Home Cell Cycle CDK inhibitor PHA-767491 HCl For research use only.

PHA-767491 HCl

Synonyms: CAY10572, NMS 1116354

PHA-767491 (CAY10572, NMS 1116354) HCl is a potent ATP-competitive dual Cdc7/CDK9 inhibitor with IC50 of 10 nM and 34 nM in cell-free assays, respectively.It displays ~20-fold selectivity against CDK1/2 and GSK3-β, 50-fold selectivity against MK2 and CDK5, 100-fold selectivity against PLK1 and CHK2.

PHA-767491 HCl Chemical Structure

PHA-767491 HCl Chemical Structure

CAS No. 942425-68-5

Purity & Quality Control

PHA-767491 HCl Related Products

Signaling Pathway

CDK Signaling Pathways

CDK Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
human SF268 cells Proliferation assay 72 h Antiproliferative activity against p53 deficient human SF268 cells after 72 hrs, IC50=0.86 μM 18469809
human HCT116 cells Proliferation assay 72 h Antiproliferative activity against human HCT116 cells expressing p53 gene after 72 hrs by proliferative assay, IC50=0.97 μM 18469809
human HCT16 cells Proliferation assay 72 h Antiproliferative activity against human HCT16 cells after 72 hrs by luciferase based assay, IC50=1 μM 19115845
human SW403 cells Proliferation assay 72 h Antiproliferative activity against human SW403 cells after 72 hrs by luciferase based assay, IC50=1 μM 19115845
human A2780 cells Proliferation assay 72 h Antiproliferative activity against human A2780 cells expressing p53 gene after 72 hrs by proliferative assay, IC50=1.07 μM 18469809
human SW48 cells Proliferation assay 72 h Antiproliferative activity against human SW48 cells after 72 hrs by luciferase based assay, IC50=1.2 μM 19115845
human MCF7 cells Proliferation assay 72 h Antiproliferative activity against human MCF7 cells after 72 hrs by luciferase based assay, IC50=1.3 μM 19115845
human U2OS cells Proliferation assay 72 h Antiproliferative activity against human U2OS cells expressing p53 gene after 72 hrs by proliferative assay, IC50=1.49 μM 18469809
human COLO205 cells Proliferation assay 72 h Antiproliferative activity against human COLO205 cells after 72 hrs by luciferase based assay, IC50=1.5 μM 19115845
human OVCAR8 cells Proliferation assay 72 h Antiproliferative activity against p53 deficient human OVCAR8 cells after 72 hrs by proliferative assay, IC50=1.56 μM 18469809
human L363 cells Proliferation assay 72 h Antiproliferative activity against human L363 cells after 72 hrs by luciferase based assay, IC50=1.6 μM 19115845
human NHDF cells Proliferation assay 72 h Antiproliferative activity against human NHDF cells after 72 hrs by luciferase based assay, IC50=1.6 μM 19115845
human NCI-H929 cells Proliferation assay 72 h Antiproliferative activity against human NCI-H929 cells after 72 hrs by luciferase based assay, IC50=1.8 μM 19115845
human SF539 cells Proliferation assay 72 h Antiproliferative activity against human SF539 cells expressing p53 gene after 72 hrs by proliferative assay, IC50=2.34 μM 18469809
human SW480 cells Proliferation assay 72 h Antiproliferative activity against p53 deficient human SW480 cells after 72 hrs by proliferative assay, IC50=2.67 μM 18469809
human NCI60 cells Proliferation assay Antiproliferative activity against human NCI60 cells, IC50=3.1 μM 18469809
human Jurkat cells Proliferation assay 72 h Antiproliferative activity against p53 deficient human Jurkat cells after 72 hrs by proliferative assay, IC50=3.2 μM 18469809
human HCT15 cells Proliferation assay 72 h Antiproliferative activity against human HCT15 cells expressing p53 gene after 72 hrs by proliferative assay, IC50=3.81 μM 18469809
human OPM2 cells Proliferation assay 72 h Antiproliferative activity against human OPM2 cells after 72 hrs by luciferase based assay, IC50=4.5 μM 19115845
human HT-29 cells Proliferation assay 72 h Antiproliferative activity against human HT-29 cells after 72 hrs by luciferase based assay, IC50=5 μM 19115845
human K562 cells Proliferation assay 72 h Antiproliferative activity against p53 deficient human K562 cells after 72 hrs, IC50=5.87 μM 18469809
U937 cells Function assay Inhibition of TNFalpha production in U937 cells, IC50=19 μM 17480064
human HeLa cells Function assay 5 μM 24 h Induction of apoptosis in human HeLa cells assessed as appearance of PARP at 5 uM after 24 hrs 18469809
NHDF Function assay 5 μM 16 h Induction of cell cycle arrest in thymidine deficient NHDF assessed as DNA synthesis in S-phase at 5 uM after 16hrs FACS analysis in presence of serum 18469809
Click to View More Cell Line Experimental Data

Biological Activity

Description PHA-767491 (CAY10572, NMS 1116354) HCl is a potent ATP-competitive dual Cdc7/CDK9 inhibitor with IC50 of 10 nM and 34 nM in cell-free assays, respectively.It displays ~20-fold selectivity against CDK1/2 and GSK3-β, 50-fold selectivity against MK2 and CDK5, 100-fold selectivity against PLK1 and CHK2.
Features The first inhibitor that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication.
Targets
Cdc7 [1]
(Cell-free assay)		 CDK9 [1]
(Cell-free assay)		 GSK-3β [1]
(Cell-free assay)		 CDK2 [1]
(Cell-free assay)		 CDK1 [1]
(Cell-free assay)		 Click to View More Targets
10 nM 34 nM 220 nM 240 nM 250 nM
In vitro
In vitro

