C-178

humand and mouse STING

C-178 包括 mmSTING N 末端的一个区域，该区域穿过膜结构域，但尚不清楚。此外，C-178 通过阻断 STING 的棕榈磷脂化来阻碍这一过程。人体细胞不会受到 C-178 的显着影响。单独使用 C-178（0-1 μM；1 小时）不会显着影响 BMDM 的基因表达谱。此外，它还可以防止 CMA 引起的 TBK1[1] 磷酸化。在小鼠骨髓产生的巨噬细胞中，C-178（1 μM；1 小时）可降低由 cdG、dsDNA、CMA 和 LPS 触发的 Ifnb1 表达 [1]。在小鼠胚胎成纤维细胞中，C-178（1 μM；0.5-4 小时）以时间依赖性方式抑制 CMA 产生的 p-TBK1 和刺蛋白的产生[1]。

因为我们注意到C-176相对于C-178的溶解度有所提高，所以我们选择该化合物进行体内研究。首先，我们使用体内点击化学方法验证了这些化合物靶向STING，并评估了单剂量腹膜内注射C-176的药代动力学特征。接下来，我们评估了C-176是否可以抑制由CMA给药引发的I型IFN的诱导。值得注意的是，用C-176预处理显著降低了CMA介导的血清I型IFN和IL-6水平的诱导。因此，C-176对小鼠有效，正如共价抑制剂所预期的那样，短的血清半衰期不会限制其体内抑制能力。为了评估C-176在自身炎症性疾病模型中拮抗STING的潜力，我们研究了其在Trex1−/-小鼠中的疗效。Trex1−/-小鼠表现出由环状GMP–AMP合酶–STING通路的持续激活引起的严重多器官炎症的迹象，并概括了人类Aicardi–Goutières综合征的某些致病特征。在验证了C-178抑制Trex1−/-小鼠细胞中干扰素刺激的基因后，我们对C-176进行了为期两周的体内疗效研究。值得注意的是，用C-176治疗Trex1−/-小鼠导致血清I型IFN水平显著降低，并强烈抑制心脏炎症参数。用C-176治疗两周的野生型小鼠没有明显的明显毒性迹象。接下来，我们在Trex1−/−小鼠中用C-176进行了为期三个月的试验，结果表明，系统炎症的各种迹象都得到了显著改善。因此，C-176可减轻小鼠STING相关的自身炎症疾病[1]。

竞争分析[1]
将表达Flag–STING的HEK293T细胞与所示化合物孵育，1小时后，加入C-176-AL 1小时。将细胞收集在PBS中，并通过C-176-AL-介导的STING标记的凝胶内分析进行分析（见“化合物与STING结合的基于凝胶的分析”）

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基于凝胶的化合物与STING结合的分析[1]
将表达Flag–STING的HEK293T细胞与C-176-AL、C-175-AZ、叠氮化碘乙酰胺（Thermo Fisher）或H-151-AL在无血清培养基中孵育，收集在PBS中，并通过重复冷冻和解冻裂解。用新制备的“点击试剂”混合物处理43微升裂解的细胞，该混合物含有三（苄基三唑基甲基）胺（TBTA）（每个样品3μl，在1:4 DMSO:t-ButOH中3 mM）、四甲基罗丹明（TAMRA）叠氮化物、SiR叠氮化物（Spichrome）或SiR炔烃（Spichrochrome）减少样品缓冲液。使用Fusion FX（Vilber-Lourmat）对凝胶内荧光进行可视化，并通过Fusion-capt高级采集软件进行分析

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与辛二酸二琥珀酰亚胺酯交联[1]
将表达Flag–mmSTING的HEK293T细胞与C-176（1μM）一起或不与C-176一起孵育1小时，并用DMSO或CMA（250μg ml−1）处理2小时。在PBS中与室温下在DMSO中新制备的1 mM二琥珀酰亚胺基亚油酸酯（DSS）（赛默飞世尔）进行交联1小时。

蛋白质印迹分析[1]
细胞类型： 小鼠骨髓源性巨噬细胞 (BMDM)
测试浓度： 0 μM； p-TBK1 和刺痛湿度纤维形成细胞蛋白表达随着时间的推移而发生[1]。 0.125μM； 0.25μM； 0.5μM； 1 μM
孵育时间： 1 小时
实验结果： 以剂量依赖性方式抑制 CMA 诱导的 p-TBK1 表达。

