cGAS/dsDNA complex (Kd = 36.2 nM); dsDNA (IC50 = 0.7 μM); cGAS (700 nM)

In macrophages, RU.521 (0.1 nM-1000 μM; 72 h) links signaling pathways triggered by dsDNA [1]. In macrophages, dsDNA-induced signaling pathways are coupled by RU.521 (0-100 μM; 24 h) [1]. 】.

In mice, RU.521 (5 mg/kg; intraperitoneally given once) reduces the signs of hunger [2].

High-throughput screening [1]
Screening reactions were carried out in 20 µl volumes in 384 small volume deep well polypropylene plates. The final concentration of cGAS enzyme, dsDNA, ATP, and GTP were 60 nM, 300 nM, 300 µM, and 300 µM, respectively. Five microliters of reaction buffer composed of 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 0.01% Tween-20 were dispensed per well using a Thermo Multidrop Combi dispenser. The liquid was collected at the well bottom using centrifugation for 30 s at 180×g. Compounds were dissolved in DMSO and 0.05 µl of 5 mM were dispensed with a Janus 384 MDT NanoHead. Final concentration of the compounds in the assay was 12.5 µM. A concentration of 0.5% DMSO did not interfere with cGAMP production from recombinant cGAS. Next, 10 µl of a master mix containing reaction buffer supplemented with 0.6 mM ATP, 0.6 mM GTP, and 0.6 µM dsDNA was added to wells in columns 1–23 using a Thermo Multidrop Combi dispenser, while 10 µl of the master mix devoid of dsDNA (control for no enzymatic activity) was added to wells in column 24. Plates were centrifuged for 30 s at 180×g to collect all liquid at the bottom of the wells. The reaction was started by adding 5 µl of 0.24 µM recombinant full-length mouse cGAS in reaction buffer to each well of the plate followed by centrifugation for 30 s at 180×g and incubation for 120 min at room temperature. The reaction was stopped by addition of 65 µl of 0.5% (v/v) formic acid per well. The plates were centrifuged for 30 s at 180×g and sealed with a Velocity11 PlateLoc thermal plate sealer. Compounds that inhibited cGAS activity by ≥ 60% were retested in concentration response experiments to determine half maximal inhibitory concentration (IC50). Compounds were serially diluted by half for a total of ten dilutions where the highest final concentration in the assay was 25 µM. The compounds selected for follow-up studies were reordered from the vendors, dissolved in DMSO to a concentration of 10 mM and re-tested in concentration response experiments. The IC50 values for the enzymatic assay were calculated using GraphPad Prism (7.01), from three replicate experiments; the error bars represent SD.

Small-molecule compounds were serially diluted to concentrations spanning the range tested in the response curves were added to 6.7 × 105 RAW-Blue macrophages plated 16 h prior in 96-well dishes, then harvested 72 h after compound addition. ATP was measured using CellTiter Glo Viability Assay (Promega) using 50 µM Tamoxifen (Sigma) as a positive control for cytotoxicity. Viability values were generated using vehicle (DMSO) or the first dose (RU.521) as 100% and Tamoxifen as 0%. Outliers were removed as described previously [1].

Animal/Disease Models: LPS injection into 8weeks old male mice [2]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection; 5 mg/kg One-time
Experimental Results: Enhance mouse cardiac function, reduce cardiac inflammatory response, oxidative stress and Apoptosis.

[1]. Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice. Nat Commun. 2017 Sep 29;8(1):750.

[2]. Small molecule inhibition of cyclic GMP-AMP synthase ameliorates sepsis-induced cardiac dysfunction in mice. Life Sci. 2020 Nov 1;260:118315.

Cyclic GMP-AMP synthase is essential for innate immunity against infection and cellular damage, serving as a sensor of DNA from pathogens or mislocalized self-DNA. Upon binding double-stranded DNA, cyclic GMP-AMP synthase synthesizes a cyclic dinucleotide that initiates an inflammatory cellular response. Mouse studies that recapitulate causative mutations in the autoimmune disease Aicardi-Goutières syndrome demonstrate that ablating the cyclic GMP-AMP synthase gene abolishes the deleterious phenotype. Here, we report the discovery of a class of cyclic GMP-AMP synthase inhibitors identified by a high-throughput screen. These compounds possess defined structure-activity relationships and we present crystal structures of cyclic GMP-AMP synthase, double-stranded DNA, and inhibitors within the enzymatic active site. We find that a chemically improved member, RU.521, is active and selective in cellular assays of cyclic GMP-AMP synthase-mediated signaling and reduces constitutive expression of interferon in macrophages from a mouse model of Aicardi-Goutières syndrome. RU.521 will be useful toward understanding the biological roles of cyclic GMP-AMP synthase and can serve as a molecular scaffold for development of future autoimmune therapies.Upon DNA binding cyclic GMP-AMP synthase (cGAS) produces a cyclic dinucleotide, which leads to the upregulation of inflammatory genes. Here the authors develop small molecule cGAS inhibitors, functionally characterize them and present the inhibitor and DNA bound cGAS crystal structures, which will facilitate drug development.[1]

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Aims: Cardiac dysfunction is the main cause of multi-organ failure following sepsis within critical care units. The present study aimed to investigate the effects of the small molecule inhibition of cyclic GMP-AMP synthase (cGAS), RU.521, on cardiac function in mice with sepsis.[2]
Materials and methods: Sepsis was induced in mice via intraperitoneal lipopolysaccharide (LPS) injection (10 mg/kg, i.p.). Mice subsequently received 5 mg/kg RU.521 within 10 min form LPS injection. The cardiac function, inflammatory factor and oxidative stress of mice were examined for 24 h following LPS injection.[2]
Key findings: RU.521 was indicated to significantly increase the cardiac function of mice with sepsis. In addition, the inflammatory responses, oxidative stress and apoptosis in hearts of sepsis mice were markedly mitigated by RU.521. Moreover, inhibition of Sirt3 inhibited the protective effects of RU.521 on mice with sepsis.[2]
Significance: The current study indicated that RU.521 alleviated the inflammatory response and alleviated the damage induced by oxidative stress, leading to cardiac protection via increased Sirt3 expression in the hearts of mice with sepsis.

C19H12CL2N4O3

415.2296

414.03

C, 54.96; H, 2.91; Cl, 17.07; N, 13.49; O, 11.56

2262452-06-0

135397653

Off-white to yellow solid

4.1

2

5

2

28

732

0

VIQXILLOJLATEF-UHFFFAOYSA-N

InChI=1S/C19H12Cl2N4O3/c1-8-13(16-9-4-2-3-5-10(9)18(27)28-16)17(26)25(24-8)19-22-12-7-6-11(20)14(21)15(12)23-19/h2-7,16,26H,1H3,(H,22,23)

3-[1-(6,7-Dichloro-1H-benzimidazol-2-yl)-5-hydroxy-3-methyl-pyrazol-4-yl]-3H-isobenzofuran-1-one

RU320521; RU-521; RU-320521; RU521; RU 320521; RU 521; 2-(4,5-dichloro-1H-benzimidazol-2-yl)-5-methyl-4-(3-oxo-1H-2-benzofuran-1-yl)-1H-pyrazol-3-one; CHEMBL4567157; 3-(1-(6,7-Dichloro-1H-benzo[d]imidazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)isobenzofuran-1(3H)-one; 3-[1-(6,7-dichloro-1H-benzimidazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl]-1(3H)-isobenzofuranone; RU.521

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 : ~83.33 mg/mL (~200.68 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.01 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 2: ≥ 2.08 mg/mL (5.01 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 (5.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)