cGAS/dsDNA complex (Kd = 36.2 nM); dsDNA (IC50 = 0.7 μM); cGAS (700 nM)
RU.521 (RU320521) targets cyclic GMP-AMP synthase (cGAS) (IC50 = 0.12 μM for human cGAS enzymatic activity; Ki = 0.08 μM, competitive inhibition mode against dsDNA substrate) [1]
RU.521 (RU320521) shows no significant inhibition against other nucleic acid sensors (STING, TLR9, RIG-I) at 10 μM [1]

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]. 】.

---

- cGAS enzyme inhibitory activity: RU.521 (RU320521) potently and selectively inhibited recombinant human cGAS-mediated cGAMP synthesis in a dose-dependent manner, with IC50 = 0.12 μM and Ki = 0.08 μM. It competed with double-stranded DNA (dsDNA) for binding to cGAS, as confirmed by binding assays [1]
- Inhibition of interferon (IFN) expression in autoimmune mice-derived macrophages: Primary macrophages isolated from MRL/lpr autoimmune mice were stimulated with dsDNA (1 μg/mL) + lipopolysaccharide (LPS, 10 ng/mL). RU.521 (RU320521) (0.1-2 μM) dose-dependently reduced IFN-β mRNA levels by 45% (0.5 μM), 68% (1 μM), and 75% (2 μM), and IFN-α protein levels by 40% (0.5 μM), 62% (1 μM), and 70% (2 μM) compared to stimulated control [1]
- Reduction of cGAMP production: In dsDNA-stimulated RAW 264.7 macrophages, RU.521 (RU320521) (1 μM) decreased intracellular cGAMP concentration by 65% [1]
- Amelioration of sepsis-induced macrophage activation: LPS (1 μg/mL) + ATP (5 mM)-stimulated RAW 264.7 macrophages were treated with RU.521 (RU320521) (0.5-2 μM). At 2 μM, it reduced pro-inflammatory cytokine levels (TNF-α: 58% reduction, IL-6: 63% reduction) and inhibited phosphorylation of NF-κB p65 (by 60%) [2]
- No significant cytotoxicity: RU.521 (RU320521) at concentrations up to 10 μM showed no obvious cytotoxicity to primary macrophages or RAW 264.7 cells (cell viability > 90%) [1][2]

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

---

- Reduction of IFN expression in autoimmune mice: MRL/lpr mice were intraperitoneally administered RU.521 (RU320521) (10 mg/kg, 20 mg/kg) once daily for 14 consecutive days. Serum IFN-β and IFN-α levels were reduced by 48%/62% and 42%/58% respectively at 10 mg/kg and 20 mg/kg. Splenic macrophages from treated mice showed 55% (20 mg/kg) reduction in dsDNA-induced IFN-β mRNA expression [1]
- Amelioration of sepsis-induced cardiac dysfunction: C57BL/6 mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. RU.521 (RU320521) (15 mg/kg) was intraperitoneally administered 1 hour after CLP and once daily for the next 2 days. At 72 hours post-CLP, left ventricular ejection fraction (LVEF) and fractional shortening (FS) were increased by 35% and 38% respectively compared to vehicle-treated sepsis mice. Cardiac tissue levels of TNF-α, IL-6, and cGAMP were reduced by 52%, 55%, and 60% respectively [2]
- Tolerability in animals: No significant body weight loss (< 7%) or obvious toxic signs (lethargy, organ damage) were observed in treated mice at effective doses [1][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.

---

- cGAS enzymatic activity assay: Recombinant human cGAS protein was mixed with dsDNA substrate (45 bp), ATP, GTP, and RU.521 (RU320521) at gradient concentrations (0.01-2 μM) in reaction buffer (pH 7.5). The mixture was incubated at 37°C for 2 hours, and cGAMP production was detected by high-performance liquid chromatography (HPLC). IC50 was calculated by plotting inhibition rate against drug concentration. Kinetic analysis with varying dsDNA concentrations confirmed competitive inhibition [1]
- Nucleic acid sensor selectivity assay: Recombinant STING, TLR9, and RIG-I proteins were separately mixed with their corresponding substrates and RU.521 (RU320521) (10 μM) in optimal reaction buffers. After 37°C incubation for 2-3 hours, enzyme activity or downstream signaling activation was detected to evaluate selectivity [1]

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].

---

- Primary macrophage isolation and IFN expression assay: Splenic macrophages were isolated from MRL/lpr mice, seeded into 6-well plates (5×10⁵ cells/well), and incubated overnight. Cells were pre-treated with RU.521 (RU320521) (0.1-2 μM) for 1 hour, then stimulated with dsDNA (1 μg/mL) + LPS (10 ng/mL) for 6 hours (mRNA) or 24 hours (protein). IFN-β mRNA levels were detected by RT-PCR, and IFN-α protein levels by ELISA [1]
- cGAMP detection assay: RAW 264.7 macrophages were seeded into 12-well plates, pre-treated with RU.521 (RU320521) (0.5-2 μM) for 1 hour, then stimulated with dsDNA (1 μg/mL) for 8 hours. Cells were lysed, and cGAMP concentration was measured by HPLC [1]
- Sepsis-related macrophage activation assay: RAW 264.7 macrophages were seeded into 96-well plates (for cytokines) or 6-well plates (for western blot), pre-treated with RU.521 (RU320521) (0.5-2 μM) for 1 hour, then stimulated with LPS (1 μg/mL) + ATP (5 mM) for 24 hours. TNF-α/IL-6 levels were detected by ELISA, and p-NF-κB p65 protein by western blot [2]

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.

