Molecular Weight (MW)

462.59

Formula

C25H34N8O

CAS No.

1227911-45-6

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 90 mg/mL (194.6 mM)

Water: <1 mg/mL

Ethanol: 90 mg/mL (194.6 mM)

SMILES

O=C([C@@H]1CN(C2=NC(NC)=NC(C3=CC4=C(C=C3)C(N)=NN4)=C2)[C@H](C)CC1)NC5CCCCC5

Synonyms

GSK 2334470; GSK2334470; GSK-2334470

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

InChi Key: QLPHOXTXAKOFMU-WBVHZDCISA-N

InChi Code: 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

In Vitro

In vitro activity: 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. GSK2334470 also similarly inhibits PDK1 from phosphorylating the PDKtide peptide substrate with IC50 of ~10 nM. GSK2334470 (0.1 μM–0.3 μM) induces significant dose-dependent inhibition of endogenous NDRG1 with over 50% reduction in phosphorylation in HEK-293 cells. GSK2334470 (30 nM) induces a significant dose-dependent inhibition of the T-loop phosphorylation of each SGK isoform in HEK-293 cells. GSK2334470 (1 μM) inhibits hydrophobic motif phosphorylation of S6K1 to a similar extent as T-loop phosphorylation in HEK-293 cells. GSK2334470 (3 μM) also suppresses S6K1 activity and phosphorylation induced by IGF1 stimulation of serum-starved HEK-293 cells. GSK2334470 (3 μM) markedly inhibits the phosphorylation of several Akt substrates [FoxO (forkhead box O), GSK3 and PRAS40]. GSK2334470 (3 μM) also induces near maximal inhibition of Akt1 activity and phosphorylation within 5 min, and Akt substrate phosphorylation (FoxO, GSK3 and PRAS40) is inhibited at a slightly later time point (10 min). GSK2334470 (0.3 μM) significantly inhibits phosphorylation of Akt or PRAS40/GSK3 in PDK1K465E/K465E knock-in but not wild-type ES cells. GSK2334470 (1 μM) effectively suppresses SGK1 activity as judged by the inhibition of NDRG1 phosphorylation in U87 glioblastoma cells. GSK2334470 (1 μM) also potently suppresses activation of S6K1 (Figure 7B) as well as SGK1 in MEF (mouse embryonic fibroblast) cells. GSK2334470 (0.1 μM) induces ~50% inhibition of RSK2 activity in HEK-293 cells. 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+]. 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.

Kinase Assay: GSK2334470 is a highly specific and potent inhibitor of PDK1 with an IC50 of 10 nM.

Cell Assay: GSK2334470 is dissolved in DMSO and diluted with appropriate medium before use. To study the inhibitory effect of GSK2334470 on mTOR-S6K pathway, non-resistant cells and the resistant sublines are treated with GSK2334470 at 5 μM for 1.5 and 12 h in 10 % FBS medium with/without MK-2206 (5 μM).

In Vivo

The efficacy of the PDK1 inhibitor (PDKi) GSK2334470 is tested in newborn BrafV600E::Pten–/–mice subjected to systemic administration of 4-HT. Twice weekly administration of PDK1 results in marked inhibition of pigmented lesions and concomitant melanomagenesis, as well as significant inhibition of lung metastases, seen by H&E staining-based quantification (~80%), and lymph node metastases as by S100 immunostaining, similar to the phenotype seen upon genetic ablation of Pdk1.

Animal model

Mice

Formulation & Dosage

Mice is dissolved in DMSO and then diluted with PBS or saline. BrafV600E::Pten–/– are generated as previously described. Cohorts of six animals per group are used in each experimental group. GSK2334470 is administered through IP injection (100 mg/kg) 3 times per week starting the same day of topical administration of 4-hydroxytamoxifen and ending at the time of mouse collection, based on earlier studies

References

Biochem J. 2011 Jan 15;433(2):357-69; J Cell Sci. 2012 Jul 1;125(Pt 13):3153-63; PLoS One. 2013;8(3):e58703; Oncogene. 2014 Aug 21;33(34):4330-9.