| 名稱 | Flavopiridol |
| 描述 | Flavopiridol (Alvocidib) (Alvocidib) competes with ATP to inhibit CDKs including CDK1, CDK2, CDK4 and CDK6 with IC50 of ~ 40 nM. It is 7.5-fold more selective for CDK1, 2, 4, 6 versus CDK7. Flavopiridol is initially found to inhibit EGFR and PKA. Phase 1/2. |
| 細(xì)胞實驗 | Cells are exposed to various concentrations of Flavopiridol for 72 hours at which time the tetrazolium dye, MTS in combination with phenazine methosulfate, is added. After 3 hours, the absorbency is measured at 492 nm, which is proportional to the number of viable cells. The results are expressed as IC50 values. For cell Cycle analysis, cells are fixed in paraformaldehyde and ethanol, washed, resuspended in staining solution of TdT enzyme and FITC-dUTP, washed, stained with PI following RNase treatment, and then analyzed by flow cytometry. (Only for Reference) |
| 激酶實驗 | CDK kinase assay: For CDK1/cyclin B1 kinase assay, kinase reactions consist of 100 ng of baculovirus expressed GST-CDK1/cyclin B1 (human) complex, 1 μg histone HI, 0.2 μCi [γ-33P]ATP, 25 μM ATP in 50 μL kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT). For CDK2/cyclin E kinase assay, kinase reactions consist of 5 ng of baculovirus expressed GST-CDK2/cyclin E (human) complex, 0.5 μg GST-RB fusion protein (amino acids 776-928 of retinoblastoma protein), 0.2 μCi [γ-33P]ATP, 25 μM ATP in 50 μL kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT). For CDK4/cyclin D1 kinase assay, kinase reactions consist of 150 ng of baculovirus expressed GST-CDK4/cyclin D1 (human), 280 ng of Stag-cyclin D1, 0.5 μg GST-RB fusion protein (amino acids 776-928 of retinoblastoma protein), 0.2 μCi [γ-33P]ATP, 25 μM ATP in 50 μL kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT). Reactions are incubated for 45 minutes for CDK1 and CDK2, or 1 hour for CDK4 at 30 °C and stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration 15%. TCA precipitates are collected onto GF/C unifilter plates using a Filtermate universal harvester and the filters are quantitated using a TopCount 96-well liquid scintillation counter. Flavopiridol is dissolved at 10 mM in dimethylformamide (DMF) and evaluated at six concentrations, each in triplicate. The final concentration of DMF in the assay = 2%. IC50 values are derived by nonlinear regression analysis and have a coefficient of variance = 16%. To assay Flavopiridol activity on CDK6, a filter-binding assay is established. The following are combined in the reaction mixture: 2 μL of CDK6 (0.7 mg/μL), 5 μL of histone H1 (6 mg/mL), 14 μL of kinase buffer (60 mM β-glycerophosphate, 30 mM p-nitrophenyl phosphate, 25 mM MOPS (pH 7.0), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 0.1 mM Na-vanadate), 3 μL of increasing concentrations of Flavopiridol diluted in 50% DMSO, and 6 μL of 33P-ATP (1 mCi/mL) in nonradioactive