PF-06726304

Catalog No.S8494 Batch:S849401

Technical Data

Formula	C₂₂H₂₁Cl₂N₃O₃
Molecular Weight	446.33	CAS No.	1616287-82-1
Solubility (25°C)*	In vitro	Ethanol	22 mg/mL (49.29 mM)
		DMSO	10 mg/mL (22.4 mM)
		Water	Insoluble
* <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. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

PF-06726304 is a selective EZH2 inhibitor with Ki values of 0.7 nM and 3 nM for WT EZH2 and Y641N respectively; also inhibits H3K27me3 with the IC50 value of 15 nM.

Targets

Ezh2 (wild-type) ^[1] (cell-free)	EZH2 Y641N ^[1] (cell-free)
0.7 nM(Ki)	3 nM(Ki)

In vitro

PF-06726304 displays very potent EZH2 biochemical inhibition as well as good activity in the cell H3K27Me3 reduction and antiproliferation effect^[1].

In vivo

PF-06726304 displays robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes. PF-06726304 displays good efficacy in a diffuse large B-cell lymphoma Karpas-422 tumor model and exhibited on-target pharmacodynamic effects in vivo^[1].

Protocol (from reference)

Cell Assay:^{^[1]}	Cell lines Karpas-422 cell line Concentrations -- Incubation Time 72 h Method Karpas-422 cells are plated in complete cell culture medium (100 μL/well) in a 96-well clear, V bottom polystyrene cell culture plate at a density of 2500 cells/well. The cells are then incubated for 2−3 h at 37 °C and 5% CO2. Compound dilution plates are prepared in 96-well clear, U-bottom polypropylene plates in 100% DMSO at 10 mM stock concentration in duplicate wells using an 11-point serial dilution (1:3 dilutions). Compounds are further diluted in growth medium, and 25 μL is added to each well of the cell plates. The highest compound concentration tested is 50 μM final, with a 0.5% final DMSO concentration. The plates are then incubated for 72 h at 37 °C and 5% CO2. At the end of the incubation period with compound, the plates are centrifuged at 2000 rpm for 5 min at room temperature, and the medium is removed. Then 100 μL of acid-extracted solution is added to each well. The plates are shaken for 50 min at 4 °C to lyse the cells, and then 38 μL of neutralization buffer is added. The plates are then frozen at −80 °C. The next day, the plates are shaken at room temperature to thaw. Cell lysates (50 μL/well) are then transferred to ELISA plates. The plates are covered and incubated for 2.5 h at room temperature with constant slow-speed shaking. After incubation, the plates are washed seven times (300 μL/well) with 1× wash buffer. Then 100 μL of biotinylated trimethylhistone H3K27 detection antibody is added to each well and incubated for 2 h at room temperature with constant slow-speed shaking. After incubation, the plates are washed as described above. Horseradish peroxidase-linked antibody (100 μL) is added to each well and incubated for 60 min at room temperature with constant slow-speed shaking. After incubation, the plates are washed. Finally, 100 μL of TMB substrate reagent is added to each well and incubated for 5 min at room temperature in the dark with slow shaking, after which 100 μL of stop solution is added to stop the reaction.
Animal Study:^{^[1]}	Animal Models Female Scid beige mice Dosages 30, 100, 300 mg/kg Administration oral

Cell Assay:^{^[1]}

Cell lines
Karpas-422 cell line
Concentrations
--
Incubation Time
72 h
Method
Karpas-422 cells are plated in complete cell culture medium (100 μL/well) in a 96-well clear, V bottom polystyrene cell culture plate at a density of 2500 cells/well. The cells are then incubated for 2−3 h at 37 °C and 5% CO2. Compound dilution plates are prepared in 96-well clear, U-bottom polypropylene plates in 100% DMSO at 10 mM stock concentration in duplicate wells using an 11-point serial dilution (1:3 dilutions). Compounds are further diluted in growth medium, and 25 μL is added to each well of the cell plates. The highest compound concentration tested is 50 μM final, with a 0.5% final DMSO concentration. The plates are then incubated for 72 h at 37 °C and 5% CO2. At the end of the incubation period with compound, the plates are centrifuged at 2000 rpm for 5 min at room
temperature, and the medium is removed. Then 100 μL of acid-extracted solution is added to each well. The plates are shaken for 50 min at 4 °C to lyse the cells, and then 38 μL of neutralization buffer is added. The plates are then frozen at −80 °C. The next day, the plates are shaken at room temperature to thaw. Cell lysates (50 μL/well) are then transferred to ELISA plates. The plates are covered and incubated for 2.5 h at room temperature with constant slow-speed shaking. After incubation, the plates are washed seven times (300 μL/well) with 1× wash buffer. Then 100 μL of biotinylated trimethylhistone H3K27 detection antibody is added to each well and incubated for 2 h at room temperature with constant slow-speed shaking. After incubation, the plates are washed as described above. Horseradish peroxidase-linked antibody (100 μL) is added to each well and incubated for 60 min at room temperature with constant slow-speed shaking. After incubation, the plates are
washed. Finally, 100 μL of TMB substrate reagent is added to each well and incubated for 5 min at room temperature in the dark with slow shaking, after which 100 μL of stop solution is added to stop the reaction.

Animal Study:^{^[1]}

Animal Models
Female Scid beige mice
Dosages
30, 100, 300 mg/kg
Administration
oral

References

[1] Pei-Pei Kung, et al. J Med Chem. 2016, 59 (18): 8306-8325.

Selleck's PF-06726304 has been cited by 2 publications

Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation [ Nat Commun, 2023, 10.1038/s41467-023-42930-y]	PubMed: 37923799
Quantitative control of noise in mammalian gene expression by dynamic histone regulation [ Elife, 2021, 10e65654]	PubMed: 34379055

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