C-170

human and mouse STING

用 STING-IN-2（C-170；0.5 μM）预处理，然后使用 cGAMP 刺激 THP-1 细胞。 STING-IN-2 (C-170) 治疗可降低 TNF 和 IFNB1 mRNA 水平以及 p-TBK1 水平 [1]。

C-170可以减轻小鼠自身炎症性疾病的病理特征[1]。首先，我们通过体内点击化学方法验证了化合物靶向STING，并评估了单剂量腹腔注射C-176的药代动力学特征（扩展数据图6a，b）。接下来，我们评估了C-176是否可以抑制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I型干扰素和IL-6血清水平的诱导。（图4a和扩展数据图6c）。因此，C-176对小鼠有效，并且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，我们研究了它在Trex1-/-小鼠中的疗效。Trex1−/-小鼠表现出由环状GMP-AMP合酶-STING通路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（扩展数据图7a），我们用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−/-小鼠后，I型干扰素的血清水平显著降低，心脏炎症参数受到强烈抑制（扩展数据图7b，C）。用C-176治疗两周的野生型小鼠没有明显的明显毒性迹象（扩展数据图6d–g）。接下来，我们在Trex1−/-小鼠中进行了为期三个月的C-176试验，结果表明，全身炎症的各种症状明显改善（图4b、C和扩展数据图7e）。因此，C-176可以减轻小鼠STING相关的自身炎症疾病。

Competition assay/竞争实验[1]
将表达Flag-STING的HEK293T细胞与指定化合物一起孵育，1小时后加入C-176-AL 1小时。将细胞收集在PBS中，通过C-176-AL-介导的STING标记的凝胶内分析进行分析（见“基于凝胶的化合物与STING结合的分析”）。

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基于凝胶的化合物与STING结合分析[1]
表达Flag-STING的HEK293T细胞在无血清培养基中与C-176-AL、C-175-AZ、叠氮碘乙酰胺或H-151-AL一起孵育，收集在PBS中，通过反复冷冻和解冻裂解。用新制备的“点击试剂”混合物处理43微升裂解细胞，该混合物含有三（苄基三唑甲基）胺（TBTA）（每个样品3μl，3 mM在1:4 DMSO:t-ButOH中）、四甲基罗丹明（TAMRA）叠氮化物）、SiR叠氮化物或SiR炔烃（每个样品2μl，1.25 mM在DMSO中），以及新制得的CuSO4（每个样品1μl）和三-（2-羧乙基）盐酸膦（TCEP）（每个样本1μl/），并在室温下孵育30分钟。通过加入还原性样品缓冲液淬灭反应。使用Fusion-FX可视化凝胶内荧光，并使用Fusion-capt高级采集软件进行分析。

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与亚琥珀酸二琥珀酰亚胺酯交联[1]
表达Flag-mmSTING的HEK293T细胞在有或没有C-176（1μM）的情况下孵育1小时，并用DMSO或CMA（250μg ml-1）处理2小时。在室温下，在PBS中用DMSO中新鲜制备的1 mM二琥珀酰亚胺基琥珀酸盐（DSS）进行交联1小时。

基于细胞的IFNβ启动子-报告荧光素酶测量[1]
将HEK293T细胞接种在96孔板中，并使用GeneJuice（Millipore）与IFN-β启动子-报告质粒（pIFNβ-GLuc）结合指定的表达构建体进行转染。16小时后，使用腔肠素作为底物在上清液中测量gaussia萤光素酶活性。[1]

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高通量化合物筛[1]
使用GeneJuice转染表达具有N端mCherry tag30的小鼠STING的HEK293T细胞，该GeneJuice具有编码环二GMP合酶的构建体，并与IFN-β萤火虫荧光素酶报告质粒结合。三小时后，将转染细胞接种在涂有文库化合物的384孔板中。对于每种化合物，选择40 nl的体积，以在测定板中获得10μM的浓度。将每个孔中DMSO的量归一化为0.1%。过夜处理后，将细胞在裂解缓冲液（25 mM Tris-磷酸盐（pH 7.8）、2 mM DTT、2 mM 1,2-二氨基环己烷-N，N，N′，N′-四乙酸、10%甘油、1%Triton X-100）中裂解20分钟，然后加入萤火虫荧光素酶底物。使用帝肯Infinite平板读数器测量报告活性。对EPFL BSF核心设施可用的化学多样性化合物集合中的约20000种化合物进行了筛选。为了鉴定hsSTING特异性化合物，表达人STING构建体的HEK293T细胞用编码小鼠环GMP-AMP合酶的构建体与IFN-β萤火虫荧光素酶报告质粒转染。如上所述进行了进一步分析。对EPFL BSF核心设施可用的化学多样性化合物集合中的约30000种化合物进行了筛选。[1]