PHA-767491 displays approximately 20-fold selectivity for Cdk1, Cdk2 and GSK3-β, 50-fold selectivity for MK2 and Cdk5 and 100-fold selectivity for PLK1 and CHK2. PHA-767491 inhibits cell proliferation in a variety of human cell lines with IC50 of 0.86 μM for SF-268 to 5.87 μM for K562, and significantly induces apoptosis in a p53-independent manner in almost all cell lines in contrast with 5-FU  which only works in a few of cell lines. Unlike current DNA synthesis inhibitors, PHA-767491 treatment at 5 μM blocks the initiation of DNA replication but not replication fork progression, due to specific inhibition of Cdc7 kinase and Mcm2 phosphorylation at the Cdc7-dependent Ser40 site. [1] The up-regulated Mcl-1 levels in ABT-737-resistant OCI-LY1 and SU-DHL-4 cells can be significantly decreased by PHA-767491 treatment at 3 μM possibly due to the inhibition of Cdk9, leading to the restoration of the sensitivity to ABT-737. [2] The direct mitochondrial dependent pro-apoptosis effect of PHA-767491 is also observed when applied at 1 μM in quiescent chronic lymphocytic leukemia (CLL) cells through the similar mechanism with EC50 of 0.34-0.97 μM. While in proliferating CLL cells stimulated by CD154 and interleukin-4, PHA-767491 treatment at 5 μM abolishes DNA synthesis by inhibiting Cdc7 rather than triggering cell death. [3]
Kinase Assay In vitro kinase assays
The inhibition of Cdc7 and Cdk9 by PHA-767491 (IC50) is determined using the strong anion exchanger (Dowex 1-X8 resin, formate form)-based assay. For each enzyme, the absolute Km values for ATP and the specific substrate are initially determined, and each assay is then run at optimized ATP/33P-γ-ATP mix (2Km) and substrate (5Km) concentrations. Cdc7 kinase assay is performed in a buffer containing 50 mM Hepes pH 7.9, 15 mM MgCl2, 2 mM β- glycerylphosphate, 0.2 mg/mL BSA, 1 mM DTT, 3 μM Na3VO4, 2Km ATP/33P-γ-ATP mix, 5Km Mcm2 (aa 10-294), 37 nM of recombinant Cdc7/Dbf4 and increasing concentration of PHA-767491 in a final volume of 30 μL, and incubated for 1 hour at 25 °C. Cdk9 kinase assay is performed using 50 nM of recombinant Cdk9/cyclin T in 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 3 μM Na3VO4, 2Km ATP/33P-γ-ATP mix, 5Km RNA polymerase CDT peptide and increasing concentration of PHA-767491 in a final volume of 30 μL, and incubated for 1 hour at 25 °C. After incubation, an amount of 150 μL of resin/formate (pH 3.0) is added to stop the reaction and capture unreacted 33P-γ-ATP, separating it from the phosphorylated substrate in solution. After 1 hour of rest, a volume of 50 μL supernatant is transferred to Optiplate 96-well plates. After the additon of 150 μL of Microscint 40, the radioactivity is counted in the TopCount.
Cell Research Cell lines HeLa, MCF7, HCT-116, U2OS, A2780, K562, SF-539, SF-268, Ovcar8, SW480, COLO205, HCT-15, Jurkat, PC3, and NHDF
Concentrations Dissolved in DMSO, final concentrations ~ 20 μM
Incubation Time 24 or 72 hours
Method

Cells are exposed to PHA-767491 for 24 or 72 hours. Cells are lysed and the ATP content in the well, used as a measure of viable cells, is determined using a thermostable firefly luciferase–based assay. Activation of caspase-3 and caspase-7 is measured as a ratio between treated sample and untreated control with a luciferase-based assay, containing a specific proluminescent substrate. DNA replication is measured as incorporation of nucleotide analog BrdU into DNA by flow cytometry.
Experimental Result Images Methods Biomarkers Images PMID
Western blot p-MCM2 / CDC7 RNA Pol II / p-RNA Pol II / Caspase-3 / PARP / Mcl-1 / XIAP / Bcl-xL / Bcl-2 / NOXA 24902048
In Vivo
In vivo

Administration of PHA-767491 twice a day for 5 days significantly inhibits the growth of HL60 xenograft in a dose-dependent manner with TGI of 50% and 92% at dose of 20 mg/kg and 30 mg/kg, respectively, the effect of which is also marked in A2780, Mx-1, and HCT-116 xenograft models as well as the mammary carcinomas, and correlates with Cdc7 inhibition and subsequently decreased phosphorylation of Mcm2 at the Cdc7-dependent site Ser40 [1]
Animal Research Animal Models Female SCID mice subcutaneously implanted with HL60 cells, male Hsd, athymic nu-nu mice subcutaneously implanted with HCT116 cells, A2780 or Mx-1 cells, and female Sprague-Dawley rats with mammary carcinomas
Dosages ~50 mg/kg
Administration Intravenous or oral administration twice a day

Chemical Information & Solubility

Molecular Weight 249.7 Formula

C12H11N3O.HCl
CAS No. 942425-68-5 SDF Download PHA-767491 HCl SDF
Smiles C1CNC(=O)C2=C1NC(=C2)C3=CC=NC=C3.Cl
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 24 mg/mL ( (96.11 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble


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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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