CRAC channel
GSK-7975A targets ORAI1 (the pore-forming subunit of the Ca2+ release-activated Ca2+ channel). In murine pancreatic acinar cells, it inhibits thapsigargin-induced SOCE with an IC50 of ~3.4 μM [3].

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].

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GSK-7975A concentration-dependently inhibits toxin-induced SOCE and cytosolic Ca2+ overload in isolated murine and human pancreatic acinar cells. In murine cells, TLCS (500 μM)-induced [Ca2+]c plateau was suppressed by ~80% using 30 μM GSK-7975A, and CCK (1 nM)-induced [Ca2+]c was suppressed by >95% using 15 μM GSK-7975A. At 50 μM, GSK-7975A retained efficacy, but at 100 μM loss of effect was observed via an unknown mechanism [3].
In human pancreatic acinar cells, GSK-7975A (10–50 μM) inhibited thapsigargin-induced SOCE, and 30 μM GSK-7975A significantly reduced TLCS (500 μM)-induced necrotic cell death pathway activation as measured by propidium iodide uptake [3].
GSK-7975A also protected isolated murine pancreatic acinar cells from TLCS-induced necrosis: normalized PI uptake was reduced from 100 (TLCS alone) to ~20 with GSK-7975A (30 μM) [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].
In three mouse models of acute pancreatitis (TLCS-AP, CER-AP, FAEE-AP), subcutaneous osmotic minipump administration of the prodrug GSK-6288B (which rapidly liberates GSK-7975A in vivo) at low (28 mg/kg/h) and high (110 mg/kg/h) doses significantly reduced local and systemic features of disease. In TLCS-AP, both doses reduced serum amylase, IL-6, pancreatic MPO; lung MPO was reduced only by the low dose. Histopathology (oedema, inflammation, necrosis, total score) was improved, with greater effect at the high dose. In CER-AP, both doses reduced serum amylase, pancreatic trypsin, MPO; low dose also reduced IL-6 and lung MPO. High dose markedly reduced all histopathological parameters nearly to control levels. In FAEE-AP, low dose reduced pancreatic and lung MPO; high dose reduced serum amylase, IL-6, pancreatic trypsin, and histopathology (oedema, inflammation, necrosis, total score). Treatment was started 30 min (TLCS-AP), with the third cerulein injection (CER-AP), or 1 h after the second FAEE injection (FAEE-AP) [3].
When GSK-7975A (high dose) administration was delayed until 6 h after disease induction in TLCS-AP and FAEE-AP, it was less effective than early treatment, with significant differences in serum amylase, IL-6 (TLCS-AP), oedema, inflammatory infiltrate (TLCS-AP), and total histopathology score [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.

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GSK-7975A effects on cytosolic calcium concentration ([Ca2+]c) were measured in isolated murine or human pancreatic acinar cells using Fura-2 fluorescence. Cells were loaded with 5 μM Fura-2, excited at 340 and 380 nm, emission >490 nm. Ratio (340/380) was recorded. For SOCE experiments, cells were treated with thapsigargin (to empty Ca2+ stores) in zero external Ca2+, then external Ca2+ (1.8 mM or 5 mM) was reintroduced; GSK-7975A was added at indicated concentrations. Alternatively, cells were perfused with TLCS (500 μM) or supramaximal CCK (1 nM) to induce sustained [Ca2+]c elevation, and GSK-7975A was added after a stable plateau was reached [3].
Necrotic cell death pathway activation was assessed by propidium iodide (PI) uptake. Isolated pancreatic acinar cells were treated with TLCS (500 μM) ± GSK-7975A for 30 min with gentle shaking at 1000 rpm at room temperature. After washing, cells were stained with PI (1 μM) and Hoechst 33342 (50 μg/ml). Hoechst stained nuclei for total cell count, PI identified plasma membrane rupture. Cells were imaged using confocal microscopy (excitation 488 nm for PI, emission 630–693 nm; excitation 364 nm for Hoechst, emission 405–450 nm). Percentage of PI-positive cells was counted in ≥3 wells and ≥12 random fields per treatment group [3].

