BX-912 is a direct, selective, and ATP-competitive PDK1 inhibitor (IC₅₀=26 nM). BX-912 blocks PDK1/Akt signaling in tumor cells and inhibits the anchorage-dependent growth of a variety of tumor cell lines in culture or induces apoptosis^[1].

IC50: 26 nM (PDK1)^[1]

BX-912 promotes a block at the G2/M phase of the cell cycle in MDA-468 cells^[1].
BX-912 binds to the ATP binding site of PDK1, and 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].
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^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

471.35

C₂₀H₂₃BrN₈O

702674-56-4

Room temperature in continental US; may vary elsewhere.

DMSO : ≥ 100 mg/mL (212.16 mM)

* “≥” means soluble, but saturation unknown.

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.75 mg/mL (5.83 mM); Clear solution

  此方案可获得 ≥ 2.75 mg/mL (5.83 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 27.5 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.75 mg/mL (5.83 mM); Clear solution

  此方案可获得 ≥ 2.75 mg/mL (5.83 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 27.5 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.75 mg/mL (5.83 mM); Clear solution

  此方案可获得 ≥ 2.75 mg/mL (5.83 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 27.5 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Feldman RI, et al. Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1. J Biol Chem. 2005 May 20;280(20):19867-74.

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].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

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].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Feldman RI, et al. Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1. J Biol Chem. 2005 May 20;280(20):19867-74.