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细胞刺激[1]
BMDM（1×106个细胞ml-1）用DMSO、C-178（0.5μM，除非另有说明）或H-151（0.5μM）预处理1小时，然后用CMA（250μg ml-1）、dsDNA（90 mer，1.33μg ml－1）、环二GMP或cGAMP（1.5μg ml~1）刺激。使用三磷酸核糖核酸（166 ng ml-1）或LPS（1μg ml-1）作为对照。THP-1细胞（1×106个细胞ml-1）用PMA（100 ng ml-1）分化至少3小时，用C-170（0.5μM）或h-151（0.5μM或如所示）处理2小时，然后用环GAMP（375 ng ml-1）或三磷酸核糖核酸（133 ng ml−1）刺激2-3小时（用于mRNA表达分析和p-TBK1免疫印迹）或过夜（用于IP-10产生）。或者，用C-178（0.5μM）预处理WI-38细胞（0.15×106个细胞ml-1）和THP-1细胞（1×106个电池ml-1），并用cGAMP（1.5μg ml-1）刺激2-3小时。为了转染dsDNA、三磷酸RNA和环二核苷酸，使用Lipofectamine 2000。90-mer DNA的有义链序列如下：5′-TACAGATC、TACTAG、GAT、TACT、TACT和TACA-3′。

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. 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, and plasma was isolated after centrifugation at 4 °C and then stored at −80 °C. Plasma parameters were measured using DimensionXpand Plus. For the peripheral blood cell profile, 100 μl of blood was collected in EDTA-K-coated tubes. Complete blood counts were analysed with an ADVIA120 haematology system. For the detection of luciferase activity, Trex1−/−Ifnb1Δβ-luc/Δβ-luc reporter mice (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 in isotonic sodium chloride. Photon flux was quantified two minutes after injection on an In-vivo Xtreme II imaging device 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 or by the Landesdirektion Dresden (Germany) and were performed in accordance with the respective legal regulations.

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

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway1,2. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.[1]

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Western blot analysis Cells were lysed in 2× Laemmli buffer, immobilized protein on beads in sample reducing buffer followed by denaturing at 95 °C for 5 min. Cell lysates were separated by SDS–PAGE and transferred onto PVDF membranes. Blots were incubated with anti-STING (D2P2F), phospho-TBK1 (D52C2), TBK1 (D1B4), anti-transferrin receptor anti-calnexin or anti-Flag M2. As secondary antibodies, anti-rabbit-IgG-HRP or anti-mouse-IgG-HRP (1:2000) were used. Anti-β-actin was used as control. ECL signal was recorded on the ChemiDoc XRS Biorad Imager and data were analysed with Image Laboratory.[1]

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Quantitative RT–qPCR Total RNA was isolated using the RNAeasy Mini Kit (Qiagen) and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis kit. Quantitative RT–qPCR was performed in duplicates using Maxima SYBR Green Master Mix on a QuantStudio 5 machine. GAPDH was used as an endogenous normalization control to obtained relative expression data. Primer sequences are as follows: mmGapdh forward, 5′-GTCATCCCAGAGCTGAACG-3′; mmGapdh reverse, 5′-TCATACTTGGCAGGTTTCTCC-3′; mmIfnb1 forward, 5′-CTCCAGCTCCAAGAAAGGAC-3′; mmIfnb1 reverse, 5′-TGGCAAAGGCAGTGTAAC TC-3′; mmTnf forward, 5′-TATGGCCCAGACCCTCACA-3′, mmTnf reverse, 5′-GGAGTAGACAAGGTACAACCCATC-3′, mmIsg15 forward, 5′-AAGAAGCAGATTGCCCAGAA-3′; mmIsg15 reverse, 5′-TCTGCGTCAGAAAGACCTCA-3′; mmCxcl10 forward, 5′-AAGTGCTGCCGTCATTTTCT-3′; mmCxcl10 reverse, 5′-GTGGCAATGATCTCAACACG-3′; hsGAPDH forward, 5′-GAGTCAACG GATTTGGTCGT-3′; hsGAPDH reverse, 5′- GACAAGCTTCCCGTTCTCAG-3′; hsIFNB1 forward, 5′-CAGCATCTGCTGGTTGAAGA-3′; hsIFNB1 reverse, 5′-CATTACCTGAAGGCCAAGGA-3′; hsTNF forward, 5′-CCCGAGT GACAAGCCTGTAG–3′; hsTNF reverse, 5′- TGAGGTACAGGCCCTCTGAT-3′.[1]

C15H16N2O4EXACTMASS

288.299

288.11

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

346691-38-1

2059265

Light yellow to yellow solid powder

1.3±0.1 g/cm3

359.3±37.0 °C at 760 mmHg

171.1±26.5 °C

0.0±0.8 mmHg at 25°C

1.602

4.01

88.1Ų

1

4

5

21

361

0

CCCCC1=CC=C(NC(C2=CC=C([N+]([O-])=O)O2)=O)C=C1

QMVOHFICEFYHMK-UHFFFAOYSA-N

InChI=1S/C15H16N2O4/c1-2-3-4-11-5-7-12(8-6-11)16-15(18)13-9-10-14(21-13)17(19)20/h5-10H,2-4H2,1H3,(H,16,18)

N-(4-Butylphenyl)-5-nitrofuran-2-carboxamide

C170; C 170; STING inhibitor C-170; N-(4-butylphenyl)-5-nitrofuran-2-carboxamide; STING-IN-2; N-(4-butylphenyl)-5-nitro-2-furancarboxamide; N-(4-butylphenyl)-5-nitro-2-furamide; C-170; CBMicro_018692; STING inhibitor C-170

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 : ~125 mg/mL (~433.58 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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