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]

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Animals: 10-week-old male C57BL/6J mice (∼25 g). GSK-7975A was administered via its phosphate prodrug GSK-6288B using subcutaneous ALZET osmotic minipumps (model 2001D) to achieve continuous delivery. Three doses were tested: 2, 28 (low), and 110 (high) mg/kg/h of GSK-6288B. Control mice received vehicle (saline or appropriate solvent) via minipump [3].
TLCS-AP model: Acute biliary pancreatitis was induced by retrograde pancreatic ductal infusion of taurolithocholic acid 3‑sulphate (TLCS, 3 mM) at 5 μl/min for 10 min using an infusion pump. Minipumps were implanted subcutaneously 30 min after TLCS infusion. Mice were humanely killed 6 h or 24 h later [3].
CER-AP model: Hyperstimulation pancreatitis was induced by seven hourly intraperitoneal injections of cerulein (50 μg/kg). Minipumps were implanted at the time of the third cerulein injection. Mice were killed 12 h after the first cerulein injection [3].
FAEE-AP model: Alcoholic pancreatitis was induced by two hourly intraperitoneal injections of palmitoleic acid (150 mg/kg) plus ethanol (1.35 g/kg). Minipumps were implanted 1 h after the second injection. Mice were killed 6 h or 24 h later [3].
For delayed treatment experiments, GSK-6288B (high dose, 110 mg/kg/h) minipump implantation was performed 6 h after disease induction in TLCS-AP and FAEE-AP models, and parameters were assessed at 24 h [3].
Endpoints measured: serum amylase (Roche analyzer), serum IL-6 (ELISA), pancreatic trypsin activity (fluorometric assay using Boc-Gln-Ala-Arg-MCA substrate), myeloperoxidase (MPO) activity in pancreas and lung (colorimetric assay with 3,3,5,5-tetramethylbenzidine), and pancreatic histopathology (H&E staining, scoring of oedema, inflammation, necrosis on a 0–3 scale by blinded investigators) [3].

GSK-7975A was administered as the phosphate prodrug GSK-6288B via subcutaneous osmotic minipump. Steady-state blood concentrations: low dose (28 mg/kg/h GSK-6288B) achieved ∼4.3 μM GSK-7975A; high dose (110 mg/kg/h) achieved ∼13.3 μM. Pancreatic concentrations: low dose ∼8.9 μM, high dose ∼49.3 μM. No detectable prodrug was found in blood or pancreas at any dose or time point, indicating rapid conversion to GSK-7975A. Steady state was reached by 1 h (low dose) or 4 h (high dose) after minipump implantation [3].
Protein binding: In blood and pancreas homogenate, GSK-7975A binding was determined by rapid equilibrium dialysis (RED). The free fraction was <10% (i.e., >90% bound) as stated in the discussion [3].
The IC50 of GSK-7975A for SOCE inhibition in murine pancreatic acinar cells is ∼3.4 μM; in T lymphocytes it is ∼1 μM [3].

Plasma protein binding of GSK-7975A was assessed using rapid equilibrium dialysis (RED) with a dialysis Teflon block and membrane strips. Blood was collected into heparinized tubes, diluted 1:1 with PBS (pH 7.4). Pancreas homogenate was diluted 1:10. Samples were dialyzed against PBS at 37 °C for 5 h (blood) or 4 h (pancreas). After equilibrium, concentrations in sample and buffer chambers were measured by LC-MS/MS. The percentage bound was calculated as ([C]sample – [C]buffer)/[C]sample × 100% [3]. No overt toxicity or LD50 data were reported. At 100 μM in vitro, GSK-7975A exhibited loss of efficacy via an unknown mechanism, but no such loss was seen at 50 μM [3].

[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]

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GSK-7975A (CAS# 1253186-56-9) is a selective ORAI1 inhibitor. Its chemical name is 2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide. The phosphate prodrug GSK-6288B is rapidly cleaved in vivo to liberate GSK-7975A. The compound was developed by GlaxoSmithKline and shown to prevent cytosolic calcium‑associated injury in human and mouse pancreatic acinar cells, and to ameliorate acute pancreatitis in three mouse models (biliary, hyperstimulation, and alcoholic). The therapeutic window is early after disease onset; delayed administration (6 h) reduces efficacy. GSK-7975A inhibits SOCE in immune cells (e.g., T lymphocytes) with an IC50 of ∼1 μM, which may contribute to its anti‑inflammatory effects [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.)