BX795 is an effective and selective PDK1 inhibitor (IC50: 6 nM), and its selectivity is 140- and 1600-fold for PDK1 over PKA and PKC in cell-free assays, respectively. Meanwhile, the selectivity for PDK1 is 100-fold than GSK3β.

PDK1:6 nM

BX795（1 μM）对以下酪氨酸蛋白激酶无抑制作用: 肝配蛋白受体A2和B3,Bruton's酪氨酸激酶,Syk和FGFR1。但BX795对抑制血管内皮生长因子受体的抑制效果低于TBK1。BX795对IRF3 Ser396位点磷酸化（TBK1-催化）的抑制作用随着ATP浓度上升而下降,意味着BX795是ATP竞争性抑制剂。BX795也抑制NUAK1,MARK1/2/4,VEGFR和MLK1/2/3,IC50分别为5,55,53,19,157,50,46和42 nM。BX795对IRF3依赖的基因转录具有抑制作用。BX795对巨噬细胞的IFN-β分泌也有抑制作用。BX795阻断IKKε和TBK1调节的IRF3激活,并对IFN-β的产量有抑制效果。BX795对TBK1/IKKε的抑制不会引起p38α MAPK和JNK1/2的激活。BX795可抑制聚(I:C)处理后IRF3在核中的积累。BX795不影响脂多糖刺激的p70核糖体S6激酶1Thr229位点的磷酸化,该位点是PDK1的作用靶点。BX795不影响NFκB依赖的基因转录激活（由IKKα/β复合体或脂多糖、聚(I:C)、IL-1α或TNFα促进）。BX795对IL-1α或TNFα刺激的MEFs有效,并阻断p38α MAPKJ和NK1/2磷酸化。

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 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 could sensitively detect inhibitors of AKT2 activation as well as direct inhibitors 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 h at room temperature, we add 25 mM EDTA and spotted a portion of the reaction mixture on Whatman P81 phosphocellulose paper. 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 Fuji phosphorimager.

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 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. Results are reported as the average ± S.E. of two or more replicates.(Only for Reference)