RU.521 (RU320521)

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

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]。

在小鼠中，RU.521（5 mg/kg；腹腔注射一次）可减少饥饿迹象 [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。

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。

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

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。