PHA-767491 (CAY10572) is a potent ATP-competitive dual Cdc7/CDK9 inhibitor with IC50 of 10 nM and 34 nM, respectively.

CDC7:10 nM, CDK9:34 nM

PHA-767491降低Chk1磷酸化并增加从裸鼠HCC异种移植物切片的肿瘤组织中的原位细胞凋亡.

PHA-767491抑制两种细胞系的增殖,在HCC1954细胞中的IC50为0.64 μM,在Colo-205细胞中的IC50为1.3 μM。PHA-767491（2 μM）在HCC1954细胞中24小时完全消除Mcm2磷酸化。PHA-767491与5-FU联用对HCC细胞表现出更强的细胞毒性并诱导显著的凋亡,表现为显著增加的胱天蛋白酶3激活和聚（ADP-核糖）聚合酶片段化。 PHA-767491直接抵消5-FU诱导的Chk1的磷酸化并降低抗凋亡蛋白髓性白血病细胞1ine的表达。 PHA-767491（0-10 μM）以时间和剂量依赖方式降低成胶质细胞瘤细胞活力,U87-MG和U251-MG细胞的IC50约为2.5 μM。

20 ng of purified human DDK, together with increasing concentrations of each DDK inhibitor is pre-incubated for 5 min. Then 10 μCi (γ)-32P ATP and 1.5 μM cold ATP are added in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT and incubated for 30 min at 30°C. The proteins are denatured in 1X Laemmli buffer at 100°C followed by SDS-PAGE and autoradiography on HyBlot CL film. DDK kinase activity is indicated by Auto-phosphorylation of DDK. 32P-labeled bands are quantified using ImageJ and the IC50 values are calculated using GraphPad.

For assays in 96 well plates 2500 cells are plated per well. After 24 hours, cells are treated with small molecule inhibitors and incubated for 72 hours at 37°C. Subsequently the cells are lysed and the ATP content is measured as an indicator of metabolically active cells using the CellTiter-Glo assay. IC50 values are calculated using the GraphPad software. For assays in six well plates, 100,000 cells are plated per well. After 24 hours, cells are treated with small molecule inhibitors and incubated for varying time points. Cells are trypsinized and a suspension is made in 5 mL of phosphate buffered saline. 30 μL of this suspension is mixed with 30 μL of CellTiter-Glo reagent followed by a 10-minute incubation at room temperature. Luminescence is measured using EnVision 2104 Multilabel Reader and BioTek Synergy Neo Microplate Reader.