PHA-767491 hydrochloride (CAY-10572 hydrochloride) is an effective ATP-competitive dual Cdc7/CDK9 inhibitor (IC50: 10/34 nM). It has ~20-fold selectivity against GSK3-β and CDK1/2, 50-fold selectivity against CDK5 and MK2, 100-fold selectivity against CHK2 and PLK1.

CDC7:10 nM, CDK9:34 nM

PHA-767491对HCC1954细胞（IC50：0.64μM）和Colo-205细胞（IC50：1.3μM）的增殖具有抑制作用，并且是体外DDK抑制剂（IC50：18.6nM）。PHA-767491（2μM）在24小时内完全消除HCC1954细胞中Mcm2的磷酸化[1]。PHA-767491与5-FU联合使用时，表现出更强的细胞毒性，并通过显著增加caspase 3激活和poly(ADP-Ribose) polymerase分裂来诱导HCC细胞中显著的凋亡。PHA-767491直接抵消了5-FU诱导的Chk1磷酸化，并减少了抗凋亡蛋白myeloid leukemia cell line的表达[2]。PHA-767491（0-10μM）以时间和剂量依赖的方式减少了U87-MG和U251-MG细胞的活性（IC50：约2.5μM）。

PHA-767491 降低了 Chk1 磷酸化，并在裸鼠 HCC 移植瘤组织切片中增加了原位细胞凋亡。

20 ng of purified human DDK is pre-incubated with increasing concentrations of each DDK inhibitor 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. Auto-phosphorylation of DDK is used as an indicator of its kinase activity. 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.