化合物SB203580 T1764

Product Introduction
Bioactivity


英文名: Adezmapimod

描述: Adezmapimod (SB 203580) 是一种 p38 MAPK 抑制剂 (IC50=0.3-0.5 μM)，具有选择性和 ATP 竞争性。Adezmapimod 具有自噬和线粒体自噬的激活活性。Adezmapimod 显示出比 PKB、LCK 和 GSK-3β 高 100 倍以上的选择性。

细胞实验: The luciferase reporter plasmid pIL6luc(-122) and the CAT reporter plasmid p(TRE)5CAT were transfected into TF-1 cell line by means of electroporation. Prior to transfection, cells were cultured for 16?h at a density of 0.5×10^6 cells/ml in the appropriate medium, washed twice and resuspended in RPMI 1640 at a density of 10×10^6 in 200?μl. When transfected with a single plasmid, 25?μg of DNA was added and the mixture was left at room temperature for 15?min. Cotransfections were performed with 15?μg of the reporter plasmid pIL6luc(-122) together with 15?μg of the dominant-negative expression plasmids (pRSV-MKK3(Ala), pcDNA3-MKK6(K82A), pRSV-NΔRaf1, pcDNA3-MKK4(Ala), pcDNA3-Flag-JNK1, or pcDNA3 (empty vector). Cotransfections of pGAL4tkluc (5?μg) with either pGAL4p65 (5?μg) or pGAL4dbd (5?μg) were performed under similar conditions. In addition, cells were cotransfected with 2?μg of a CMV-CAT plasmid, to normalize for transfection efficiency. Electroporation, in 0.4?cm electroporation cuvettes, was performed at 240?V and 960?μF with Gene Pulser electroporator. After electroporation, the cells were replated in RPMI 1640 containing 2% FBS. Six hours after transfection cells were stimulated for 24?h with medium or OA (30?ng/ml) or SB203580 for 30?min prior to OA stimulation. The cells were then harvested and lysed by commercially available luciferase lysis buffer. One-hundred μl of lysis product was added to 100?μl of luciferase assay reagents and luciferase activity was measured with the Anthos Lucy1 luminometer. CAT reporter activity of 100?μl lysis product plus 100?μl CAT dilution buffer was determined with a commercially available CAT Elisa kit [3].

激酶实验: Cells were lysed in Buffer A for Western blotting and PKB kinase assays. Kinase assays were performed according to the manufacturer's instructions. Briefly, 4 μg of sheep anti-PKBα was immobilized on 25 μl of protein G-Sepharose overnight (or 1.5 h) and washed in Buffer A (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mMEGTA, 0.5 mM Na3VO4, 0.1% β-mercaptoethanol, 1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, pepstatin, leupeptin, and 1 μM microcystin). The immobilized anti-PKB was then incubated with 0.5 ml of lysate (from 5 × 10^6 cells) for 1.5 h and washed three times in 0.5 ml of Buffer A supplemented with 0.5 M NaCl, two times in 0.5 ml of Buffer B (50 mM Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1 mM EGTA, and 0.1% β-mercaptoethanol), and twice with 100 μl of assay dilution buffer; 5× assay dilution buffer is 100 mM MOPS, pH 7.2, 125 mMβ-glycerophosphate, 25 mM EGTA, 5 mM sodium orthovanadate, 5 mM DTT. To the PKB enzyme immune complex was added 10 μl of assay dilution buffer, 40 μM protein kinase A inhibitor peptide, 100 μM PKB-specific substrate peptide, and 10 μCi of [γ-32P]ATP, all made up in assay dilution buffer. The reaction was incubated for 20 min at room temperature with shaking, then samples were pulse spun, and 40 μl of reaction volume were removed into another tube to which was added 20 μl of 40% trichloroacetic acid to stop the reaction. This was mixed and incubated for 5 min at room temperature, and 40 μl was transferred onto P81 phosphocellulose paper and allowed to bind for 30 s. The P81 pieces were washed three times in 0.75% phosphoric acid then in acetone at room temperature. γ-32P incorporation was then measured by scintillation counting [1].

