| 名稱 | PLX-4720 |
| 描述 | PLX-4720 is a potent and selective B-Raf (V600E) inhibitor designed to block the ATP-binding site of oncogenic B-Raf with IC50 of 13 nM. |
| 細(xì)胞實(shí)驗(yàn) | Cells are treated with various concentrations PLX-4720 for 24, 48, and 72 hours. Cell proliferation is measured by using the CellTiter-Glo Luminescent Cell Viability Assay or MTT assay. For cell cycle analysis, supernatant and cells are collected, pelleted, and fixed with 70% ethanol. Before staining with propidium iodide (10 μg/mL), cells are incubated for 1 hour at 37 °C in 0.5 mg/mL RNase I to rid samples of residual RNA contamination. Samples are then analyzed by using the EPICS XL apparatus. For the assessment of apoptosis, media and cells are harvested and pelleted before staining with annexin-FITC and propidium iodide. Samples are subsequently analyzed by using the EPICS XL apparatus [1]. |
| 激酶實(shí)驗(yàn) | In vitro Raf kinase activities: The in vitro kinase activities of wild type Raf and mutants are determined by measuring phosphorylation of biotinylated-MEK protein using Perkin-Elmer's AlphaScreen Technology. For each enzyme (0.1 ng), 20-μL reactions are carried out in 20 mM Hepes (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 100 nM biotin-MEK protein, various ATP concentrations, and increasing concentrations of PLX-4720 at room temperature. Reactions are stopped at 2, 5, 8, 10, 20, and 30 minutes with 5 μL of a solution containing 20 mM Hepes (pH 7.0), 200 mM NaCl, 80 mM EDTA, and 0.3% BSA. The stop solution also includes phospho-MEK Antibody, Streptavidin-coated Donor beads and Protein A Acceptor beads. The antibody and beads are preincubated in stop solution in the dark at room temperature for 30 minutes. The final dilution of antibody is 1/2,000, and the final concentration of each bead is 10 μg/mL. The assay plates are incubated at room temperature for one hour then are read on a PerkinElmer AlphaQuest reader [1]. |
| 動物實(shí)驗(yàn) | Female athymic mice (NCr nu/nu) were implanted s.c. on day 0 with 30–60 mg COLO205 tumor fragments. Treatments began on day 11, when the mean estimated tumor mass was 104 mg (range, 95–113 mg). All animals were dosed with vehicle (5% DMSO, 1% methylcellulose) or PLX4720 suspended in vehicle by gavage daily for 14 consecutive days. Tumor burden (mg) was estimated from caliper measurements [1]. |
| 體外活性 | 方法：1205Lu 和 C8161 細(xì)胞用 PLX4720（0.1,1,10μM，24小時） 并用膜聯(lián)蛋白 V/FITC 和碘化丙啶 （PI） 染色以分析細(xì)胞凋亡。
結(jié)果：PLX4720誘導(dǎo)1205Lu細(xì)胞周期停滯并引起細(xì)胞凋亡，且呈現(xiàn)濃度依賴性。[1]
方法：8505c、TPC-1 和 NT 細(xì)胞用PLX4720（1μM或10μM）處理72小時后，加入溴脫氧尿苷（BrdU，10μM）1小時，隨后進(jìn)行細(xì)胞周期分析、BrdU測定和凋亡測定。
結(jié)果：8505c細(xì)胞用 1 μM PLX4720處理導(dǎo)致 1 小時后磷酸化 ERK-1/ERK-2 蛋白水平降低 >90%；即使在 72 小時后，細(xì)胞增殖也沒有顯著差異，而用 10 μM PLX4720處理 8505c 細(xì)胞 1 小時或 72 小時可降低磷酸化 ERK-1/ERK-2 BrdU攝取降低；在 TPC-1 細(xì)胞中，用 1 μM PLX4720處理 1 小時導(dǎo)致磷酸化 ERK-1/ERK-2 蛋白水平升高（約 80%）;用 10 μM 處理 1 小時PLX4720磷酸化 ERK-1/ERK-2 降低約 45%；1 μM 和 10 μM PLX4720 都引起較低的細(xì)胞增殖；只有 10 μM PLX4720 在處理 72 小時后減少了 TPC-1 細(xì)胞的遷移；10 μM PLX4720處理的NT細(xì)胞顯示出顯著降低的細(xì)胞增殖。[3] |
| 體內(nèi)活性 | 方法：利用攜帶BRAF COLO205細(xì)胞（BRAFV600E系列突變）的裸鼠服用PLX4720(5 ,20,1000mg/kg,每日一次,口服),觀察小鼠體內(nèi)腫瘤生長情況。
結(jié)果：較低劑量的 5 mg/kg PLX4720對腫瘤生長的影響非常有限；20mg/kg的PLX4720治療荷瘤小鼠導(dǎo)致腫瘤生長的大量阻滯；PLX4720治療耐受性良好，將PLX4720劑量增加至1,000mg / kg導(dǎo)致血漿水平增加（高達(dá)600μM），而沒有任何不良反應(yīng)的證據(jù)。[1] |
| 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)
DMSO : 60 mg/mL (144.99 mM)
H2O : < 1 mg/mL (insoluble or slightly soluble)
|
| 關(guān)鍵字 | Inhibitor | inhibit | PLX-4720 | Raf kinases | Raf | PLX 4720 |
| 相關(guān)產(chǎn)品 | Pelitinib | Doramapimod | Vemurafenib | Dabrafenib | Sorafenib tosylate | Exarafenib | Regorafenib | Sorafenib | GW 441756 | Regorafenib monohydrate | Sulindac sulfide | LY3009120 |
| 相關(guān)庫 | 抑制劑庫 | 經(jīng)典已知活性庫 | 抗癌活性化合物庫 | 已知活性化合物庫 | 激酶抑制劑庫 | NO PAINS 化合物庫 | 臨床前化合物庫 | 神經(jīng)退行性疾病化合物庫 | 疼痛相關(guān)化合物庫 | 酪氨酸激酶分子庫 |