| 名稱 | AZ3146 |
| 描述 | AZ3146 is a selective Mps1 inhibitor with IC50 of ~35 nM. |
| 細(xì)胞實(shí)驗(yàn) | AZ3146 is disolved in DMSO (100 mM) and diluted into 100 μM, 10 μM, 1 μM and 0.1 μM sequentially with DMEM containing 10% FBS before use[2]. The TTK inhibitor AZ3146 is disolved in DMSO at a concentration in 100 mM and diluted into 100 μM, 10 μM, 1 μM and 0.1 μM sequentially with DMEM containing 10% FBS before use. In vitrocytotoxicity assays are performed. HCC cells are plated into 96-well plates at the density of 3×103 per well. AZ3146 is added in the indicated concentrations the next day. The inhibitor treated cells are cultured and tested at a 24-hour intervals for 3-4 days using CCK-8[2]. |
| 激酶實(shí)驗(yàn) | His-tagged human Mps1Cat encoding amino acids 510-857 is generated. For kinase assays, 500 ng is added to buffer (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 μg/mL BSA, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 10 mM MgCl2, and 0.5 μg/mL myelin basic protein), AZ3146, and 100 μM γ-[32P]ATP (2 μCi/assay). Reactions are incubated at 30°C for 20 min, spotted onto P81 paper, washed in 0.5% phosphoric acid, and immersed in acetone. Phosphate incorporation is determined by scintillation counting. For immunoprecipitation kinase assays, HeLa cells are treated with nocodazole for 14 h, mitotic cells isolated, washed in PBS, and lysed for 30 min in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% NP-40, 5 mM EDTA, 5 mM EGTA, 40 mM β-glycerophosphate, 0.2 mM PMSF, 1 mM DTT, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 1 μM okadaic acid, and complete EDTA-free protease inhibitor cocktail. Full-length Mps1 is immunoprecipitated. Purified complexes are washed with lysis buffer containing 100 mM NaCl and assayed as described for the recombinant protein. To quantify 32P incorporation, reactions are stopped with SDS sample buffer and separated by SDS-PAGE followed by phosphorimaging. The plate is analyzed using a phosphorimager using AIDA software. To assess the specificity of AZ3146, a single-point screen is carried using kinase profiling service. 50 kinases are selected and assayed with 1 μM AZ3146[1]. |
| 體內(nèi)活性 | AZ3146不影響Aurora B和BubR1的有絲分裂特異性磷酸化形式。HeLa細(xì)胞用諾考達(dá)唑和2 μM AZ3146處理,僅短暫延遲有絲分裂,隨后重新復(fù)制它們的基因組,表明AZ3146能夠覆蓋SAC。值得注意的是,90% AZ3146處理的HeLa細(xì)胞發(fā)生異常有絲分裂,~50%進(jìn)入分裂后期的細(xì)胞沒有調(diào)整它們的染色體,~30%完成有絲分裂的細(xì)胞沒有進(jìn)行明顯的染色體分離。AZ3146也抑制FAK,JNK1,JNK2 和 Kit。AZ3146明顯抑制細(xì)胞中Mps1的磷酸化作用。AZ3146也會(huì)抑制已經(jīng)建立的SAC信號(hào),如從諾考達(dá)唑阻斷釋放后,AZ3146明顯加速有絲分裂結(jié)束。AZ3146對(duì)Mad2的著絲粒定位有顯著影響,將其降至~15%,但是它對(duì)Mad1的作用不明顯,其水平保持在~60%。進(jìn)入有絲分裂期前,Mps1被AZ3146抑制,隨后Mad1和Mad2對(duì)著絲粒的聚集被阻止。然而,如果Mps1在有絲分裂進(jìn)入后被AZ3146抑制,則Mad1-C-Mad2核心復(fù)合物仍然與著絲粒結(jié)合,但O-Mad2不能聚集到核心。在其他未受到干擾的有絲分裂中,AZ3146使完成有絲分裂的時(shí)間從對(duì)照組的90分鐘減少到32分鐘。 |
| 存儲(chǔ)條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 11.3 mg/mL (25 mM)
Ethanol : 33.9 mg/mL (75 mM)
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| 關(guān)鍵字 | Inhibitor | inhibit | AZ3146 | Monopolar spindle 1 | AZ-3146 | Mps1 |
| 相關(guān)產(chǎn)品 | S-trityl-L-Cysteine | Paprotrain | Eg5-IN-1 | Empesertib | UMK57 | BRD9876 | CW-069 | SB-743921 hydrochloride | Eg5 Inhibitor V, trans-24 | BAY1217389 | AZD-4877 | MPI-0479605 |
| 相關(guān)庫 | 抑制劑庫 | 經(jīng)典已知活性庫 | 已知活性化合物庫 | 激酶抑制劑庫 | 細(xì)胞骨架化合物庫 | NO PAINS 化合物庫 | 表型篩選靶點(diǎn)鑒定庫 |