BX-912 is a specific inhibitor of 3-Phosphoinositide-dependent Kinase-1 (PDK1, IC50: 12 nM). The selectivity of BX-912 is more 10 fold than C-Kit, EGFR, PKA, PKC etc.

PDK1:12 nM

BX-912对ChcK1（IC50：0.83 μM）,PKA（IC50：0.11 μM）,c-kit（IC50：0.85 μM）和KDR（IC50：0.41 μM）有抑制作用。在肿瘤细胞中,BX-912可阻断PDK1/Akt信号,能抑制多种肿瘤细胞的贴壁依赖型生长(比如PC-3细胞)或者诱导凋亡。许多Akt活性升高的癌细胞株在软琼脂中对BX-912引起的生长抑制的敏感性比在组织培养塑料中高30倍,与PDK1/Akt信号通路的细胞生存功能一致,这对未贴壁细胞尤为重要。BX-912对PDK1酶活有显著的抑制效果, 但不影响AKT2活性（IC50>10 μM）,故BX-912是PDK1的直接抑制剂。BX-912使含4 NDNA的MDA-468细胞数显著增多,并使细胞停在G2/M期。BX-912有效抑制细胞生长（IC50：0.32 μM）。BX-912（1 μM）对HCT-116细胞生长的抑制也十分有效（抑制率达96%）。

Kinase assays: 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) contained: 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 hours 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 could sensitively detect inhibitors of AKT2 activation as well as direct inhibitor BX912 of PDK1 or AKT2. To measure PDK1 activity directly, the final assay mixture (60 μL) contained 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1 mM EDTA, 0.1% β-mercaptoethanol, 1 mg/mL bovine serum albumin, 10 mM MgOAc, 10 μM ATP, 0.2 μCi of [γ-33P]ATP, 7.5 μM substrate peptide (H2N-ARRRGVTTKTFCGT), and 60 ng of purified recombinant human PDK1. After 4 hours at room temperature, 25 mM EDTA is added and a portion of the reaction mixture on P81 phosphocellulose paper is spotted. The filter paper is washed three times with 0.75% phosphoric acid and once with acetone. After drying, the filter-bound labeled peptide is quantified using a phosphorimager.

Cells such as MDA-468, MDA-453 are seeded at a low density (1.5-3 × 103 cells/well, 0.1 mL/well, 96-well plates) and are incubated overnight. BX912 treatments are made by adding 10 μL/well of the compound in 1% dimethyl sulfoxide and growth medium (final concentration of dimethyl sulfoxide, 0.1%), followed by brief shaking. Treated cells are incubated for 72 hours, 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. (Only for Reference)