PF-477736

Name: PF-477736
Brand: Selleck Chemicals
SKU: S2904
Rating: 5 (10 reviews)

For research use only.

Catalog No.S2904 Synonyms: PF-736,PF-00477736

10 publications

CAS No. 952021-60-2

PF-477736 is a selective, potent and ATP-competitive Chk1 inhibitor with K_i of 0.49 nM in a cell-free assay and also inhibits VEGFR2, Aurora-A, FGFR3, Flt3, Fms (CSF1R), Ret and Yes. It shows ~100-fold selectivity for Chk1 than Chk2. Phase 1.

Selleck's PF-477736 has been cited by 10 publications

Cancer Discov, 2017, 7(10):1168-1183

PubMed: 28801307 ( click the link to review the publication )

mBio, 2020, 11(1)

PubMed: 32071277 ( click the link to review the publication )

Cancers (Basel), 2020, 4;12(4)

PubMed: 32260355 ( click the link to review the publication )

Oncogenesis, 2019, 8(7):38

PubMed: 31209198 ( click the link to review the publication )

PLoS One, 2019, 14(11):e0221288

PubMed: 31721781 ( click the link to review the publication )

Nat Commun, 2018, 9(1):2622

PubMed: 29977027 ( click the link to review the publication )

Nat Commun, 2018, 9(1):4514

PubMed: 30375513 ( click the link to review the publication )

Nat Commun, 2017, 8(1):1697

PubMed: 29167438 ( click the link to review the publication )

BMC Cancer, 2015, 15:196

PubMed: 25884494 ( click the link to review the publication )

BMC Cancer, 2014, 14(1):570

PubMed: 25104095 ( click the link to review the publication )

1 Customer Review

Cells were treated at the same concentrations as in Caspase3/7 assay for 16 hours and total cell lysates were prepared for Western blotting. GAPDH was used as a loading control.

BMC Cancer, 2015, 10.1186/s12885-015-1231-z. PF-477736 purchased from Selleck.

Purity & Quality Control

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Biological Activity

Description

Targets

Chk1 ^[1] (Cell-free assay)	VEGFR2 ^[1] (Cell-free assay)	Fms ^[1] (Cell-free assay)	YES ^[1] (Cell-free assay)	Chk2 ^[1] (Cell-free assay)
0.49 nM(Ki)	8 nM(Ki)	10 nM(Ki)	14 nM(Ki)	47 nM(Ki)

In vitro

PF-477736 (128 nM) abrogates the camptothecin-induced DNA damage checkpoint in a dose-dependent manner in CA46 and HeLa cells. PF-477736 effectively abrogates the gemcitabine-induced S-phase arrest with a corresponding increase in apoptotic cell populations in HT29 cells. PF-477736 (540 nM) enhances gemcitabine-induced cytotoxicity in a time- and dose-dependent manner in HT29 cells. PF-477736 potentiates the growth-inhibitory activity of a panel of chemotherapeutic agents across a broad spectrum of p53-deficient human cancer cell lines in the MTT assay. Addition of PF-477736 (360 nM) to gemcitabine-arrested cells induces a dramatic increase in the intensity of H2AX phosphorylation, reflecting a greater number of γ-H2AX molecules near sites of DNA damage. ^[1] PF-477736 (0.5 nM) selectively blocks p73 and P53 phosphorylation in presence of curcumin in HL-60 cells. ^[2] PF-477736 (360 nM) suppresses docetaxel-induced phosphorylation of histone H3 (Ser10) and Cdc25C (Ser216) and potentiates apoptosis in COLO205 cells. ^[3] PF-477736 (250 nM) combined with MK-1775 has marked synergistic cytotoxic activity in OVCAR-5 cells. PF-477736 (250 nM) combined with MK-1775 causes accumulation of cells with a DNA content between 2N and 4N in OVCAR-5 cells. PF-477736 (250 nM) combined with MK-1775 causes premature mitosis before the end of DNA replication, with damaged DNA leading to apoptotic cell death in OVCAR-5 cells. ^[4]

