SR12343

两种优化的领先 NBD 类似物 SR12343 和 SR12460 通过阻断 IKKβ 和 NEMO 之间的相互作用抑制肿瘤坏死因子 α (TNF-α) 和脂多糖 (LPS) 诱导的 NF-κB 活化，并抑制小鼠中 LPS 诱导的急性肺部炎症。[1]
SR12343 显示出与 8K-NBD 肽相似的特性，与 NBD 肽 (400 μM) 相比，在低得多的浓度 (50 μM) 下显示出对 COX-2、IL-6 和 iNOS 表达的显着抑制。[1]
用指示药物预处理原始 264.7 细胞 30 分钟，然后用 1 μg/ml LPS 刺激 2 小时。然后收获细胞进行 RNA 提取和 qRT-PCR 分析。所用药物浓度如下：IKKi VII (2 μM)、8K-NBD 肽 (200 μM)、SR12343 (50 μM)、SR12460 (50 μM)、SR12454 (50 μM) 和 SR11481 (50 μM)。[1]

使用 SR12343 和 SR12460 对杜氏肌营养不良症 (DMD) 小鼠模型进行长期治疗可减轻炎症浸润、坏死和肌肉退化，表明这些小分子 NBD 模拟物是治疗炎症和退行性疾病的潜在疗法。[1]

NBD 类似物靶向 NEMO/IKKβ 相互作用来抑制 NF-κB 信号传导
为了确定新型 NBD 类似物是否靶向体内 NEMO-IKKβ 相互作用，使用来自 Raw 264.7 巨噬细胞的提取物进行共免疫沉淀。所有 4 种类似物降低 IKKβ-NEMO 相互作用的效果与 NBD 肽一样好甚至更好，其中 SR12343 最有效。SR12343 还以剂量依赖性方式降低了 Raw 264.7 细胞中 NEMO 和 IKKβ 之间的结合。在这些条件下未观察到 NEMO 与 TNF 受体相关因子 2 (TRAF2) 或 IκBα 之间的相互作用。先前的研究表明 NBD 肽也抑制了 NEMO 与 IKKα 的相互作用。然而，SR12343 仅在最高剂量下对 NEMO/IKKα 结合仅有边际影响，这表明这些抑制剂影响 NEMO/IKKα 相互作用的效率要低得多。为了证明 SR12343（在体内破坏 NEMO/IKKβ 相互作用的最有效模拟物）直接影响 NEMO/IKKβ 相互作用，使用重组 GST-NEMO 和 FLAG-IKKβ 进行了体外谷胱甘肽 S-转移酶 (GST) 下拉试验。如图 5C 所示，SR12343 即使在 12.5 μM 的剂量下也能够破坏 GST-NEMO 和 FLAG-IKKβ 之间的相互作用。为了证明 TNF-α 和 LPS 刺激后 NF-κB 介导的转录减少不是由于脱靶效应，我们通过蛋白质印迹分析检查了 NF-κB 信号和丝裂原活化蛋白激酶 (MAPK) 通路的激活。SR12343 降低了 TNF-α 和 LPS 刺激后磷酸化 IKK 复合物、IκBα 和 p65 的水平。IκBα 的降解持续减少。然而，在 TNF-α 或 LPS 刺激后，用 SR12343 (150 μM) 处理后，磷酸化 c-Jun N 端激酶 (p-JNK)、p-p38MAPK、总 JNK 和 p38MAPK 的水平没有变化。这些结果表明，观察到的 SR12343 抑制作用是由 IKK/NF-κB 直接介导的，而不是由于脱靶效应（例如通过 JNK 或 p38MAPK 通路）引起的。SR12343 治疗对抗淋巴毒素 β 受体 (LTβR) 激活非经典 NF-κB 通路也没有影响，如从 p100 到 p52 的加工未发生改变所示。[1]

