HBX 19818 is a specific inhibitor of ubiquitin-specific protease 7 (USP7), with an IC₅₀ of 28.1 μM.

IC50: 28.1 μM (USP7)^[1]

HBX 19818 is an inhibitor of USP7, with an IC₅₀ of 28.1 μM. HBX 19818 shows no effects on USP8, USP5, USP10, CYLD, UCH-L1, UCH-L3 or on SENP1, a SUMO protease, with IC₅₀s of > 200 μM. HBX 19818 selectively inhibits USP7 with IC₅₀ of ∼6 μM in in human cancer cells. In addition, HBX 19818 (1.5, 4, 12, 36, or 100 μM) inhibits USP7 deubiquitination of polyubiquitinated p53. HBX 19818 (30 μM) also causes significantly higher levels of Mdm2 polyubiquitinated forms in USP7-overproducing HEK293 cells than those in DMSO-treated control cells. HBX 19818 inhibits HCT116 proliferation in a dose-dependent manner, with an IC₅₀ of ∼2 μM^[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.

421.96

C₂₅H₂₈ClN₃O

1426944-49-1

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

DMSO : 20 mg/mL (47.40 mM; Need ultrasonic)

*

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。

In Vivo:

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 mg/mL (4.74 mM); Clear solution

  此方案可获得 ≥ 2 mg/mL (4.74 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.0 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% corn oil

  Solubility: ≥ 2 mg/mL (4.74 mM); Clear solution

  此方案可获得 ≥ 2 mg/mL (4.74 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 20.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Reverdy C, et al. Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme. Chem Biol. 2012 Apr 20;19(4):467-77.

The ability of HBX 19818 and HBX 28,258 to inhibit a panel of deubiquitinating enzymes, including UCH-L3 (13 pM), USP7 (100 pM), USP8 (1.36 nM), UCH-L1 (2.5 nM), USP5 (10 nM), USP20 (10 nM), and USP2 (500 pM), is tested using the UbAMC substrate (300 nM). The potential effects of HBX 19818 and HBX 28,258 are also tested on the enzymatic activities of SENP1 (80 pM), cathepsin-B (100 pM), and caspase-3 (100 pM) using the SUMO1-AMC (750 nM), ZRR-AMC (3 μM), and DEVD-AMC (250 nM) substrates, respectively. All enzymes are tested in USP7 reaction buffer (50 mM Tris-HCl [pH 7.6], 0.5 mM EDTA, 5 mM DTT, 0.01% Triton X-100, and 0.05 mg/mL serum albumin), except for two enzymes, USP8 (same buffer but pH 8.8) and caspase-3 (100 mM HEPES [pH 7.5], 10% sucrose, and 0.1% CHAPS). All enzymes are pre-incubated with DMSO or compounds (including HBX 19818) for 30 min at room temperature, and the enzymatic reaction is initiated by adding the substrate of interest. The reaction mixture is incubated at room temperature for 1 hr, and the reaction is stopped by adding acetic acid (100 mM). The reactions are monitored using the PHERAstar^[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.

HCT116 cell proliferation is evaluated by incubating HCT116 cells for 30 min in culture medium containing 10 µM 5- bromo-2-deoxyuridine (BrdU), which is incorporated into the DNA of proliferating cells. Cells are then harvested by trypsin treatment, collected by centrifugation, and the pellet is resuspended and incubated in 70% ethanol for 30 min at 4°C. After centrifugation and supernatant removal, DNA is denaturated by incubating it in 2 N HCl for 30 min at room temperature. The percentage of BrdU-containing cells is then determined by flow cytometry, making it possible to quantify proliferating cells. Cell cycle is evaluated after treatment with HBX 19818 for 24 hr, followed by fixing detached cells and trypsinized cells in 70% ethanol for 30 minutes at 4°C. Cells are then incubated in PBS supplemented with 1% BSA, 0.5% Tween 20, 50 µg/mL RNase A and 50 µg/mL propidiumiodide for 30 minutes at 37°C. Samples are analyzed on a FACSort fluorocytometer. The percentage of cells in the different phases of the cell cycle is calculated using Multicycle software^[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]. Reverdy C, et al. Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme. Chem Biol. 2012 Apr 20;19(4):467-77.