PHA-767491 845714-00-3

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Molecular Weight:

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


Biological Activity


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 or gemcitabine 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.


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. 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.


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 DMBA-induced mammary carcinomas, and correlates
with Cdc7 inhibition and subsequently decreased phosphorylation of Mcm2
at the Cdc7-dependent site Ser40


Protocol(Only for Reference)


Kinase Assay: [1]

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 Assay: [1]

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.

Animal Study: [1]

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 DMBA-induced mammary carcinomas
Formulation	Dissolved in DMSO, and diluted in saline
Dosages	~50 mg/kg
Administration	Intravenous or oral administration twice a day
Solubility	1% DMSO/30% polyethylene glycol/1% Tween 80, 30 mg/mL
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Baboon	Dog	Monkey	Rabbit	Guinea pig	Rat	Hamster	Mouse
Weight (kg)	12	10	3	1.8	0.4	0.15	0.08	0.02
Body Surface Area (m2)	0.6	0.5	0.24	0.15	0.05	0.025	0.02	0.007
Km factor	20	20	12	12	8	6	5	3

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×	mouse Km(3)	= 11.2 mg/kg
	rat Km(6)

Chemical Information

Molecular Weight (MW)	213.24
Formula	C12H11N3O
CAS No.	845714-00-3

Storage	3 years -20℃Powder
	6 months-80℃in solvent (DMSO, water, etc.)
Synonyms	CAY10572

Solubility (25°C) *	In vitro	DMSO	24 mg/mL (112.54 mM)
		Water	<1 mg/mL (
		Ethanol	<1 mg/mL (
	In vivo	1% DMSO/30% polyethylene glycol/1% Tween 80	30 mg/mL
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Name	2-(pyridin-4-yl)-6,7-dihydro-1H-pyrrolo[3,2-c]pyridin-4(5H)-one

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