PF-4708671 Licensed and Manufactured by Pfizer

Catalog No.S2163

PF-4708671 is a cell-permeable inhibitor of p70 ribosomal S6 kinase (S6K1 isoform) with Ki/IC50 of 20 nM/160 nM in cell-free assays, 400-fold greater selectivity for S6K1 than S6K2, and 4- and >20-fold selectivity for S6K1 than MSK1 and RSK1/2, respectively. First S6K1-specific inhibitor to be reported.

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PF-4708671 Chemical Structure

PF-4708671 Chemical Structure
Molecular Weight: 390.41

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Related Compound Libraries

PF-4708671 is available in the following compound libraries:

Product Information

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Product Description

Biological Activity

Description PF-4708671 is a cell-permeable inhibitor of p70 ribosomal S6 kinase (S6K1 isoform) with Ki/IC50 of 20 nM/160 nM in cell-free assays, 400-fold greater selectivity for S6K1 than S6K2, and 4- and >20-fold selectivity for S6K1 than MSK1 and RSK1/2, respectively. First S6K1-specific inhibitor to be reported.
Targets p70 S6K1 [1]
(Cell-free assay)
IC50 160 nM
In vitro PF-4708671 is a piperazinyl-pyrimidine analogue and the first S6K1-specific inhibitor. PF-4708671 does not significantly inhibit the activity of the closely related S6K2 isoform or a number of other AGC kinases (Akt1, Akt2, PKA, PKCα, PKCϵ, PRK2, ROCK2, RSK1, RSK2 or SGK1) . PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 is also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 does not affect activity of mTORC1. [1]
Cell Data
Cell LinesAssay TypeConcentrationIncubation TimeFormulationActivity DescriptionPMID
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MC-IXCM3vxOWdzd3e2aDDJcohq[mm2aX;uJGF{e2G7M2nYVGlEPTB;MUCwMlYyPSEQvF2=NF;4VpFUSU6JRWK=

... Click to View More Cell Line Experimental Data

In vivo
Features

Protocol(Only for Reference)

Kinase Assay: [1]

Protein kinase activity assays For selectivity IC50 assays, purified active GST–S6K1, GST–S6K2, His–MSK1 (residues 2–802), His–RSK1 (residues 1–735) and His–RSK2 (residues 2–740) (0.5 units/mL) are assayed for 30 min at 30 °C in a 50 μL assay mixture in buffer A containing either 30 μM Crosstide (GRPRTSSFAEG, for S6K1, S6K2 and MSK1) or 30 μM Long S6 (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, for RSK1 and RSK2), 10 mM magnesium acetate and 100 μM [γ- 32P]ATP. Reactions are terminated and the incorporation of [γ-32P]phosphate into the peptide substrate is determined by applying the reaction mixture on to P81 phosphocellulose paper and scintillation counting after washing the papers in phosphoric acid. One unit of activity is defined as that which catalysed the incorporation of 1 nmol of [32P]phosphate into the substrate. To determine the Ki for PF-4708671, full-length recombinant S6K1 is added to a final concentration of 5 nM to Omnia assay buffer containing various concentrations of compound. The reaction is run for 60 min at 30 °C in a 50 μL assay volume. The fluorescence of the peptide is monitored at an excitation wavelength of 360 nm and an emission wavelength of 485 nm. The rate of the reaction at each compound concentration is normalized to the DMSO control rate, and this normalized rate against concentration is fitted to the Morrison tight-binding equation for a competitive inhibitor to provide the true Ki. In order to assay S6K activity in HEK-293 cell lysates, cells are lysed in Tris lysis buffer. Lysate (0.5 mg) is incubated with 5 μg of S6K antibody conjugated to Protein G–Sepharose for 1 h at 4 °C on a vibrating platform. Immunoprecipitates are washed twice with lysis buffer and twice with buffer A, and kinase activity is assayed exactly as described above using the Crosstide peptide.

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

SpeciesMouseRatRabbitGuinea pigHamsterDog
Weight (kg)0.020.151.80.40.0810
Body Surface Area (m2)0.0070.0250.150.050.020.5
Km factor36128520
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)
1

References

[1] Pearce LR, et al. Biochem J, 2010, 431(2), 245-255.

Chemical Information

Download PF-4708671 SDF
Molecular Weight (MW) 390.41
Formula

C19H21F3N6

CAS No. 1255517-76-0
Storage 3 years -20℃powder
6 months-80℃in solvent
Synonyms N/A
Solubility (25°C) * In vitro DMSO 30 mg/mL (76.84 mM)
Ethanol 8 mg/mL (20.49 mM)
Water <1 mg/mL (<1 mM)
In vivo 30% PEG400+0.5% Tween80+5% propylene glycol 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 1H-Benzimidazole, 2-[[4-(5-ethyl-4-pyrimidinyl)-1-piperazinyl]methyl]-6-(trifluoromethyl)-

Customer Product Validation(2)


Click to enlarge
Rating
Source Mol Cancer Ther, 2015, 14(3): 799-809. PF-4708671 purchased from Selleck
Method Apoptosis & Proliferation Assays
Cell Lines Nude mice tumor tissue
Concentrations 60 mg/kg
Incubation Time 15 d
Results The tumor tissues from OSI-906+PF-4708671–treated mice showed the most TUNEL-positive staining and the least Ki-67–positive staining.

Click to enlarge
Rating
Source PLoS One 2014 9(2), e90388. PF-4708671 purchased from Selleck
Method Western blot
Cell Lines QBC939, RBE, HCCC-9810 cells
Concentrations 10 uM
Incubation Time 48 h
Results It is notable that p70S6K inhibitor PF-4708671, which had no demonstrable effects on ATF4 and GRP78 accumulation, enhanced the decreasing of ATF4 and GRP78 in 4EGI-1-treated QBC939, RBE and HCCC-9810 cells.

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.

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
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