| 名稱 | PP2 |
| 描述 | PP2 (AG 1879,AGL 1879) is a effective inhibitor of Lck/Fyn (IC50:4/5 nM) , ~100-fold less potent to EGFR, inactive for ZAP-70, PKA and JAK2. |
| 細(xì)胞實(shí)驗(yàn) | Cell viability is determined using an in vitro toxicology assay kit following the manufacturer's instructions. Cells are seeded in 96-well plates at day 0. Starting at day 1, cells are treated for 2 days with each of a series of increasing concentrations of PP2 (1 μM, 10 μM, and 100 μM). At the end of this period, cell proliferation is evaluated by a colorimetric assay based on the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by mitochondria dehydrogenase in viable cells, leading to formazan formation. This experiment is repeated three times with 10 determinations/tested concentration.(Only for Reference) |
| 激酶實(shí)驗(yàn) | Immune complex enzyme assays: The acid-treated enolase is diluted 1:20 with 1× PBS before aliquoting 100 mL/well into a Nunc 96-well high protein binding assay plate. Assay wells are then aspirated; blocked with 0.5% bovine serum, 1× PBS for 1 h at 37 ℃;and then washed five times with 300 mL of 1× PBS/well. The source of Lck is either LSTRA cells or Lck expressed in HeLa cells using a vaccinia expression system. FynT is expressed in HeLa cells using the vaccinia system. Cells (12.5× 106/mL) are lysed in lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, and 23 trypsin inhibitory units/mL aprotinin), and the lysates are clarified by centrifugation at 14,000 cpm for 15 min at 4 ℃ in an Eppendorf tube. The clarified lysates are then incubated with the appropriate anti-kinase antibody at 10 μg/mL for 2 h at 4 ℃. Protein A-Sepharose beads are added to the antibody/lysate mixture at 250 μL/mL and allowed to incubate for 30 min at 4 ℃. The beads are then washed twice in 1 mL of lysis buffer and twice in 1 mL of kinase buffer (25 mM HEPES, 3 mM MnCl2, 5 mM MgCl2, and 100 μM sodium orthovanadate) and resuspended to 50% (w/v) in kinase buffer. Twenty-five microliters of the bead suspension is added to each well of the enolase-coated 96-well high protein binding plate together with an appropriate concentration of compound and [γ-32P]ATP (25 μL/well of a 200 μCi/mL solution in kinase buffer). After incubation for 20 min at 20 ℃, 60 μLl of boiling 2× solubilization buffer containing 10 mM ATP is added to the assay wells to terminate the reactions. Thirty microliters of the samples is removed from the wells, boiled for 5 min, and run on a 7.5% SDS-polyacrylamide gel. The gels are subsequently dried and exposed to Kodak X-AR film. For quantitation, films are scanned using a Molecular Dynamics laser scanner, and the optical density of the major substrate band, enolase p46, is determined.In companion experiments for measuring the activity of compounds against Lck, the assay plate is washed with two wash cycles on a Skatron harvester using 50 mM EDTA, 1 mM ATP. Scintillation fluid (100 μL) is then added to the wells, and 32P incorporation is measured using a micro-β-counter. |
| 體外活性 | 在局灶性腦缺血損傷的大鼠中,PP2（）降低約50%梗死面積.在局灶性腦缺血損傷的大鼠中,與對(duì)照組相比,PP2（1.5 mg/kg,i.p.）具有更好的神經(jīng)功能評(píng)分.在脾臟接種HT29細(xì)胞的SCID小鼠中,與對(duì)照組相比,PP2（5 mg/kg/day）降低原代腫瘤的生長(zhǎng)率,且明顯降低相對(duì)肝重和肝轉(zhuǎn)移量. |
| 體內(nèi)活性 | 1-100 mM PP2劑量依賴性抑制人類結(jié)腸癌細(xì)胞(SW480,HT29和PMCO1),肝癌細(xì)胞(KYN-2,Li7,PLC/PRF/5和HepG2)和乳腺癌細(xì)胞(MDA-MB-468,MCF-7,和 BT-474)生長(zhǎng)。在NIH3T3 和NIH-RET/PTC3 細(xì)胞裂解液中,5 μM PP2可抑制體內(nèi)RET/PTC1腫瘤蛋白的磷酸化和信號(hào)。在轉(zhuǎn)化RET/PTC1的NIH3T3成纖維細(xì)胞、攜帶自發(fā)性RET/PTC1重排的兩種人類甲狀腺乳頭狀癌細(xì)胞系TPC1 和 FB2中,5 μM PP2抑制血清非依賴性的生長(zhǎng)。5 μM PP2抑制TPC1細(xì)胞I型膠原基質(zhì)入侵。在HeLa 和 SiHa 細(xì)胞中,10 μM PP2下調(diào)pSrc-Y416,pEGFR-Y845和-Y1173 表達(dá)水平。10 μM PP2可以調(diào)節(jié)細(xì)胞周期停滯,這是通過(guò)上調(diào)HeLa和SiHa細(xì)胞的p21(Cip1) 和 p27(Kip1),及下調(diào)HeLa細(xì)胞的cyclin A和細(xì)胞周期蛋白依賴性激酶-2,-4 (Cdk-2,-4)的表達(dá),和下調(diào)SiHa細(xì)胞的cyclin B 和 Cdk-2表達(dá)實(shí)現(xiàn)的。20 μM PP2抑制40-50% HT29 細(xì)胞生長(zhǎng),處理1小時(shí),該濃度降低Src活性,并維持抑制35％Src活性達(dá)2天。20 μM PP2明顯促進(jìn)大部分癌細(xì)胞(HT29, SW480, PMCO1, PLC/PRF/5, KYN-2, Li7, MCF-7和 MDA-MB-468)聚合,該作用有E-鈣粘蛋白依賴性。在癌細(xì)胞中,20 μM PP2增強(qiáng)E-鈣粘蛋白表達(dá),且強(qiáng)促進(jìn)E-鈣粘蛋白與肌動(dòng)蛋白細(xì)胞骨架的關(guān)聯(lián)。在HT29細(xì)胞中,20 μM PP2增強(qiáng)α-catenin,β-catenin和 γ-catenin表達(dá),而在PLC/PRF/5 和 MCF-7細(xì)胞,α-catenin的總蛋白水平?jīng)]有改變,但β-catenin 和 γ-catenin水平稍微增長(zhǎng)。 |
| 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 : 56 mg/mL (185.57 mM), Sonication is recommended.
Ethanol : 2 mg/mL (6.63 mM), Sonication is recommended.
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| 關(guān)鍵字 | ZAP70 | TyrosineKinases | Tyrosine Kinases | Src | PP-2 | PP2 | PP 2 | Lck | JAK2 | Fyn | EGFR | AGL-1879 | AGL1879 | AG 1879,AGL-1879 | AG 1879,AGL1879 |
| 相關(guān)產(chǎn)品 | Lapatinib | Neratinib | Chalcone | Tofacitinib Citrate | Gefitinib | Erlotinib | Osimertinib | Erlotinib hydrochloride | Lidocaine Hydrochloride hydrate | Genistein | Nintedanib | Ruxolitinib phosphate |
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