H 151 (H-151)

STING (stimulator of interferon genes)

在 HEK293T 细胞中，H-151 (0.02-2 μM) 降低 IFNβ 荧光素酶报告基因测量值 [1]。在 THP-1 细胞中，H-151（0.5 μM；2 小时）抑制 TBK1 磷酸化 [1]。 hsSTING 掌化被 H-151（1 μM；3 小时）抑制 [1]。

在 CMA 治疗的小鼠中，H-151（每只小鼠 750 nmol；单次腹腔注射）可显着降低全身细胞因子反应 [1]。在 CMA 治疗的小鼠中，H-151（每只小鼠 750 nmol；单次腹腔注射）可显着降低全身细胞因子反应 [1]。 d) Trex1®/？表达生物发光 IFNβ 报告基因（750 nmol/小鼠；单次腹腔注射）的 H-151 小鼠达到很强的全身水平，显示出显着的血清半衰期，并且功能与野生型小鼠相似。在小鼠中表现出显着的效果[1]。 mmSTING 加合物形成 [1]。

高通量化合物筛选
使用GeneJuice（Millipore）转染表达具有N-末端mCherry标签的小鼠STING的HEK293T细胞，该细胞具有编码环状二GMP合酶的构建体和IFN-β萤火虫萤光素酶报告质粒。三小时后，将转染的细胞接种在涂有文库化合物的384孔板（Corning）中。对于每种化合物，选择40nl的体积以在测定板中获得10μM的浓度。将每个孔中DMSO的量标准化为0.1%。过夜处理后，将细胞在裂解缓冲液（25mM磷酸三钠（pH 7.8），2mM DTT，2mM 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，然后加入萤火虫萤光素酶底物。使用Tecan Infinite平板读数器测量报告活性。对来自EPFL BSF核心设施的约20000种化学多样化合物进行了筛选。为了鉴定hsSTING特异性化合物，用编码小鼠环状GMP–AMP合酶的构建体和IFN-β萤火虫萤光素酶报告质粒转染表达人STING构建体的HEK293T细胞。如上所述进行了进一步的分析。对来自EPFL BSF核心设施的约30000种化学多样化合物进行了筛选。[1]
竞争分析
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”）。[1]

免疫沉淀
Flag–STING在HEK293T细胞中通过多西环素诱导表达过夜。将细胞与或不与C-178或C-176（1μM）一起孵育1小时，并用DMSO或CMA（250μg ml−1）处理2小时。将细胞在PBS中洗涤，并在裂解缓冲液（50mM HEPES、150mM NaCl、10%甘油、1mM MgCl、1mM CaCl、1%Brij-58和蛋白酶抑制剂混合物（Sigma P8340））中裂解30分钟。使用抗Flag M2亲和凝胶琼脂糖凝胶（Sigma）在4 °C。在裂解缓冲液和PBS中严格洗涤后，完全去除上清液，并在进行SDS–PAGE之前在样品缓冲液中煮沸树脂。对于内源性STING的免疫沉淀，将脾细胞在上述裂解缓冲液中裂解，并与抗STING（RD System AF6516）和G琼脂糖珠（GE Healthcare，17-0618-01）孵育过夜。在PBS中洗涤珠，并对C-176-AL与STING的结合进行基于凝胶的分析
基于凝胶的化合物与STING结合的分析
将表达Flag–STING的HEK293T细胞与C-176-AL、C-175-AZ、碘代叠氮化物或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（Thermo Fisher）、SiR叠氮化物（Spichrochrome）或SiR炔烃（Spichrome）（每个样本2μl，DMSO中1.25 mM）和新制备CuSO4（每个样本1μl）和三-（2-羧乙基）膦盐酸盐（TCEP）通过加入还原性样品缓冲液而淬灭。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
与二琥珀酰亚胺亚油酸酯交联
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。

Mice and in vivo studies
C57BL/6J mice (stock number 000664) were purchased from Jackson Laboratories. TREX1-deficient mice were a gift from T. Lindahl 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 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 (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.
Formulation and doses: intraperitoneally injection; 750 nmol H-151 per mouse in 200 μl 10% Tween-80 in PBS.

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

C17H17N3O

279.343

279.1372

C, 73.10; H, 6.13; N, 15.04; O, 5.73

941987-60-6

941987-60-6;

White to off-white solid powder

3.4

56.9Ų

O=C(NC1=CNC2=C1C=CC=C2)NC3=CC=C(CC)C=C3

UJZDIKVQFMCLBE-UHFFFAOYSA-N

InChI=1S/C17H17N3O/c1-2-12-7-9-13(10-8-12)19-17(21)20-16-11-18-15-6-4-3-5-14(15)16/h3-11,18H,2H2,1H3,(H2,19,20,21)

N-(4-Ethylphenyl)-N'-1H-indol-3-yl-urea

H 151 H-151 H151

2934999001

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 : ~100 mg/mL (~357.99 mM)

配方 1 中的溶解度: 2.5 mg/mL (8.95 mM) in 5% DMSO 5% Tween80 + 90% PBS (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (7.45 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 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 3 中的溶解度: 2.08 mg/mL (7.45 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 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 4 中的溶解度: ≥ 2.08 mg/mL (7.45 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
≥ 2.08 mg/mL (7.45 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.08 mg/mL (7.45 mM) in 10% DMSO + 90% Corn oil

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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。