生物活性

GSK 2334470 is a potent 3-phosphoinositide-dependent protein kinase (PDPK1) inhibitor (IC50 ~ 10 nM). This compound exhibits no effect on a variety of other kinases, including Aurora, ROCK, p38 MAPK and PI3K. Suppresses T-loop phosphorylation and subsequent activation of PDK1 substrates S6K1, SGK and RSK in vitro; exhibits limited inhibitory effect on Akt activation. The selectivity of GSK 2334470 allows for determination of the role of PDK1 in various cell lines, including cancerous cell lines. This compound also has a limited inhibitory effect on Akt activation.

体外研究

体内研究

激酶实验

Specificity kinase panel[1]

All assays were carried out robotically atroom temperature (21^oC), and were linear with respect to time andenzyme concentration under the conditions used. Assays were performed for 30min using Multidrop Micro reagent dispensers in a 96-well format. Theconcentration of magnesium acetate in the assays was 10 mM and [γ -³²P]ATP (∼800 c.p.m./pmol)was used at 5 μM for Aurora A, CK2α, DYRK3, EF2K, ERK1, ERK8, GSK3β, HER4, HIPK2,IGF1R, IKKβ, IRR, MARK3, MKK1, p38γ MAPK, p38δ MAPK, PAK4, PIM2, Akt1 (S473D),PLK1, PKCζ and PRK; 20 μM for Aurora B, BRSK1, CaMKKβ, CDK2-cyclinA2, CHK1,CHK2, CK1δ, CSK, EPH-B3, ERK2, FGFR1, GCK, HIPK1, HIPK3, IR, IRAK4, JNK1α1,JNK2α2, JNK3α1, LKB1, MAPKAP-K2, MAPKAP-K3, MARK2, MLK1, MLK3, MSK1, MST2,MST4, NUAK1, p38βMAPK, PAK2 (T402E), PAK5, PAK6, PDK1, PIM1, PIM3, PKA, PKCα,PKCγ , PRAK, RIPK2, ROCKII, S6K1 (T412E), SGK1 (S422D), SYK, TTK and YES1; and50 μM forBRSK2, BTK, CaMK1,DYRK1a, DYRK2, EPHA2, IKKε, LCK, MARK4, MELK, MINK1,MNK1, MNK2α, NEK2A, NEK6, p38αMAPK, Akt2 (S474D), PKD1, RSK1, RSK2, Src, SRPK1and TBK1, in order to be at or below the Km for ATP for each enzyme.

Kinaseactivity assays

Endogenous Akt, S6K and RSK wereimmunoprecipitated from 0.1 to 1 mg of cell lysate for 2 h at 4 ^oCon a vibrating platform using 3–5 μg of the indicated antibodies. For the SGKactivity assays, 150 μg of transfected lysate was incubated with 5 μg ofglutathione–Sepharose for 3 h at 4^oC. The immunoprecipitates werewashed twice with lysis buffer containing 0.5 mM NaCl, followed by two washeswith kinase buffer. Kinase reactions were initiated by a reaction mixture tobring the final concentrations of the reaction components to 0.1 mM [γ -³²P]ATP(∼200 c.p.m./pmol), 5 mM magnesium acetate,0.1% 2-mercaptoethanol and 30 mM Crosstide peptide (GRPRTSSFAEGKK). Reactions werecarried out for 20 min at 30^oC on a vibrating platform and stoppedby spotting the reactions on to P81 phosphocellulose paper. Cerenkov countingwas done after washing the papers in phosphoric acid, rinsing in acetone andair-drying. One unit of activity was defined as that which catalysed the incorporationof 1 nmol of [32P]phosphate into the substrate over 1 h.

细胞实验

Cell culture[2]

MDA-MB-231 and HEK293 were cultured inDMEM; A375M and PC3 were cultured in RPMI 1640. Media were supplemented with10% (v/v) foetal bovine serum (Invitrogen), sodium pyruvate1% (v/v), 1xL-glutamine/penicillin/streptomycin (Invitrogen) and 0.1% gentamycin (v/v).

Invasionassay

MDA-MB-231 and A375M were serum-starvedovernight before the experiment. The following day Matrigel pre-coatedTranswell inserts (8.0μm pores, 10 mm diameter) were rehydrated. Where indicated, cellswere pretreated with 1μM GSK22334470 for 30 minutes and then allowed to invade in presenceof the inhibitor. After 36 hours the upper face of the porous membrane wascleaned with a cotton bud and cells that invaded the Matrigel were fixed withparaformaldehyde and stained with 0.1% Crystal Violet solution for 10 minutes.A Leica phase-contrast light microscope using a 10 x magnification objectivewas used for manual counting. A minimum of five fields was counted per insert.Each invasion experiment was performed in duplicate and the average of invadingcells/fields was calculated.

动物实验

Animal studies[3]

RPMI8226 cells (5×10⁶ cells/μlper site) in their logarithmic growth phase were implanted subcutaneously intothe right flank of 3-4 weeks-old female severe combined immunodeficient (SCID)mice. Tumor volume was measured and calculated. When tumors reached a volume of80-100 mm³, mice were randomly assigned to one of the treatmentgroups: 5 days of GSK2334470 (GSK-470) (40 mg/ kg/d), 5 days of PP242 (20mg/kg/d), or 5 days of GSK-470 combined with PP242 as an intraperitonealinjection once every day. The untreated control group received DMSO. After 5days of treatment, one mouse of each groups were sacrificed, and tumors wereharvested for immunohistochemistry.

Immunohistochemistry

Tumors were fixed in 4% paraformaldehyde, embeddedin paraffin, and then cut in 4-mm sections. Detection of p-mTOR (Ser2448) andp-mTOR (Ser2481) in primary tumor samples was performed using the correspondingspecific antibodies. Apoptotic cells in tumor samples were assessed by TUNEL stainingwith an In Situ Cell Death Detection kit according to the manufacturer’sprotocol. All tissue sections were counterstained with hematoxylin.

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] Najafov A, Sommer EM, Axten JM, Deyoung MP, Alessi DR. Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem J. 2011;433(2):357-369.
[2] Raimondi C, Chikh A, Wheeler AP, Maffucci T, Falasca M. Correction: A novel regulatory mechanism links PLCgamma1 to PDK1. J Cell Sci. 2017;130(5):1016.
[3] Yang C HX, Liu H, Xiao F, Wei J, You L, Qian W. PDK1 inhibitor GSK2334470 exerts antitumor activity in multiple myeloma and forms a novel multitargeted combination with dual mTORC1/C2 inhibitor PP242. Oncotarget. 2017;8(24):39185-39197.

体内溶解度

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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