H 151 (H-151)

STING (stimulator of interferon genes)

In HEK293T cells, H-151 (0.02-2 μM) lowers IFNβ luciferase reporter gene measurements [1]. In THP-1 cells, H-151 (0.5 μM; 2 h) suppresses TBK1 phosphorylation [1]. hsSTING palmification is inhibited by H-151 (1 μM; 3 hours) [1].

In CMA-treated mice, H-151 (750 nmol per mouse; single intraperitoneal injection) dramatically lowers systemic cytokine responses [1]. In CMA-treated mice, H-151 (750 nmol per mouse; single intraperitoneal injection) dramatically lowers systemic cytokine responses [1]. d) Trex1®/? H-151 mice that express a bioluminescent IFNβ reporter (750 nmol/mouse; single intraperitoneal injection) reach strong systemic levels, show a notable serum half-life, and function similarly to wild-type mice. demonstrated notable effectiveness in mice [1]. mmSTING adduct formation [1].

High-throughput chemical compound screen
HEK293T cells that expressed mouse STING with an N-terminal mCherry tag were transfected using GeneJuice (Millipore) with a construct encoding for cyclic di-GMP synthase in conjunction with an IFN-β firefly luciferase reporter plasmid. Three hours later, transfected cells were seeded in 384-well plates (Corning) coated with library compounds. For each compound, a 40-nl volume was selected to obtain a concentration of 10 μM in the assay plates. The amount of DMSO in each well was normalized to 0.1%. After overnight treatment, cells were lysed in lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, 1% Triton X-100) for 20 min followed by the addition of firefly luciferase substrate. Reporter activity was measured using a Tecan Infinite plate reader. The screen was performed on ~20,000 compounds from a chemically diverse compound collection available at the BSF core facility at EPFL. For the identification of hsSTING-specific compounds, HEK293T cells expressing a human STING construct were transfected with a construct encoding mouse cyclic GMP–AMP synthase in conjunction with an IFN-β firefly luciferase reporter plasmid. Further analysis was performed as described above. The screen was performed on ~30,000 compounds from a chemically diverse compound collection available at the BSF core facility at EPFL.[1]
Competition assay
HEK293T cells expressing Flag–STING were incubated with the indicated compounds and after 1 h, C-176-AL was added for 1 h. Cells were collected in PBS and analysed by in-gel analysis of C-176-AL-mediated labelling of STING (see ‘ Gel-based analysis of compound binding to STING’).[1]

Immunoprecipitation
Flag–STING expression was induced in HEK293T cells overnight by doxycycline. Cells were incubated with or without C-178 or C-176 (1 μM) for 1 h and treated with DMSO or CMA (250 μg ml−1) for 2 h. Cells were washed in PBS and lysed in lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerin, 1 mM MgCl, 1 mM CaCl, 1% Brij-58 and protease inhibitor cocktail (Sigma P8340)) for 30 min. Flag–STING was immunoprecipitated using anti-Flag M2 affinity gel agarose gel (Sigma) for 2 h at 4 °C. After stringent washing in lysis buffer and PBS, the supernatant was completely removed and the resin was boiled in sample buffer before SDS–PAGE was performed. For immunoprecipitation of endogenous STING, splenocytes were lysed in the above-mentioned lysis buffer and incubated with anti-STING (RD System AF6516) and G sepharose beads (GE Healthcare, 17-0618-01) overnight. Beads were washed in PBS and gel-based analysis of C-176-AL binding to STING was performed.
Gel-based analysis of compound binding to STING
HEK293T cells expressing Flag–STING were incubated with C-176-AL, C-175-AZ, iodoacetamide azide or H-151-AL in serum-free medium, collected in PBS and lysed by repetitive freezing and thawing. Forty-three microlitres of lysed cells was treated with a freshly prepared ‘click reagent’ mixture containing tris(benzyltriazolylmethyl)amine (TBTA) (3 μl per sample, 3 mM in 1:4 DMSO:t-ButOH), tetramethylrhodamine (TAMRA) azide (Thermo Fisher), SiR azide (Spirochrome) or SiR alkyne (Spirochrome) (2 μl per sample, 1.25 mM in DMSO), and freshly prepared CuSO4 (1 μl per sample) and tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 μl per sample) and incubated at room temperature for 30 min. The reaction was quenched by addition of reducing sample buffer. In-gel fluorescence was visualized using Fusion FX (Vilber Lourmat) and analysed by Fusion capt advance acquisition software.
Crosslinking with disuccinimidyl suberate
HEK293T cells expressing Flag–mmSTING were incubated with or without C-176 (1 μM) for 1 h and treated with DMSO or CMA (250 μg ml−1) for 2 h. Crosslinking was performed in PBS with 1 mM disuccinimidyl suberate (DSS) (Thermo Fisher) freshly prepared in DMSO at room temperature for 1 h.

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)

Solubility in Formulation 1: 2.5 mg/mL (8.95 mM) in 5% DMSO 5% Tween80 + 90% PBS (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.45 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 20.8 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 3: 2.08 mg/mL (7.45 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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 4: ≥ 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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 (Please use freshly prepared in vivo formulations for optimal results.)