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RT-PCR[1]
细胞类型：小鼠骨髓源性巨噬细胞 (BMDM)
测试浓度： 1 μM
孵育时间：1小时
实验结果：Ifnb1表达在BMDM中下调。

C57BL/6J mice (stock number 000664) were purchased from Jackson Laboratories. TREX1-deficient mice were a gift from T. Lindahl31 and were backcrossed for >10 generations to C57BL/6NJ. Mice were maintained under specific-pathogen-free (SPF) conditions at EPFL. For the pharmacokinetic studies, wild-type mice were injected intraperitoneally with 750 nmol C-176 per mouse in 200 μl corn oil (Sigma). Blood was collected at 30 min, 2 h and 4 h and serum C-176 levels were measured by mass spectrometry (liquid chromatography–high-resolution mass spectrometry). To assess the in vivo inhibitory effect of C-176, wild-type mice (8–12 weeks of age) were injected either with vehicle or C-176. After 1 h or 4 h, CMA was administered at a concentration of 224 mg kg−1. Four hours later, mice were euthanized and the serum was collected to measure CMA-induced cytokine levels. To assess the in vivo inhibitory effect of H-151, wild-type mice were injected intraperitoneally with 750 nmol H-151 per mouse in 200 μl 10% Tween-80 in PBS. After 1 h CMA (112 mg kg−1) was administered, and after 4 h mice were euthanized and the serum was collected. The efficacy study in Trex1−/− mice was conducted as follows: mice (2–5 weeks of age) were injected with 7.5 μl of C-176 or DMSO dissolved in 85 μl corn oil twice per day for 11 consecutive days. Mice were euthanized by anaesthetization in a CO2 chamber followed by cervical dislocation. For toxicology studies, 8-week-old mice were injected daily with 562.5 nmol of C-176 for 2 weeks. At day 14, blood samples were collected in lithium-heparin-coated tubes (Microvette CB 300 Hep-Lithium), and plasma was isolated after centrifugation at 4 °C and then stored at −80 °C. Plasma parameters were measured using DimensionXpand Plus (Siemens Healthcare Diagnostics AG). For the peripheral blood cell profile, 100 μl of blood was collected in EDTA-K-coated tubes (Microvette CB 300 EDTA K2). Complete blood counts were analysed with an ADVIA120 haematology system (Siemens Healthcare Diagnostics AG). For the detection of luciferase activity, Trex1−/−Ifnb1Δβ-luc/Δβ-luc reporter mice32 (aged 4–7 weeks) were injected intraperitoneally daily for 7 days with 750 nmol H-151 or DMSO in 200 μl PBS 0.1% Tween-80. For in vivo imaging, mice were anaesthetized with isofluran and injected intravenously with 15 mg kg−1 XenoLight D-luciferin (Perkin Elmer) in isotonic sodium chloride. Photon flux was quantified two minutes after injection on an In-vivo Xtreme II imaging device (Bruker) with binning set to 8 × 8 pixels and an integration time of 3 min. Animal experiments were approved either by the Service de la Consommation et des Affaires Vétérinaires of the canton of Vaud (Switzerland) or by the Landesdirektion Dresden (Germany) and were performed in accordance with the respective legal regulations [1].

[1]. Haag SM, et al. Targeting STING with covalent small-molecule inhibitors. Nature. 2018 Jul;559(7713):269-273.

C17H10N2O5

322.28

322.059

C, 62.49; H, 5.59; N, 9.72; O, 22.20

329198-87-0

329198-87-0

Light yellow to yellow solid

4.1

101Ų

O=C(C1=CC=C([N+]([O-])=O)O1)NC2=CC=C3C(OC4=CC=CC=C34)=C2

URUVDCCYSJEGQQ-UHFFFAOYSA-N

InChI=1S/C17H10N2O5/c20-17(14-7-8-16(24-14)19(21)22)18-10-5-6-12-11-3-1-2-4-13(11)23-15(12)9-10/h1-9H,(H,18,20)

N-(Dibenzo[b,d]furan-3-yl)-5-nitrofuran-2-carboxamide

C178 C 178 C-178

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 : ~41.67 mg/mL (~129.30 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。