生物活性

PF 4708671 is a membrane permeable p70 ribosomal S6 kinase inhibitor. This compound has been shown to decrease the S6K1-mediated phosphorylation of S6, Rictor and mTOR in response to IGF1.Cell-permeable inhibitor of p70 ribosomal S6 kinase (S6K1 isoform) (Ki = 20 nM; IC50 = 160 nM). Displays no effect on the activity of RSK and MSK in vivo. Exhibits no significant inhibition of S6K2 or other AGC kinases (e.g. Akt, PKA and ROCK) in vitro. 

The activity of AGC kinases[1]

 Impact on LTB4 metabolism in neutrophil suspensions[2]

The IC50 of PF-4708671 in inhibiting mitochondrial Complex I is 5.2 μM.[6]

体外研究

体内研究

30% PEG400+0.5% Tween80+5% Propylene glycol

激酶实验

mTORC1 activity assays[1]

HEK-293 cells were lysed in Hepes lysis buffer and 3mg of lysate was pre-cleared by incubation with 5μl of Protein G-Sepharose conjugated to pre-immune IgG. The lysates were then incubated with 5μl of Protein G-Sepharose covalently conjugated to either5 μg of anti-Raptor antibody, or 5μg of pre-immune IgG for 1.5 h at 4OC on a vibrating platform. The immunoprecipitates were washed four times with Hepes lysis buffer, followed by two washes with Hepes kinase buffer. For maximal mTORC1 activity, the lysis buffer for the initial two wash steps contained 0.5 M NaCl. Kinase reactions were initiated by adding 0.1 mMATP and 10 mM magnesium in the presence or absence of PF-4708671 and inactive GST-S6K1 (0.5μg). Reactions were carried out for 30 min at 30OC on a vibrating platform and stopped by the addition of SDS sample buffer. Reactions were filtered through a0.22μm Spin-X filter and samples subjected to electrophoresis and immunoblot analysis.

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 30OC in a 50μl assay mixture in buffer A containing either 30μM Crosstide (GRPRTSSFAEG, for S6K1, S6K2 and MSK1) or 30μM Long S6 (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, for RSK1 and RSK2), 10 mM magnesium acetate and 100μM [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 1nmol 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 30OC in a 50 μl assay volume. The fluorescence of the peptide was monitored at an excitation wavelength of 360 nm and an emission wave length 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◦C 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.

细胞实验

Cell culture[5]

Mouse embryonic fibroblast (MEF) and green fluorescent protein (GFP)-conjugated LC3 (GFP-LC3) expressing HeLa cells (GFPLC3/HeLa) cells were maintained under 5% CO2at 37oC in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin.

Cell cytotoxicity assay

Cells were seeded at a density of 2x103cells/well in a final volume of 100 mL onto 96 well plates. After 24 h, the cells were treated with PF-4708671 (50mM) or an equal volume of DMSO for 24 h. Cell viability was estimated using a WST-1 cell proliferation assay kit. The live cell number was expressed as the absorbance at 450 nm, which was averaged from triplicate wells after subtracting turbidity measured at 600 nm.

Measurement of ROS

Intracellular ROS production was assessed using 5,6-chloromethyl-20,70-dichlorodihydro- fluorescein diacetate. In brief, cells (3 x105) were plated on 35 mm dishes. After 24 h, the cells were treated with PF-4708671 in phenol red-free media. The cells were then rinsed once with 2 mL of Hank's balanced salt solution and incubated for 5 min with CM-H2DCFDA. The cells were then washed with Hank's balanced salt solution and fluorescence images were obtained with an Axiovert 200 fluorescence microscope. Relative DCF fluorescence was calculated by averaging the levels of fluorescence from 50 to 80 cells after subtracting background fluorescence.

动物实验

Drugs[3]

10 mg of PF–4708671 were fully dissolved in 1 ml DMSO and stored at -80°C for in vitro experiments.

For in vivo assays, PF-4798671 was dissolved in 10% DMSO first and further diluted in 30% PEG400, 0.5% Tween 80 and 5% propylene glycol, to achieve a final DMSO concentration of 1%.

In vivo effects of PF-4708671 in a nude mouse xenograft model established with H460 cells

H460 cells, which showed the highest growth rate, were selected for in vivo experiments.

Female nude mice (4 weeks, 18 to 20g) were housed with a 12h light/12h dark cycle. All experiments were carried out in SPF (Special Pathogen Free) conditions. Mice were divided into three groups randomly, and each group contained three mice (n = 3/each group). The end points of observation were as follows: 1) the diameter of tumors > 3cm; 2) transplanted tumors had necrosis and/or decay; 3) natural death. The method of sacrifice at the end of the vivo research was decapitated after anesthesia under the premise of without other mice could see. In Group 1 (H460 group), 1×107H460 cells were subcutaneously injected into mice. Group 2 (negative control, NC group) animals were injected cells as in Group1; three days later, they were intraperitoneally administered 200μl of solvent mix (1% DMSO, 30% PEG400, 0.5% Tween80 and 5% propylene glycol) daily for 1 week. In Group 3 (H460+PF4708671 group), mice were treated as described for Group 2, with 200μl PF4708671 (50 mg/kg) instead of solvent mix. Tumors sizes were measured daily for more than one week (long diameter = a; short diameter = b), and volumes were derived as follows: V = ab2/2. Tumor inhibition rate = [(control tumor weight—experimental tumor weight)/control tumor weight] x 100%.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Pearce LR, Alton GR, Richter DT, et al. Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1). Biochem J. 2010;431(2):245-255.
[2] Archambault AS, Turcotte C, Martin C, et al. Leukotriene B(4) Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the omega-Oxidation of Leukotriene B(4) In Vitro and In Cellulo. PLoS One. 2017;12(1):e0169804.
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体内溶解度
约23 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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