PDK1 (IC50 = 10 nM)
PDK1 (3-phosphoinositide-dependent protein kinase 1) with IC50 of ~10 nM [1].
PDK1 [3]

GSK2334470 inhibits PDK1 from activating full-length Akt1 in the presence of PtdIns(3,4,5)P3-containing lipid vesicles or a mutant of Akt1 lacking the PH domain (ΔPH-Akt1) with IC50 of ~10 nM. With an IC50 of less than 10 nM, GSK2334470 similarly prevents PDK1 from phosphorylating the PDKtide peptide substrate. In HEK-293 cells, GSK2334470 (0.1 M-0.3 M) causes a significant dose-dependent inhibition of endogenous NDRG1 with a phosphorylation reduction of over 50%. In HEK-293 cells, GSK2334470 (30 nM) causes a significant, dose-dependent inhibition of each SGK isoform's T-loop phosphorylation. GSK2334470 (30 µM) suppresses U46619 induced Ca2+-sensitized force in α-toxin permeabilized rabbit pulmonary artery SM. GSK2334470 (30 µM) results in a significant decrease in the contractile force in response to [Ca2+]. [2]GSK2334470 (1 μM) results in total abrogation of the EGF-induced intracellular calcium increase and inositol phosphates accumulation in MDA-MB-231 cells. GSK2334470 (1 μM) inhibits PLCγ1 Tyr783 phosphorylation in MDA-MB-231 cells. [3]

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In MK2206-resistant neuroblastoma (NB) sublines (LAN-1-MK, KP-N-SIFA-MK, SK-N-DZ-MK), GSK2334470 attenuated cell growth in a dose-dependent manner, alone or combined with MK2206 (5 μM). IC50 values in resistant sublines ranged from 1.64 μM (SK-N-DZ-MK) to 5.1 μM (KP-N-SIFA-MK), which were lower than in non-resistant cells (IC50 from 2.90 μM (SK-N-DZ) to 17.4 μM (KP-N-SIFA)). [1]
GSK2334470 (5 μM) induced G0-G1 accumulation in LAN-1 cells (from 39.33% to 54.52%) and in LAN-1-MK cells (from 36.10% to 44.75% without MK2206, and from 40.91% to 48.51% with MK2206). [1]
GSK2334470 (5 μM for 1.5 and 12 h) decreased phosphorylation of PDK1, mTOR, and S6K (p-PDK1, p-mTOR, p-S6K) in both MK2206 non-resistant and resistant NB cells. MK2206-resistant sublines had higher levels of p-PDK1 than non-resistant cells. GSK2334470 suppressed p-mTOR and p-S6K in all tested cell lines. [1]
In LAN-1-MK and NB-19-MK, pS9-GSK3β was suppressed by GSK2334470, indicating compensation of PDK1 in absence of AKT activity. [1]
In MDA-MB-231 breast cancer cells, treatment with the PDK1 inhibitor GSK2334470 (1 μM) resulted in total abrogation of EGF-induced intracellular calcium increase and inositol phosphates accumulation. [3]
In MDA-MB-231 cells, GSK2334470 (1 μM) inhibited EGF-induced PLCγ1 Tyr783 phosphorylation. [3]
In PC3 prostate cancer cells, GSK2334470 (1 μM) strongly inhibited EGF-induced but not ATP-mediated calcium increase. [3]
In MDA-MB-231 and A375M cells, treatment with GSK2334470 (1 μM) significantly inhibited cell invasion on Matrigel. [3]

GSK2334470 is a highly specific and potent PDK1 inhibitor.

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Endogenous Akt, S6K, and RSK are immunoprecipitated using 3 g to 5 g of the designated antibodies from 0.1 mg to 1 mg of cell lysate for two hours at 4 °C on a vibrating platform. In order to measure the SGK activity, 150 g of transfected lysate is incubated with 5 g of glutathione-Sepharose at 4 °C for 3 hours. The immunoprecipitates are then washed twice with kinase buffer and twice with lysis buffer containing 0.5 mM NaCl. A reaction mixture is used to start kinase reactions, resulting in final concentrations of 0.1 mM [-32P]ATP (200 c.p.m./pmol), 5 mM magnesium acetate, 0.1% 2-mercaptoethanol, and 30 mM Crosstide peptide (GRPRTSSFAEGKK).

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Not explicitly described for GSK2334470 in the provided literature. [1][3]

GSK2334470 is dissolved in DMSO and diluted with appropriate medium before use. GSK2334470 is administered to non-resistant cells and resistant sublines for 1.5 and 12 hours in 10% FBS medium with/without MK-2206 (5 M) to investigate its inhibitory effect on the mTOR-S6K pathway.

