SU9516 is a potent CDK2 inhibitor, with an IC50 of 22 nM, and also has inhibitory effects on CDK1 and CDK4, with IC50s of 40 nM and 200 nM, respectively.

CDK1:40 nM, CDK4:200 nM, CDK2:22 nM

SU9516降低了视网膜母细胞瘤蛋白pRB的cdk2特异性磷酸化作用，增加了caspase-3的活化，并改变了RKO和SW480细胞的细胞周期。此外，SU9516还抑制了这两种细胞系的细胞增殖，并诱导了细胞凋亡。[1] SU9516通过抑制RNA Pol II CTD的磷酸化、氧化损伤以及Mcl-1的转录下调，杀死了白血病细胞。[2] 在人T细胞白血病Jurkat细胞中，SU9516显著提高了对甲氨蝶呤的敏感性。[3] 此外，SU9516还抑制了Aurora-A的着丝粒定位和随之发生的着丝粒增倍。[4]

CDK kinase assay: Kinase assays are performed in 96-well polypropylene plates. Each reaction contained 2 μg of histone H1 at a final concentration of 10 μM [γ-33P]ATP (0.2 μCi/well; approximately twice the experimentally determined Km), 10 mM MgCl2, 1 mM DTT, 0.01% Triton X-100, and 10% glycerol in a 40 μL volume. The reaction is initiated with the addition of 20 μL enzyme (6 ng cdk2/well resulting in a final concentration of 1.6 nM), which is previously diluted 1:50–1:200 in the same buffer, and allowed to proceed for 1 h at room temperature. Reaction is stopped by the addition of 0.01 mL 10% phosphoric acid, and 25 μL of reaction mixture is transferred to P30 phosphocellulose filter mat paper. The filter mat is washed three times with 1.0% phosphoric acid, air dried, and then counted for radioactivity in a liquid scintillation counter. The cdk4 kinase assay for cyclin D1-cdk4 is carried out in a polypropylene 96-well microtiter plate format measuring the incorporation of radioactive phosphate into GST-Rb. Purified cyclin D1-cdk4 is incubated with 1 μg GST-Rb in 20 mM HEPES (pH 7.5) in the presence of 10 mM MgCl2, 1 mM DTT, 0.01% Triton X-100, and 10% glycerol. The final cdk4 concentration is 10 ng/well, or 1.6 nM. Kinase reaction is initiated by the addition of ATP at a final concentration of 10 μM ATP (twice the experimentally determined Km) and [γ-33P]ATP (1.0 μCi per well) in a 60-μL volume and allowed to proceed at room temperature for 1 h. Reaction is stopped by the addition of 0.01 ml 10% phosphoric acid, and 25 μL of reaction mixture is transferred to P30 phosphocellulose filter mat paper. The filter mat is treated as for Cdk1/Cdk2 assays.

Method: Cells are seeded at 2×103/well of white bottom 96-well plates, treated with INCB018424 from DMSO stocks (0.2% final DMSO concentration), and incubated for 48 hours at 37 ℃ in an atmosphere containing 5% CO2. Viability is measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent or viable cell counting. Values are transformed to percent inhibition relative to vehicle control, and IC50 curves are fitted according to nonlinear regression analysis of the data using PRISM GraphPad.