---

- Autoimmune mouse model: Female MRL/lpr mice (8-10 weeks old) were randomly divided into control, 10 mg/kg, and 20 mg/kg RU.521 (RU320521) groups (n=8 per group). The compound was dissolved in a mixture of DMSO and sterile saline (volume ratio 1:9) and administered intraperitoneally once daily for 14 days. Control group received equal volume of vehicle. Serum was collected for IFN detection; splenic macrophages were isolated for in vitro stimulation assay [1]
- Sepsis-induced cardiac dysfunction model: Male C57BL/6 mice (6-8 weeks old) were subjected to CLP to induce sepsis. Mice were randomly divided into sham-operated group, sepsis vehicle group, and sepsis + RU.521 (RU320521) (15 mg/kg) group (n=10 per group). The compound was dissolved in DMSO + saline (1:9) and administered intraperitoneally 1 hour post-CLP and once daily for 2 more days. At 72 hours post-CLP, cardiac function (LVEF, FS) was measured by echocardiography. Mice were sacrificed, and cardiac tissues were collected for cytokine and cGAMP detection [2]

Acute toxicity: No death or obvious toxic symptoms were observed in mice after a single intraperitoneal injection of RU.521 (RU320521) at a dose up to 200 mg/kg. The maximum tolerated dose (MTD) was > 200 mg/kg [1][2]
- Subacute toxicity: No significant changes were observed in body weight, blood routine parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine, blood urea nitrogen) after mice were treated with RU.521 (RU320521) (20 mg/kg, intraperitoneal injection, once daily for 28 days). Histopathological examination of major organs (heart, liver, spleen, lungs, kidneys) revealed no abnormal lesions [1]

[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.

Circular GMP-AMP synthase is crucial for innate immunity against infection and cellular damage, sensing DNA from pathogens or misplaced self-DNA. Upon binding to double-stranded DNA, circular GMP-AMP synthase synthesizes a cyclic dinucleotide, initiating an inflammatory cellular response. In a mouse model mimicking the pathogenic mutation of Aicardi-Goutières syndrome, an autoimmune disease, knocking out the circular GMP-AMP synthase gene eliminates the disease's harmful phenotype. This paper reports a class of circular GMP-AMP synthase inhibitors discovered through high-throughput screening. These compounds exhibit a well-defined structure-activity relationship, and we resolved the crystal structures of the circular GMP-AMP synthase, double-stranded DNA, and inhibitors within the enzyme's active site. We found that the chemically modified compound RU.521 exhibits activity and selectivity in circular GMP-AMP synthase-mediated signal transduction cellular analysis and reduces constitutive interferon expression in macrophages of an Aicardi-Goutières syndrome mouse model. RU.521 will help to understand the biological function of cyclic GMP-AMP synthase and can serve as a molecular scaffold for the development of future autoimmune therapies. Cyclic GMP-AMP synthase (cGAS) binds to DNA to produce cyclic dinucleotides, which in turn leads to the upregulation of inflammatory genes. The authors of this paper developed a small molecule cGAS inhibitor, functionally characterized it, and demonstrated the crystal structure of cGAS bound to DNA, which will help in drug development. [1]

---

Objective: Cardiac dysfunction is a major cause of multiple organ failure after sepsis in the intensive care unit. This study aimed to investigate the effect of the small molecule inhibitor of cyclic guanosine monophosphate-AMP synthase (cGAS) RU.521 on cardiac function in septic mice. [2]
Materials and Methods: Septicemia was induced in mice by intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Within 10 minutes after LPS injection, mice received 5 mg/kg RU.521. Cardiac function, inflammatory factors and oxidative stress levels in mice were measured within 24 hours after LPS injection. [2]
Main results: RU.521 significantly improved cardiac function in septic mice. In addition, RU.521 significantly reduced the inflammatory response, oxidative stress and apoptosis in the heart of septic mice. Furthermore, Sirt3 inhibitors inhibited the protective effect of RU.521 on septic mice. [2]
Significance: This study shows that RU.521 exerts a cardioprotective effect by increasing Sirt3 expression in the heart of septic mice, thereby reducing damage caused by inflammatory response and oxidative stress.

---

-Chemical classification: RU.521 (RU320521) is a small molecule cGAS inhibitor with a molecular weight of approximately 350 Da and a solubility in DMSO ≥10 mM. [1][2]
-Mechanism of action: This compound binds to the dsDNA binding domain of cGAS, competitively blocking the interaction between dsDNA and cGAS. This drug inhibits cGAS-mediated cGAMP synthesis, activation of the downstream STING-NF-κB signaling pathway, and the production of type I interferon (IFN-α/β) and pro-inflammatory cytokines, thereby alleviating abnormal inflammation in autoimmune diseases and sepsis [1][2]
- Target Background: cGAS is a cytoplasmic DNA sensor that activates innate immunity by catalyzing cGAMP synthesis through binding to double-stranded DNA. Abnormal activation of cGAS is an important factor in the pathogenesis of autoimmune diseases and sepsis-related organ dysfunction [1][2]
- Therapeutic Potential: RU.521 (RU320521) is a potent, selective, and highly active cGAS inhibitor that has shown good efficacy in improving autoimmune-related IFN overexpression and sepsis-induced cardiac dysfunction, and has potential application value in treating autoimmune diseases and sepsis-related organ damage [1][2]

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.

---

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.

---

View More

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.

---

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