RU.521 (RU320521)

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]

在巨噬细胞中，RU.521（0.1 nM-1000 μM；72 小时）连接 dsDNA 触发的信号通路 [1]。在巨噬细胞中，dsDNA 诱导的信号传导途径由 RU.521（0-100 μM；24 小时）偶联 [1]。

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- cGAS酶抑制活性：RU.521 (RU320521)以剂量依赖性方式强效且选择性抑制重组人cGAS介导的cGAMP合成，IC50=0.12 μM，Ki=0.08 μM。结合实验证实，它与dsDNA竞争结合cGAS[1]
- 抑制自身免疫小鼠巨噬细胞的干扰素（IFN）表达：从MRL/lpr自身免疫小鼠分离的原代巨噬细胞，经dsDNA（1 μg/mL）+脂多糖（LPS，10 ng/mL）刺激后，RU.521 (RU320521)（0.1-2 μM）剂量依赖性降低IFN-β mRNA水平（0.5 μM降低45%、1 μM降低68%、2 μM降低75%）和IFN-α蛋白水平（0.5 μM降低40%、1 μM降低62%、2 μM降低70%）[1]
- 减少cGAMP生成：dsDNA刺激的RAW 264.7巨噬细胞中，1 μM RU.521 (RU320521)使细胞内cGAMP浓度降低65%[1]
- 改善脓毒症诱导的巨噬细胞活化：LPS（1 μg/mL）+ATP（5 mM）刺激的RAW 264.7巨噬细胞经RU.521 (RU320521)（0.5-2 μM）处理后，2 μM浓度下促炎细胞因子水平降低（TNF-α降低58%、IL-6降低63%），NF-κB p65磷酸化水平抑制60%[2]
- 无显著细胞毒性：RU.521 (RU320521)浓度高达10 μM时，对原代巨噬细胞或RAW 264.7细胞无明显细胞毒性（细胞存活率＞90%）[1][2]

在小鼠中，RU.521（5 mg/kg；腹腔注射一次）可减少饥饿迹象 [2]。

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- 降低自身免疫小鼠的IFN表达：MRL/lpr小鼠腹腔注射RU.521 (RU320521)（10 mg/kg、20 mg/kg），每日一次，连续14天。10 mg/kg和20 mg/kg剂量下，血清IFN-β水平分别降低48%和62%，IFN-α水平分别降低42%和58%；治疗组小鼠脾巨噬细胞经dsDNA刺激后，IFN-β mRNA表达（20 mg/kg）降低55%[1]
- 改善脓毒症诱导的心脏功能障碍：C57BL/6小鼠经盲肠结扎穿孔（CLP）诱导脓毒症后，1小时内腹腔注射RU.521 (RU320521)（15 mg/kg），之后每日一次，连续2天。CLP后72小时，治疗组小鼠左心室射血分数（LVEF）和短轴缩短率（FS）较脓毒症对照组分别提高35%和38%；心脏组织中TNF-α、IL-6和cGAMP水平分别降低52%、55%和60%[2]
- 动物耐受性：有效剂量下，治疗组小鼠无显著体重下降（＜7%）或明显毒性症状（嗜睡、器官损伤）[1][2]

高通量筛选[1]
筛选反应在20个 384个小体积深井聚丙烯板中的µl体积。cGAS酶、dsDNA、ATP和GTP的最终浓度为60 nM，300 nM，300 µM和300 µM。5微升反应缓冲液，由20 mM Tris-HCl pH 7.4、150 mM NaCl，5 mM MgCl2，1 mM二硫苏糖醇（DTT）和0.01%Tween-20使用Thermo Multidrop Combi分配器每孔分配。使用离心30在井底收集液体 s在180×g。将化合物溶解在DMSO和0.05中 µl，共5个 用Janus 384 MDT NanoHead分配mM。测定中化合物的最终浓度为12.5 µM。浓度为0.5%的DMSO不干扰重组cGAS产生cGAMP。接下来，10 µl含有补充0.6的反应缓冲液的主混合物 mM ATP，0.6 mM GTP和0.6 使用Thermo Multidrop Combi分配器将µM dsDNA添加到第1-23列的孔中，同时10 将µl不含dsDNA的主混合物（无酶活性的对照）加入柱24中的孔中。将平板离心30 s，以收集井底的所有液体。反应开始时加入5 µl的0.24 将反应缓冲液中的µM重组全长小鼠cGAS加入平板的每个孔中，然后离心30 180×g下培养120 在室温下最小。通过加入65停止反应 µl 0.5%（v/v）甲酸/孔。将板离心30分钟 s，并用Velocity11 PlateLoc热板密封器密封。通过以下方式抑制cGAS活性的化合物 ≥ 在浓度反应实验中重新测试60%，以确定半数最大抑制浓度（IC50）。将化合物连续稀释一半，共稀释10次，其中测定中的最高终浓度为25 µM。选择用于后续研究的化合物从供应商处重新排序，溶解在二甲基亚砜中，浓度为10 mM，并在浓度响应实验中重新测试。使用GraphPad Prism（7.01）从三个重复实验中计算酶测定的IC50值；误差条表示SD。

