[Description]:
BX-912 is a potent PDK1 inhibitors with an IC50 of 12 nM.
[Related Catalog]:
[Target]
IC50: 12 nM (PDK1), 110 nM (PKA), 410 nM (KDR), 650 nM (CDK2/cyclin E), 830 nM (Chck1), 850 nM (c-Kit), 1.25 μM (PKC), 2.1 μM (T-Fyn), 6.1 μM (Insulin receptor), 7.4 μM (GSK3β)[1]
[In Vitro]
BX-912 also inhibits PKA, KDR, CDK2/cyclin E, Chck1, c-Kit, PKC, T-Fyn, Insulin receptor, and GSK3β with IC50s of 110 nM, 410 nM, 650 nM, 830 nM, 850 nM, 1.25 μM, 2.1 μM, 6.1 μM and 7.4 μM in kinase assays, respectively. BX-912 is identified in a coupled assay measuring PDK1- and PtdIns-3,4-P2-mediated Akt activation, which can detect inhibitors of PDK1, AKT2, or other steps critical for activation of AKT2. BX-912 potently inhibits PDK1 enzyme activity in a direct kinase assay format (IC50=26), although BX-912 fails to block preactivated AKT2 activity (IC50>10 μM). BX-912 binds to the ATP binding site of PDK1. The aminopyrimidine backbone of BX-912 adopts a similar orientation in the active site of PDK1. To examine the kinase selectivity of BX-912, its effects on the in vitro activity of 10 different Ser/Thr and tyrosine kinases are determined, including the related AGC kinases PKA and PKCα. BX-912 is 9-fold selective for PDK1 relative to PKA. BX-912 blocks PDK1 activity in PTEN-negative PC-3 cells. PTEN-negative PC-3 cells display constitutive activation of Akt which is reflected in high levels of the PDK1 product, phospho-Thr308-Akt[1].
[Kinase Assay]
PDK1 is assayed in a direct kinase assay and a coupled assay format measuring PDK1 and PtdIns-3,4-P2 mediated activation of AKT2. For the coupled assay, the final assay mixture (60 μL) contains: 15 mM MOPS, pH 7.2, 1 mg/mL bovine serum albumin, 18 mM β-glycerol phosphate, 0.7 mM dithiothreitol, 3 mM EGTA, 10 mM MgOAc, 7.5 μM ATP, 0.2 μCi of [γ-33P]ATP, 7.5 μM biotinylated peptide substrate (biotin-ARRRDGGGAQPFRPRAATF), 0.5 μL of PtdIns-3,4-P2-containing phospholipid vesicles, 60 pg of purified recombinant human PDK1, and 172 ng of purified recombinant human AKT2. After incubation for 2 h at room temperature, the biotin-labeled peptide is captured from 10 μL of the assay mixture on Streptavidin-coated SPA beads, and product formation is measured by scintillation proximity in a Wallac MicroBeta counter. The product formed is proportional to the time of incubation and to the amount of PDK1 and inactive AKT2 added. PDK1 is added at suboptimal levels so that the assay can sensitively detect inhibitors of AKT2 activation as well as direct inhibitors of PDK1 or AKT2[1].
[Cell Assay]
The cell lines MDA-468, MDA-453, HCT-116, U87-MG, U2OS, PC-3, B16F10, and MiaPaCa; LOX amelanotic human melanoma cells; and HeLa cells seeded at a low density (1,500-3,000 cells/well, 0.1 mL/well, 96-well plates) are incubated overnight. Compound treatments are made by adding 10 μL/well of BX-912 (1, 10, 100 and 1000 nM) in 1% DMSO and growth medium (final concentration of DMSO, 0.1%), followed by brief shaking. Treated cells are incubated for 72 h, and viability is measured by the addition of 10 μL of the metabolic dye WST-1. The WST-1 signal is read in a plate reader at 450 nm, and a no cell, or zero time cell, background is subtracted to calculate the net signal[1].
[References]
[Related Small Molecules]