动物实验: In survival studies, C57BL/6J mice weighing 20 g to 30 g were briefly anesthetized with isoflurane and challenged with 0.05 mL of IT normal saline (NS, noninfected controls) or E. coli (15 × 10^9 CFU/kg) as previously described. One hour before NS challenge, mice (n = 24) received either intraperitoneal SB203580 (100 mg/kg in 0.25 mL) or diluent only (placebo). Infected animals received SB203580 in doses of 100, 10, 1, or 0.1 mg/kg or placebo 1 hour before IT E. coli (n = 241); SB203580 100 or 0.1 mg/kg or placebo 1 hour after E. coli (n = 121); or SB203580 100 mg/kg or placebo 12 hours after E. coli (n = 72). All animals received ceftriaxone (100 mg/kg in 0.1 mL, subcutaneously) for 4 days and NS (0.5 mL, subcutaneously) for 1 day beginning 4 hours after challenge. Animals were observed every 2 hours for the initial 48 hours, every 4 hours from 48 hours to 72 hours, every 8 hours from 72 hours to 96 hours, and then twice daily until study completion (168 hours). Sequential weekly experiments with 24 animals each compared either two to three doses of SB203580 versus placebo administered at similar times or similar doses of SB203580 versus placebo at differing treatment times. Study groups in each experiment were of equivalent sample size (i.e., 6 – 8 per group) [5].

体外活性: 方法：人肝癌细胞 HepG2 用 Adezmapimod (0.1-20 μM) 处理 30 min，使用 Western Blot 方法检测靶点蛋白表达水平。结果：Adezmapimod 剂量依赖性地激活 ERK，但不激活 p38 和 JNK。ERK 的最大活化是在 1- 10 μM 之间。[1]方法：CD4+T 细胞用 Adezmapimod (1-25 μM) 处理 72 h，使用 Flow Cytometry 方法分析 Tregs 的增殖。结果：Adezmapimod 以剂量依赖的方式抑制 TNF 诱导的 Tregs 增殖，抑制率为32.0-73.2%。Adezmapimod 处理也显著降低了培养的 CD4+T 细胞中 Foxp3+ Treg 的比例，抑制率为24.9-47.05%。[2]

体内活性: 方法：为检测体内活性，将 Adezmapimod (25 mg/kg in 4% DMSO+30% PEG 300+5% Tween 80+61% ddH2O) 单次腹腔注射给 LPS 处理的 C57BL/6J 小鼠，24 和 72 h 后，处死小鼠。结果：通过 Adezmapimod 的处理，LPS 诱导的 Tregs 上 Ki-67 和 TNFR2 表达的上调被完全消除。在 LPS 处理的小鼠中，Adezmapimod 对 Tregs 增殖扩增的抑制作用可以持续至少 72 h。[2]方法：检测对子宫内膜异位症 (EM) 发育的影响，将 Adezmapimod (1 μg/mg) 腹腔注射给诱导 EM 的 BALB/c 小鼠，每天一次，持续二十四天。结果：Adezmapimod 降低了小鼠子宫内膜异位病变的重量和大小。与 EM 组相比，Adezmapimod 组腹膜细胞的 IL-1β、TNF-α、MMP-2 和 MMP-9 水平降低。EM 组 p38 MAPK 的磷酸化水平升高，Adezmapimod 下调了磷酸化水平。[3]

存储条件: Powder: -20°C for 3 years | In solvent: -80°C for 1 year

溶解度: DMSO : 50 mg/mL (132.47 mM)
1eq. HCl : 37.7 mg/mL (100 mM)


关键字: Inhibitor | PKBα | inhibit | SAPK2a | GSK3β | Mitophagy | p38 MAPK | Mitochondrial Autophagy | Autophagy | SB-203580 | Adezmapimod | PB-203580 | SB 203580 | RWJ64809 | PB203580 | LCK | SAPK2b | RWJ-64809 | ATP-competitive

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