ATP at 90 μM concentration (final concentration: 15 μM). The assay is initiated by the addition of 33P-ATP. The reaction is incubated for 20 minutes at 30°C. A 25 μL aliquot of the supernatant is then spotted onto Whatman P81 phosphocellulose paper. Filters are washed 5 times with 1% phosphoric acid solution. Wet filters are counted in the presence of 1 mL of scintillation fluid. Cdk9 activity is measured using 50 nM of recombinant Cdk9/cyclin T in 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 3 μM Na3VO4, 150 μM RNA polymerase CDT peptide and 80 μM ATP. Cdk7 assay is performed in the same buffer using 37 nM of purified kinase in the presence of 200 μM ATP and 10 μM myelin binding protein as a substrate. The potency of Flavopiridol toward CDK9 and CDK7 is determined using either a strong anion exchanger (Dowex 1-X8 resin, formate form)-based assay or a scintillation proximity assay. IC50 values are calculated from the dose-response curves. |
| 體外活性 | Flavopiridol對于與之無關(guān)的激酶如MAP、PAK、PKC和EGFR的活性較低，其IC50值>14 μM。Flavopiridol顯著抑制HCT116、A2780、PC3和Mia PaCa-2細(xì)胞的集落生長，其IC50值分別為13 nM、15 nM、10 nM和36 nM。[1] 此外，F(xiàn)lavopiridol還能強(qiáng)效抑制糖原合成激酶-3 (GSK-3)的活性，IC50為280 nm。[2] 與其他CDKs相比，F(xiàn)lavopiridol對CDK7的抑制活性較弱，IC50為875 nM。Flavopiridol (0.5 μM) 能夠抑制pSer807/811 Rb和pThr199 NPM，而對pThr821 Rb的改變較輕。同時，F(xiàn)lavopiridol還能減少RNA聚合酶II的整體水平及其在CTD重復(fù)序列上Ser2 Ser5的磷酸化水平。[3] 作為廣譜CDK抑制劑，F(xiàn)lavopiridol可以在G1或G2階段抑制細(xì)胞周期進(jìn)程。Flavopiridol (0.3 μM) 通過抑制CDK4或CDK2激酶活性，在MCF-7或MDA-MB-468細(xì)胞中誘導(dǎo)G1阻滯。[4] Flavopiridol對多種腫瘤細(xì)胞系表現(xiàn)出強(qiáng)效的細(xì)胞毒性，IC50值范圍從LNCAP的16 nM到K562的130 nM。[5] |
| 體內(nèi)活性 | Flavopiridol以7.5 mg/kg劑量連續(xù)7天給藥, 對P388小鼠型白血病表現(xiàn)出輕度抗腫瘤活性, 并且對植入裸鼠的人類A2780卵巢癌細(xì)胞顯示出活性, 產(chǎn)生1.5 log細(xì)胞殺傷(LCK)。[5] Flavopiridol在1-2.5 mg/kg的劑量下連續(xù)10天治療，能顯著以劑量依賴性方式抑制小鼠膠原誘導(dǎo)的關(guān)節(jié)炎，通過抑制滑膜增生和關(guān)節(jié)破壞，同時血清中針對膠原II型(CII)抗體(Abs)的濃度和對CII的增殖反應(yīng)得到維持。[6] 在攜帶正常p21的Hct116異種移植裸鼠中, 管理CPT-11 (100 mg/kg)后分別在7小時和16小時給予Flavopiridol (3 mg/kg)，顯著抑制腫瘤退縮86%和82%, 相較于單獨(dú)使用CPT-11（40%抑制率）顯示出>2倍的抑制效果。此組合產(chǎn)生約30%的完全響應(yīng)率(CR)，而單獨(dú)CPT-11治療組則未見CR。[7] |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 4.02 mg/mL (10 mM), Sonication is recommended.
Ethanol : 8 mg/mL (19.9 mM)
H2O : < 1 mg/mL (insoluble or slightly soluble)
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| 關(guān)鍵字 | HMR 1275 | inhibit | L86-8275 | L-868275 | L 868275 | CDK | Cyclin dependent kinase | Inhibitor | Autophagy | Flavopiridol | Human immunodeficiency virus | Apoptosis | HIV | HMR1275 |
| 相關(guān)產(chǎn)品 | Guanidine hydrochloride | Naringin | Valproic Acid | L-Glutamic acid | Emtricitabine | Hydroxychloroquine | Lamivudine | Dextran sulfate sodium salt (MW 4500-5500) | Stavudine | L-Ascorbic acid | Paeonol | Sodium 4-phenylbutyrate |
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