Assay

Methods	Test Index	PMID
Western blot	p-53 / p-p53 / p21 / MDM2 ; PubMed: 31362335 Int J Mol Sci Immunoblot analysis of the indicated proteins in NB-39-nu cells treated for 24 h with 1 μM CHK1i (PF-47736) or DMSO (NT). β-actin was used as a loading control. p-ATM / γ-H2AX ; PubMed: 31362335 Int J Mol Sci Immunoblot analysis of the indicated proteins in NB-39-nu cells after treatment with DMSO (0, NT), 1, 5, or 10 μM CHK1i (PF-477736) for 24 h.	31362335
Growth inhibition assay	Cell viability; PubMed: 29167438 Nat Commun Dose response of Burkitt lymphoma cells lines to Chk1 inhibitor PF-477736. Survival was assessed using AnnexinV-staining and flow cytometry.	29167438
Immunofluorescence	p-p53; PubMed: 31362335 Int J Mol Sci Indirect immunofluorescence of NB-39-nu cells treated for 24 h with 1 μM PF-477736 or DMSO. Cells were stained with polyclonal anti-p-p53-Ser15 (red) and 4′,6-diamidino-2-phenylindole (DAPI, nuclear DNA, blue).	31362335

In vivo

PF-477736 (4 mg/kg i.v.) results in terminal half-life (T1/2) of 2.9 hours, AUC of 5.72 μg×hr/mL and CLp of 11.8 mL/min/kg in rats. PF-477736 dose-dependently enhances the antitumor activity of a maximum tolerated dose of gemcitabine in the Colo205 xenograft mouse model. PF-477736 (12 mg/kg) induces an increase in the phosphorylation of histone H3 (Ser10) and of phospho-histone H2AX in the Colo205 xenograft mouse model. ^[1] PF-477736 (15 mg/kg i.p.) enhances docetaxel induced tumor growth inhibition and tumor growth delay in COLO205 and MDA-MB-231 xenograft models. ^[3] PF 477736 (10 mg/kg once daily i.p.) combined with MK-1775 (30 mg/kg twice a day oral) leads to greater tumor growth inhibition in mice bearing OVCAR-5 xenografts. ^[4]

Protocol

Kinase Assay: ^[1]	- Collapse Binding assay: The assay is performed in a 96-well plate for 20 minutes at 30℃ in 0.1 mL of assay buffer containing 50 mM TRIS pH 7.5, 0.4 M NaCl, 4 mM PEP, 0.15 mM NADH, 28 units of lactate dehydrogenase/mL, 16 units of pyruvate kinase/mL, 3 mM DTT, 0.125 mM Syntide-2, 0.15 mM ATP and 25 mM magnesium chloride. Assays are initiated with 1 nM of CHK1 kinase domain. The inhibition of CHK1 activity is determined by measuring initial velocities in the presence of varying concentrations of PF-477736. The data is analyzed using Enzyme Kinetic and Excel software and fit to a kinetic model for competitive inhibition to obtain a Ki value. The kinase selectivity of PF-477736 is evaluated by screening the compound at 1 μM or 10 μM against a panel 2 of about 100 protein kinases.
Cell Research: ^[1]	- Collapse Cell lines: HT29, Colo205, PC-3, MDA-MB-231 and K562 cells Concentrations: ~1 μM Incubation Time: 96 hours Method: The IC50 assay measures the antiproliferative effects of PF-477736 on p53-defective human cancer cell lines. Cells in each line are seeded in complete medium at an exponentially growing density in 96-well assay plate and allowed to attach for 16 hours. Serial dilutions of PF-477736 are then done, and appropriate controls are added to each plate. Cells are incubated with drug for 96 hours. After incubation, MTT working stock diluted in complete medium is added to each well, and cells are incubated for 4 hours. After centrifugation and supernatant removal, DMSO is added to each well and plates are read on SpectraMax plate reader at 540 nm. (Only for Reference)
Animal Research: ^[1]	- Collapse Animal Models: Colo205 xenograft mouse model Dosages: 40 mg/kg Administration: intravenous injection (Only for Reference)

References

[4] Carrassa L, et al. Cell Cycle, 2012, 11(13), 2507-2517.

