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.

CDK6:40 nM, CDK1:40 nM, CDK2:40 nM, CDK4:40 nM

Flavopiridol对于与之无关的激酶如MAP、PAK、PKC和EGFR的活性较低，其IC50值>14 μM。Flavopiridol显著抑制HCT116、A2780、PC3和Mia PaCa-2细胞的集落生长，其IC50值分别为13 nM、15 nM、10 nM和36 nM。[1] 此外，Flavopiridol还能强效抑制糖原合成激酶-3 (GSK-3)的活性，IC50为280 nm。[2] 与其他CDKs相比，Flavopiridol对CDK7的抑制活性较弱，IC50为875 nM。Flavopiridol (0.5 μM) 能够抑制pSer807/811 Rb和pThr199 NPM，而对pThr821 Rb的改变较轻。同时，Flavopiridol还能减少RNA聚合酶II的整体水平及其在CTD重复序列上Ser2 Ser5的磷酸化水平。[3] 作为广谱CDK抑制剂，Flavopiridol可以在G1或G2阶段抑制细胞周期进程。Flavopiridol (0.3 μM) 通过抑制CDK4或CDK2激酶活性，在MCF-7或MDA-MB-468细胞中诱导G1阻滞。[4] Flavopiridol对多种肿瘤细胞系表现出强效的细胞毒性，IC50值范围从LNCAP的16 nM到K562的130 nM。[5]

Flavopiridol以7.5 mg/kg剂量连续7天给药, 对P388小鼠型白血病表现出轻度抗肿瘤活性, 并且对植入裸鼠的人类A2780卵巢癌细胞显示出活性, 产生1.5 log细胞杀伤(LCK)。[5] Flavopiridol在1-2.5 mg/kg的剂量下连续10天治疗，能显著以剂量依赖性方式抑制小鼠胶原诱导的关节炎，通过抑制滑膜增生和关节破坏，同时血清中针对胶原II型(CII)抗体(Abs)的浓度和对CII的增殖反应得到维持。[6] 在携带正常p21的Hct116异种移植裸鼠中, 管理CPT-11 (100 mg/kg)后分别在7小时和16小时给予Flavopiridol (3 mg/kg)，显著抑制肿瘤退缩86%和82%, 相较于单独使用CPT-11（40%抑制率）显示出>2倍的抑制效果。此组合产生约30%的完全响应率(CR)，而单独CPT-11治疗组则未见CR。[7]

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.

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)