Novel NBD mimetics suppress LPS-induced acute pulmonary inflammation in vivo
To determine the stability of the NBD mimetics in vivo, their levels were measured in the plasma of mice 2 h post i.p. injection with 10 mg/kg of each compound. The concentration of SR12460 was very high in plasma (>6.5 μg/mL; 20 μM), while levels in brain, muscle, spleen, and liver were lower. SR12343 and SR12454 had lower plasma concentrations, with SR12343 showing a higher level in liver and SR12454 showing higher concentrations in muscle and spleen. The level of SR11481 was undetectable in plasma and tissues . Because SR12454 and SR12460 share similar chemical structures and SR11481 demonstrates poor in vivo exposure, SR12460 and SR12343 were selected for further in vivo analysis. However, it is important to note that SR12343 was not as orally active as SR123460 .[1]
To determine their NF-κB inhibitory effects in vivo, SR12343 and SR12460 were tested in an acute model of LPS-induced systemic endotoxemia. C57BL/6J mice were pretreated with vehicle control, 8K-NBD peptide, or NBD mimetics at 10 mg/kg for 30 min, followed by LPS induction at 10 mg/kg . Lung and liver were harvested 2–4 h post treatment for qRT-PCR analysis of NF-κB target genes. SR12343 was able to significantly inhibit NF-κB transcriptional activity in lung, demonstrated by the inhibition of iNOS, IκBα, COX-2, and IL-6, while expression of TNF-α was unchanged . The NF-κB inhibitory effects of the compounds were less effective in liver compared to lung, demonstrated by significant inhibition of only COX-2 expression. Despite lower plasma and tissue concentrations of SR12343 compared to SR12460, its inhibition of NF-κB/IKK in liver and lung were greater than that of SR12460 . Similarly, acute treatment with SR12343 also affected the LPS-induced expression of NF-κB target genes at protein level. The extent of phosphorylation of IκBα and the levels of COX-2 were reduced in lung and liver tissues treated with SR12343 . Serum levels of IL-6 detected by ELISA increased significantly following LPS induction and were reduced by acute treatment with SR12343. These results are consistent with our RT-PCR analysis shown in Fig 6C. However, no significant differences in lung histopathology were observed. Finally, there was a slight reduction in the number of white blood cells and neutrophils in the SR12343-treated group. Taken together, the results demonstrate that SR12343 and SR12460 are effective at attenuating LPS-induced acute lung inflammation by suppressing NF-κB target gene expression.[1]
Novel NBD mimetics alleviate necrosis and muscle degeneration in mdx mice Because SR12343 and SR12460 both reduced LPS-induced NF-κB activation in vivo, they were further tested in mdx mice, a mouse model of DMD in which NF-κB is chronically activated. Mdx mice develop normally at birth and undergo a massive myonecrosis starting at 3 wk. Treatment of mdx mice with IKK/NF-κB inhibitors effectively reduces inflammation, blocks necrosis, and increases muscle regeneration. Initially, the effect of acute treatment of SR12343 on NF-κB DNA binding activity in tibialis anterior (TA) muscle in 9-wk-old mdx mice was examined by EMSA. Treatment with a single injection of 30 mg/kg of SR12343 resulted in a reduction in NF-κB DNA binding 2 h post injection . Subsequently, Mdx mice were chronically treated with vehicle, SR12343 (30 mg/kg), SR12460 (30 mg/kg), or 8K-NBD (10 mg/kg) starting from day 21, 3 times/wk for 4 wk, similar to the dosing regimen used with the NBD peptide. No significant weight loss was observed in chronically treated mdx mice . In addition, there was no increase in levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), or alkaline phosphatase (ALP) in chronically treated mdx mice, suggesting that treatment with SR12343 and SR12460 had no overt liver toxicity.[1]

[1]. Development of novel NEMO-binding domain mimetics for inhibiting IKK/NF-κB activation. PLoS Biol. 2018 Jun 11;16(6):e2004663.

[2]. New Trends in Aging Drug Discovery. Biomedicines. 2022 Aug 18;10(8):2006.

C15H15BRCLN3O

368.656101465225

367.008

2055101-86-3

124117414

Off-white to light yellow solid powder

3.8

54

2

3

6

21

332

0

PEOFCAIUELALCR-UHFFFAOYSA-N

InChI=1S/C15H15BrClN3O/c16-12-3-1-2-11(8-12)6-7-18-15(21)10-20-14-5-4-13(17)9-19-14/h1-5,8-9H,6-7,10H2,(H,18,21)(H,19,20)

N-(3-Bromophenethyl)-2-((5-chloropyridin-2-yl)amino)acetamide

SR 12343SR-12343 SR12343

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。