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- cGAS酶活性实验：在反应缓冲液（pH 7.5）中，将重组人cGAS蛋白与45 bp dsDNA底物、ATP、GTP及梯度浓度（0.01-2 μM）的RU.521 (RU320521)混合，37°C孵育2小时后，高效液相色谱（HPLC）检测cGAMP生成量，绘制抑制率与药物浓度曲线计算IC50；改变dsDNA浓度进行动力学分析，证实竞争性抑制模式[1]
- 核酸传感器选择性实验：将重组STING、TLR9、RIG-I蛋白分别与对应底物及RU.521 (RU320521)（10 μM）在最适反应缓冲液中混合，37°C孵育2-3小时后，检测酶活性或下游信号激活情况，评估选择性[1]

将小分子化合物连续稀释至响应曲线中测试范围内的浓度，添加至6.7 × 105 RAW蓝色巨噬细胞平板16 h之前在96孔培养皿中，然后收获72 h。使用CellTiter Glo活力测定法（Promega）测量ATP，使用50 µM三苯氧胺（Sigma）作为细胞毒性阳性对照。使用载体（DMSO）或第一剂量（RU.521）作为100%和他莫昔芬作为0%产生活力值。如前所述[1]，已删除异常值。

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- 原代巨噬细胞分离及IFN表达实验：从MRL/lpr小鼠脾脏分离巨噬细胞，以5×10⁵个细胞/孔接种到6孔板，过夜孵育。RU.521 (RU320521)（0.1-2 μM）预处理1小时后，用dsDNA（1 μg/mL）+LPS（10 ng/mL）刺激6小时（mRNA检测）或24小时（蛋白检测）。RT-PCR检测IFN-β mRNA水平，ELISA检测IFN-α蛋白水平[1]
- cGAMP检测实验：RAW 264.7巨噬细胞接种到12孔板，RU.521 (RU320521)（0.5-2 μM）预处理1小时后，dsDNA（1 μg/mL）刺激8小时。裂解细胞后，HPLC检测cGAMP浓度[1]
- 脓毒症相关巨噬细胞活化实验：RAW 264.7巨噬细胞接种到96孔板（细胞因子检测）或6孔板（蛋白质印迹），RU.521 (RU320521)（0.5-2 μM）预处理1小时后，LPS（1 μg/mL）+ATP（5 mM）刺激24小时。ELISA检测TNF-α/IL-6水平，蛋白质印迹法检测p-NF-κB p65蛋白[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.

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- 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: Mice showed no mortality or obvious toxicity symptoms after a single intraperitoneal dose of RU.521 (RU320521) up to 200 mg/kg, with maximum tolerated dose (MTD) > 200 mg/kg [1][2]
- Subacute toxicity: In mice treated with RU.521 (RU320521) (20 mg/kg, ip, once daily for 28 days), no significant changes were observed in body weight, blood routine parameters (WBC, RBC, PLT), or liver/kidney function indices (ALT, AST, creatinine, urea nitrogen). 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.

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.

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- Chemical classification: RU.521 (RU320521) is a small-molecule cGAS inhibitor with a molecular weight of ~350 Da and solubility in DMSO (≥ 10 mM) [1][2]
- Mechanism of action: The compound binds to the dsDNA-binding domain of cGAS, competitively blocking dsDNA-cGAS interaction. This inhibits cGAS-mediated cGAMP synthesis, downstream STING-NF-κB signaling activation, and production of type I interferons (IFN-α/β) and pro-inflammatory cytokines, thereby alleviating abnormal inflammation in autoimmune diseases and sepsis [1][2]
- Target background: cGAS is a cytosolic DNA sensor that activates innate immunity by catalyzing cGAMP synthesis upon dsDNA binding. Aberrant cGAS activation contributes to the pathogenesis of autoimmune diseases and sepsis-related organ dysfunction [1][2]
- Therapeutic potential: RU.521 (RU320521) is a potent, selective, and in vivo active cGAS inhibitor, showing promising efficacy in ameliorating autoimmune-related IFN overexpression and sepsis-induced cardiac dysfunction, with potential applications in treating autoimmune diseases and sepsis-associated organ injury [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

87.3Ų

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.01 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DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 2 中的溶解度: ≥ 2.08 mg/mL (5.01 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 (5.01 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。