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Cell proliferation was evaluated using MTT assay. Cells (5×10^3) were seeded in 96-well plates, pre-incubated for 6 h, then treated with GSK2334470 at indicated concentrations for 72 h. MTT (20 μl, 5 mg/ml) was added for 3.5 h, media removed, DMSO (150 μl) added, and absorbance measured at 590 nm. [1]
Cell cycle analysis was performed after treatment with/without GSK2334470 for 12 h. Cells (2×10^6) were fixed in ethanol overnight at -20°C, incubated with propidium iodide (PI) Triton X-100 solution containing RNase A for 30 min at room temperature in darkness, then DNA content analyzed by FACScan flow cytometer using ModFitLT software. [1]
For Western blotting, cells were serum-starved for 1 h, then incubated with/without GSK2334470 (5 μM) for 1.5 and 12 h. Cytoplasmic extracts (40 μg/lane) were run on SDS-PAGE, transferred to PVDF membrane, and probed with specific antibodies against p-PDK1, p-AKT, p-mTOR, p-S6K, GSK3β, p-GSK3β, N-MYC, and Actin. [1]
For intracellular calcium measurement, cells were serum-starved overnight, loaded with Fluo-4-AM (4 μM) for 45 min at 37°C, then treated with GSK2334470 (1 μM) for 30 min during de-esterification. Cells were stimulated with EGF (20 ng/ml), and fluorescence was measured for 6 minutes using a confocal microscope. [3]
Inositol phosphates accumulation was measured in cells labeled with [3H] myo-inositol for 24 h. Cells were pre-incubated with GSK2334470 (1 μM) for 30 min, then stimulated with EGF (50 ng/ml). Total inositol phosphates were separated by anion exchange chromatography. [3]
Invasion assay was performed using Matrigel pre-coated Transwell inserts (8.0 μm pores). Cells were pretreated with GSK2334470 (1 μM) for 30 min and then allowed to invade for 36 h in the presence of the inhibitor. Invaded cells were fixed, stained with Crystal Violet, and counted. [3]

In order to dilute the mice, DMSO is first dissolved in PBS or saline. As previously mentioned, BrafV600E::Pten are generated. Each experimental group utilizes cohorts of six animals. Based on previous studies[3], GSK2334470 is administered by IP injection (100 mg/kg) three times per week beginning on the day that 4-hydroxytamoxifen is topically applied and ending at the time of mouse collection.

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[1]. Biochem J. 2011 Jan 15;433(2):357-69.

[2]. Cancer Cell Int. 2015 Sep 29;15:91.

[3]. J Cell Sci. 2012 Jul 1;125(Pt 13):3153-63.

(3S,6R)-1-[6-(3-amino-1H-indazole-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide is a member of the indazole class of compounds.

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GSK2334470 is a PDK1 inhibitor. MK2206-resistant neuroblastoma cells showed lower IC50 of GSK2334470 compared to non-resistant cells, indicating reliance on PDK1-mTOR-S6K pathway. [1]
PDK1 regulates PLCγ1 activation, and GSK2334470 inhibits EGF-induced PLCγ1 tyrosine phosphorylation and calcium signaling. The PDK1-PLCγ1 pathway is important for cancer cell invasion. [3]

C25H34N8O

462.5905

462.285

C, 64.91; H, 7.41; N, 24.22; O, 3.46

1227911-45-6

46215815

white solid powder

1.3±0.1 g/cm3

1.667

2.31

4

7

5

34

688

2

O=C([C@]1([H])C([H])([H])N(C2C([H])=C(C3C([H])=C([H])C4C(N([H])[H])=NN([H])C=4C=3[H])N=C(N([H])C([H])([H])[H])N=2)[C@]([H])(C([H])([H])[H])C([H])([H])C1([H])[H])N([H])C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

QLPHOXTXAKOFMU-WBVHZDCISA-N

InChI=1S/C25H34N8O/c1-15-8-9-17(24(34)28-18-6-4-3-5-7-18)14-33(15)22-13-20(29-25(27-2)30-22)16-10-11-19-21(12-16)31-32-23(19)26/h10-13,15,17-18H,3-9,14H2,1-2H3,(H,28,34)(H3,26,31,32)(H,27,29,30)/t15-,17+/m1/s1

(3S,6R)-1-(6-(3-amino-1H-indazol-6-yl)-2-(methylamino)pyrimidin-4-yl)-N-cyclohexyl-6-methylpiperidine-3-carboxamide

GSK 2334470; GSK2334470; GSK-2334470

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~90 mg/mL (194.6 mM)
Water: <1 mg/mL
Ethanol: 90 mg/mL (194.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.40 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.40 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)