BI-D1870

RSK4 (IC50 = 15 nM); RSK3 (IC50 = 18 nM); RSK2 (IC50 = 24 nM); RSK4 (IC50 = 15 nM); RSK1 (IC50 = 31 nM)

BI-D1870 抑制 RSK1、RSK2、RSK3 和 RSK4，IC50 为 10-30 nM，但在 100 倍高浓度下测试时不会显着抑制其他 10 种 AGC 激酶成员和 40 多种其他蛋白激酶。在人胚肾 293 细胞和 Rat-2 细胞中，BI-D1870 具有细胞渗透性，并抑制 RSK 介导的佛波酯和 EGF 诱导的糖原合酶激酶激酶 3β 和 LKB1 的磷酸化。其他六种 AGC 激酶的激动剂触发的底物磷酸化不受 BI-D1870 的影响。此外，BI-D1870 不会阻断佛波酯或 EGF 引起的 CREB 磷酸化。[1]

注射 BI-D1870 (0.5 mg/kg) 的实验性自身免疫性脑脊髓炎 (EAE) 小鼠表现出迟发性神经缺陷，但没有明显的体重减轻。组织病理学分析显示对照小鼠的脊髓中有炎症细胞浸润和脱髓鞘，但 BI-D1870 治疗的小鼠则没有。 BI-D1870 可防止 TH1 或 TH17 细胞浸润至中枢神经系统。

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多发性硬化症（MS）是一种中枢神经系统（CNS）自身免疫性脱髓鞘疾病，由TH1和TH17细胞浸润中枢神经系统引起。核糖体S6激酶2（RSK2；RPS6KA3）通过减弱RORγt转录活性和IL-17A的产生来调节TH17分化。泛RSK抑制剂BI-D1870也抑制TH17分化，但BI-D1870在体内的作用尚不清楚。在这里，我们制备了患有实验性自身免疫性脑脊髓炎（EAE）的小鼠，并用BI-D1870对其进行治疗。BI-D1870给药通过减少TH1和TH17细胞向中枢神经系统的浸润以及降低TH17细胞中Ccr6的mRNA水平来保护小鼠免受EAE的侵害。这些结果表明，抑制RSK是治疗MS的一种有前景的策略[3]。

将纯化的 His6–RSK1、His6–RSK2 或 GST–RSK2 1–389:S381E (1–2 单位/mL) 在含有 30 μM 底物肽 (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) 的缓冲液 A 中的 50 μL 测定混合物中于 30 °C 测定 10 分钟)、10 mM 醋酸镁和 100 μM [γ-32P]ATP。如前所述，停止并检查反应。 1个酶单位是1分钟内催化1nmol底物肽磷酸化所需的量。为了测定 HEK-293 或 Rat-2 细胞裂解物中的 RSK 和 MSK1，从细胞裂解物中对这些激酶进行免疫沉淀（RSK 为 0.1 mg 裂解物蛋白，MSK1 为 0.3 mg），并且对于 RSK 测定，免疫沉淀物用缓冲液 A 含有 1 mM ATP，并在测定前使用缓冲液 A 两次，作为预防措施，确保 BI-D1870 从 RSK 同工型解离。

使用 MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴化物]测定法评估细胞生长，重复六次。在 96 孔平底板中，将细胞 (5 103/200 L) 接种 24 小时，然后在指定的时间内暴露于不同浓度的测试剂中。除去培养基后，加入200μL含有0.5mg/mL浓度的MTT的培养基，并将细胞在37℃下孵育2小时。除去培养基后，将每孔的还原 MTT 染料溶解在 200 L DMSO 中。 Synergy HT 多模式酶标仪在 570 nm 处用于测量吸光度。

Myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 MEVGWYRSPFSRVVHLYRNGK) (BEX) is used to induce EAE in C57/BL6J mice. Mice are given s.c. injections of 200 g of MOG peptide emulsified in 100 μL of PBS, 100 l of complete Freund's adjuvant (CFA), and five mg/mL of Mycobacterium tuberculosis. Additionally, on days 0 and 2, 500 ng of pertussis toxin is intravenously injected. Two days after receiving the MOG peptide vaccination, mice are given an intraperitoneal injection of the RSK inhibitor (BI-D1870; 0.5 mg/kg), which is repeated every other day for 11 days. As controls, mice are given only dimethyl sulfoxide (DMSO) solution. On a scale from zero (no disease) to six (death), paralysis is rated as follows: one (tail limpness), two (hind limb weakness), three (hind limb paralysis), four (fore limb weakness), five (quadriplegia), and six (no disease). Hematoxylin and eosin (H & E) staining is carried out on CNS samples after they have been cut at a thickness of 4 m and fixed with 4% paraformaldehyde for histological analysis.

[1]. Biochem J. 2007 Jan 1; 401(Pt 1): 29–38.

[2]. Biochem J . 2007 Jan 1;401(1):29-38.

[3]. Immunobiology . 2016 Feb;221(2):188-92.

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.[1]

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Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC(50) of 10-30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3beta and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.[2]

C19H23F2N5O2

391.4150

391.181

C, 58.30; H, 5.92; F, 9.71; N, 17.89; O, 8.18

501437-28-1

501437-28-1

25023738

Pale orange to brown solid powder

1.3±0.1 g/cm3

579.3±60.0 °C at 760 mmHg

304.2±32.9 °C

0.0±1.7 mmHg at 25°C

1.585

3.53

81.59

2

8

5

28

542

0

FC1C(=C(C([H])=C(C=1[H])N([H])C1=NC([H])=C2C(=N1)N(C([H])([H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])C([H])(C([H])([H])[H])C(N2C([H])([H])[H])=O)F)O[H]

DTEKTGDVSARYDS-UHFFFAOYSA-N

InChI=1S/C19H23F2N5O2/c1-10(2)5-6-26-11(3)18(28)25(4)15-9-22-19(24-17(15)26)23-12-7-13(20)16(27)14(21)8-12/h7-11,27H,5-6H2,1-4H3,(H,22,23,24)

2-((3,5-difluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one

BI D1870; BID1870; 501437-28-1; 2-((3,5-difluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one; 2-(3,5-difluoro-4-hydroxyanilino)-5,7-dimethyl-8-(3-methylbutyl)-7H-pteridin-6-one; BI-D 1870; 2-[(3,5-DIFLUORO-4-HYDROXYPHENYL)AMINO]-7,8-DIHYDRO-5,7-DIMETHYL-8-(3-METHYLBUTYL)-6(5H)-PTERIDINONE; 2-((3,5-difluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one.; MFCD11223662; BI-D1870

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: ~78 mg/mL (~199.3 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.39 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (6.39 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.39 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 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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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。