- Collapse

Solubility (25°C)

In vitro	DMSO	6 mg/mL (14.3 mM)
	Water	Insoluble
	Ethanol	Insoluble
In vivo	Add solvents to the product individually and in order(Data is from Selleck tests instead of citations): 2% DMSO+40% PEG 300 For best results, use promptly after mixing.	5 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.

Chemical Information Download PF-477736 SDF

Molecular Weight	419.48
Formula	C₂₂H₂₅N₇O₂
CAS No.	952021-60-2
Storage	3 years-20°C powder 2 years-80°C in solvent
Synonyms	PF-736,PF-00477736
Smiles	CN1C=C(C=N1)C2=C3C=NNC(=O)C4=C3C(=CC(=C4)NC(=O)C(C5CCCCC5)N)N2

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

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Step 2: Enter the in vivo formulation (Different batches have different solubility ratios, please contact Selleck to provide you with the correct ratio)

% DMSO+ % + % Tween 80+ % ddH₂O

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Calculation results:

Working concentration： mg/ml；

Method for preparing DMSO master liquid: ： mg drug pre-dissolved in μL DMSO (Master liquid concentration mg/mL， Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation：Take DMSO master liquid, next addμL PEG300， mix and clarify, next addμL Tween 80，mix and clarify, next add μL ddH₂O，mix and clarify.

Note：1.Please make sure the liquid is clear before adding the next solvent.
2.Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.

Bio Calculators

Molarity Calculator

Calculate the mass, volume or concentration required for a solution. The Selleck molarity calculator is based on the following equation:

Mass (mg) = Concentration (mM) × Volume (mL) × Molecular Weight (g/mol)

*When preparing stock solutions, please always use the batch-specific molecular weight of the product found on the via label and MSDS / COA (available on product pages).

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Calculate the dilution required to prepare a stock solution. The Selleck dilution calculator is based on the following equation:

Concentration _(start) x Volume _(start) = Concentration _(final) x Volume _(final)

This equation is commonly abbreviated as: C1V1 = C2V2 ( Input Output )

* When preparing stock solutions always use the batch-specific molecular weight of the product found on the vial label and MSDS / COA (available online).

The Serial Dilution Calculator Equation

Serial Dilutions
Concertration (start):
Dilution-folds:

Computed Result

C1=C0/X	C1:	LOG(C1):
C2=C1/X	C2:	LOG(C2):
C3=C2/X	C3:	LOG(C3):
C4=C3/X	C4:	LOG(C4):
C5=C4/X	C5:	LOG(C5):
C6=C5/X	C6:	LOG(C6):
C7=C6/X	C7:	LOG(C7):
C8=C7/X	C8:	LOG(C8):

Molecular Weight Calculator

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Tip: Chemical formula is case sensitive. C10H16N2O2 c10h16n2o2

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Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

Molarity Calculator

Clinical Trial Information (data from https://clinicaltrials.gov, updated on 2020-09-01)

NCT Number	Recruitment	interventions	Conditions	Sponsor/Collaborators	Start Date	Phases
NCT00437203	Terminated	Drug: PF-00477736\|Drug: gemcitabine	Neoplasms	Pfizer	December 2006	Phase 1

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

Tel: +1-832-582-8158 Ext:3

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Frequently Asked Questions

Question 1:
Can you advise the vehicle suggested for S2904: 2% DMSO/40% PEG 300 at 5mg/ml, is it a suspension or clear solution? I wanted to know how to dissolve this compound for an i.p. injection in mice
Answer:
S2904 can dissolve in 2% DMSO/40% PEG 300 at 5mg/ml as a clear solution. If you are going to use this kind of vehicle, please dissolve the compound in DMSO clearly first, then add PEG300, then water.

Chk Signaling Pathway Map

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PF-477736