GSK-7975A

CRAC channel

μM 时，GSK-7975A 可以降低组胺、白三烯 C4 和细胞因子（TNFα、IL-5/-8/-13 和 TNFα）的释放 50%，同时还减少消耗 FcεRI 的 Ca2+ 流入 [1]。 -7975A 在多种动物物种中增加 T 细胞炎症细胞因子的完全释放，并抑制肥大细胞介质的释放。 GSK-7975A 可抑制钙通过 CRAC 通道的流入。这导致不同浓度制剂对肥大细胞的抑制。 GSK-7975A 不会阻止小鼠和豚鼠肥大细胞释放细胞因子，但它完全阻止小鼠制剂中 T 细胞释放细胞因子 [2]。在人胰腺肺泡细胞中，以浓度悬浮液形式抑制 Ca2+ 释放和/或 Ca2+ 电流激活后毒素诱导的 ORAI1 激活（在对照细胞中观察到 >90% 的抑制水平）。在小鼠中，GSK-7975A 剪刀可阻止人胰腺肺泡细胞开始坏死 [3]。

在 TLCS-AP、CER-AP 和 FAEE-AP 中，GSK-7975A 以剂量和时间耦合的方式抑制急性胰腺炎的全身和局部症状。仅在低剂量时，GSK-7975A 才能显着降低肺 MPO；在升高的水平下，它会显着降低胰腺 MPO 水平、IL-6 和血清酶淀粉。 GSK-7975A 显着降低了 TLCS-AP、CER-AP 和 FAEE-AP 的胰腺组织病理学特征 [3]。

构成钙释放激活钙（CRAC）通道的两种关键蛋白质中的功能丧失突变表明了这种离子通道在免疫细胞功能中的关键作用。本研究的目的是证明，使用人、大鼠、小鼠和豚鼠的细胞制剂，在体外使用CRAC通道的高选择性抑制剂可以抑制免疫细胞活化。两种选择性CRAC通道小分子阻断剂；对GSK-5498A和GSK-7975A进行了测试，以证明它们在多种物种中抑制肥大细胞释放介质和T细胞释放促炎细胞因子的能力。GSK-5498A和GSK-7975A均完全抑制了通过CRAC通道的钙内流。这导致抑制了多种人类和大鼠制剂中肥大细胞介质和T细胞因子的释放。豚鼠和小鼠制备的肥大细胞不受GSK-5498A或GSK-7975A的抑制；然而，在小鼠制剂中，T细胞释放的细胞因子被完全阻断。GSK-5498A和GSK-7975A证实了CRAC通道在人类肥大细胞和T细胞功能中的关键作用，并且可以在体外实现抑制。大鼠表现出与人类相似的药理学，促进了这一物种与这一系列分子的未来体内研究。这些观察结果共同为鉴定适用于治疗炎症性疾病临床开发的CRAC阻断剂迈出了关键的一步。[2]

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膜片钳电生理学[1]
如前所述，使用膜片钳技术的全细胞变体。一些实验中的电流也是通过使用斜坡协议诱发的，该协议由-120至+120 mV的连续电压斜坡组成。本文的在线知识库www.jacionline.org提供了更多详细信息。
根据需要，将CRACM通道阻断剂GSK-7975A和Synta-6627、Gd3+和La3+直接添加到记录室中。GSK-7975A是来自专利WO 2010/1222089的化合物36。

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: Influx of extracellular Ca(2+) into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. However, the identity of the ion channels underlying this Ca(2+) influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca(2+) release-activated Ca(2+) current are candidates.[1]
Objectives: To investigate the expression and function of CRACM channels in HLMCs.[1]
Methods: CRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus.[1]
Results: CRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detectable in HLMCs. A CRACM-like current was detected following FcεRI-dependent HLMC activation and also in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca(2+)-selective current obtained under both conditions was blocked by 10 μM La(3+) and Gd(3+), known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers-GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca(2+) influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C(4), and cytokines (IL-5/-8/-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue.[1]
Conclusions: The presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca(2+) influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are therefore a potential therapeutic target in the treatment of asthma and related allergic diseases.[1]

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Background & aims: Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.[3]
Methods: Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. 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.[3]
Results: 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]
Conclusions: Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.[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

67.2Ų

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.29 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.29 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.29 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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