45.0 °C at 760 mmHg

233.3±28.7 °C

0.0±1.2 mmHg at 25°C

1.574

2.88

2

8

4

28

540

0

FC(C1C=C(C=CC=1CN1C=CC(NC(C2C(=CC=CC=2F)F)=O)=N1)O)(F)F

CPYTVBALBFSXSH-UHFFFAOYSA-N

InChI=1S/C18H12F5N3O2/c19-13-2-1-3-14(20)16(13)17(28)24-15-6-7-26(25-15)9-10-4-5-11(27)8-12(10)18(21,22)23/h1-8,27H,9H2,(H,24,25,28)

2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide

GSK-7975A; GSK 7975A; GSK7975A; GSK-7975; GSK 7975; GSK7975; GSK-7975A; 1253186-56-9; 2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide; CHEMBL4570175; 2,6-difluoro-N-[1-[[4-hydroxy-2-(trifluoromethyl)phenyl]methyl]pyrazol-3-yl]benzamide; 2,6-difluoro-N-(1-{[4-hydroxy-2-(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)benzamide; SCHEMBL705705; GSK7975A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 90 mg/mL (~226.53 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)