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) were 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 were terminated and the incorporation of [γ -32P]phosphate into the peptide substrate was 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 was 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 was added to a final concentration of 5 nM to Omnia assay buffer containing various concentrations of compound. The reaction was run for 60 min at 30℃ in a 50 μl assay volume. The fluorescence of the peptide was monitored at an excitation wavelength of 360 nm and an emissionwavelength of 485 nm. The rate of the reaction at each compound concentration was normalized to the DMSO control rate, and this normalized rate against concentration was 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 were lysed in Tris lysis buffer. Lysate (0.5 mg) was incubated with 5 μg of S6K antibody conjugated to Protein G–Sepharose for 1 h at 4℃ on a vibrating platform. Immunoprecipitates were washed twice with lysis buffer and twice with buffer A, and kinase activity was assayed exactly as described above using the Crosstide peptide.

Cell lines

HEK-293 cells, A549, SK-MES-1 and NCI-H460 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

0.1-10 μM for 16-72 h

Applications

PF-4708671 inhibited S6K1 activity and induced S6K1 phosphorylation in HEK-293 cells [1]. Moreover, PF-4708671 significantly inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, resulting in cell cycle arrest in G0-G1 phase [2].

Animal models

Nude mouse xenograft model

Dosage form

50 mg/kg; intraperitoneal administration, daily for 1 week;

Applications

PF-4708671 inhibited tumor growth in a nude mouse xenograft model established with H460 cells in vivo.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

1. Pearce, L. R., Alton, G. R., Richter, D. T., Kath, J. C., Lingardo, L., Chapman, J., Hwang, C. and Alessi, D. R. (2010) Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1). Biochem J. 431, 245-255

2. Qiu, Z. X., Sun, R. F., Mo, X. M. and Li, W. M. (2016) The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth. PLoS One. 11, e0147185