| 名稱 | BI-D1870 |
| 描述 | BI-D1870 is a highly specific, blood-brain-permeable, and ATP-competitive inhibitor of the N-terminal AGC kinase domain of RSK, with IC50 values ??of 31 nM, 24 nM, 18 nM, and 15 nM for RSK1, RSK2, RSK3, and RSK4. |
| 細胞實驗 | The rat embryo fibroblast cell line, Rat-2 cells were cultured on 10 cm-diameter dishes in Dulbecco's Modified Eagle's medium supplemented with 10% (v/v) FBS. HEK-293 cells were cultured on 10 cm-diameter dishes in Dulbecco's Modified Eagle's medium supplemented with 10% FBS and 1×antimycotic/antibiotic solution. Prior to stimulation, cells were cultured in the absence of serum for 16 h. Inhibitors were dissolved in DMSO at a 1000-fold higher concentration than they were used at. These inhibitors, or the equivalent volume of DMSO as a control, were added to the tissue culture medium 30 min prior to stimulation unless indicated otherwise. The final concentration of DMSO in the culture medium was 0.1% and had no effect on agonist-induced activation or phosphorylation of any of the substrates examined. The cells were stimulated with the indicated agonists and lysed in 1 ml of ice-cold Lysis Buffer and centrifuged at 16000 g at 4 °C for 5 min. The supernatants were frozen in liquid nitrogen and stored at ?80 °C until use. Protein concentrations were determined using the Bradford method with BSA as the standard [1]. |
| 激酶實驗 | Purified His6–RSK1, His6–RSK2 or GST–RSK21–389:S381E (1–2 units/ml) were assayed for 10 min at 30 °C in a 50 μl assay mixture in Buffer A containing 30 μM substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK), 10 mM magnesium acetate and 100 μM of [γ-32P]ATP. Reactions were terminated and analyzed as described previously. The amount of enzyme that catalyzed the phosphorylation of 1 nmol of substrate peptide in 1 min was termed one unit. In order to assay RSK and MSK1 in HEK-293 or Rat-2 cell lysates, these kinases were immunoprecipitated from the cell lysates (0.1 mg of lysate protein for RSK and 0.3 mg for MSK1) and assayed as described previously, except that for RSK assays the immunoprecipitates were washed twice with Buffer A containing 1 mM ATP and twice with Buffer A prior to the assay, as a precaution to ensure dissociation of BI-D1870 from the RSK isoforms [1]. |
| 動物實驗 | Myelin oligodendrocyte glycoprotein (MOG) peptide 35–55. (MEVGWYRSPFSRVVHLYRNGK) (BEX) was used to induce EAE in C57/BL6J mice. Mice were injecteds.c. with 200 g of MOG peptide in100 L of PBS emulsified in 100 L complete Freund's adjuvant (CFA) that was further supplemented with five mg mL?1 Mycobacterium tuberculosis (H37Ra). In addition, 500 ng pertussis toxin was injected i.p. on days zero and two. The RSK inhibitor (BI-D1870; 0.5 mg kg?1) was injected i.p. into mice two days after immunization with MOG peptide, and injection was repeated every other day for 11 days. Mice that received only dimethyl sulfoxide (DMSO) solution were used as controls. Paralysis was evaluated according to the following scale: zero, no disease; one, tail limpness; two, hind limb weakness; three, hind limb paralysis; four, forelimb weakness; five, quadriplegia; six, death. For histological analysis, CNS samples were fixed with 4% paraformaldehyde and sliced at 4 m, and then hematoxylin & eosin (H & E) staining was performed [4]. |
| 體外活性 | 方法：將HEK-293細胞與 BI-D1870（10 μM，15分鐘，總共4小時） 孵育為了檢驗BI-D1870是否能抑制細胞中的RSK活性，研究BI-D1870對HEK-293細胞中RSK催化其已知底物磷酸化的影響，使用PMA作為激動劑激活ERK1/ERK2和RSK亞型。
結(jié)果：用 BI-D1870 孵育細胞可極大地抑制 PMA 誘導(dǎo)的 GSK3α 和 GSK3β 磷酸化 ，相比之下，BI-D1870在任何時間點對PMA誘導(dǎo)的ERK1/ERK2激活（由Raf和MKK1催化）或CREB在Ser位點的磷酸化幾乎沒有影響。[1]
方法：BI-D1870（1,2,3,4,5.28或者48小時） 處理口腔癌細胞系 (SCC2095、SCC4、SCC9、Ca922 和 HSC-3) 和NHOK細胞，MTT檢測這些細胞的生長情況。
結(jié)果： BI-D1870 對 OSCC 細胞表現(xiàn)出劑量反應(yīng)性抗增殖作用。[3]
方法：用 BI-D1870 （0.5,2μM）處理轉(zhuǎn)染免疫熒光 GFP-LC3 的細胞 48 小時，并在共聚焦顯微鏡下觀察；使用 DAPI 染色定位細胞核，用 BI-D1870 處理 48 小時的觀察細胞中 LC3B 的蛋白質(zhì)印跡。
結(jié)果：BI-D1870 誘導(dǎo)自噬，LC3B-II 的蛋白質(zhì)印跡表明這種誘導(dǎo)具有劑量依賴性。[3] |
| 體內(nèi)活性 | 方法：在用 MOG 肽免疫小鼠兩天后，BI-D1870（0.5 mg/kg) 腹腔注射到小鼠體內(nèi)，每隔一天重復(fù)注射一次，持續(xù) 11 天。
結(jié)果：小鼠表現(xiàn)出延遲神經(jīng)缺陷；BI-D1870 治療對體重減輕有中等程度的保護作用。[4] |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | Ethanol : < 1 mg/mL (insoluble or slightly soluble)
H2O : < 1 mg/mL (insoluble or slightly soluble)
DMSO : 6.88 mg/mL (17.56 mM), Sonication is recommended.
10% DMSO+40% PEG300+5% Tween 80+45% Saline : 7.2 mg/mL (18.39 mM), Suspension. Please add co-solvents sequentially, clarifying the solution as much as possible before adding the next one. Dissolve by heating and/or sonication if necessary. Working solution is recommended to be prepared and used immediately.
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| 關(guān)鍵字 | BI-D 1870 | BI-D1870 | inhibit | Ribosomal S6 Kinase (RSK) | S6K | Inhibitor | BI-D-1870 | Autophagy | BI D1870 | BID1870 |
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