EOAI3402143 is a deubiquitinase (DUB) inhibitor, which inhibits dose-dependently inhibits Usp9x/Usp24 and Usp5.

Usp5^[1]
Usp9x^[1][2][3]
Usp24^[2]

EOAI3402143 retains potent Usp9x and Usp5 inhibitory activity^[1]. EOAI3402143 dose-dependently inhibits Usp9x and Usp24 activity, increases tumor cell apoptosis^[2]. Treatment of UM-2, UM-6, UM-16, and UM-76 with Usp9x inhibitor EOAI3402143 (G9) dose-dependently suppresses cell survival, while 600 nM of EOAI3402143 completely suppresses UM-2 3D colony growth when compared to untreated vehicle controls^[3].

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.

To examine this potential, the effect of EOAI3402143 (G9) treatment is investigated on human MIAPACA2 tumor xenografts. Human MIAPACA2 cells are injected subcutaneously into NSG mice. Primary tumor development is monitored by caliper measurements, and once measurable, mice are separated into two groups and are treated with either vehicle control (PEG300/DMSO) or G9 at 15 mg/kg. Tumor growth, animal weight, behavior, and mobility are monitored during treatment. In parallel, murine 8041 tumors are also established and subjected to similar G9 treatment and tumor monitoring regimen as the human MIAPACA2 xenografts. Consistent with the in vitro findings, Usp9x inhibition results in the suppression of tumor growth in human tumor xenografts, but any significant effect on the growth of 8041 tumors xenografts is not observed, although the Usp9x activity is inhibited effectively by EOAI3402143 treatment in both human MIAPACA2 and mouse 8041 xenograft tumors^[3].

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.

503.42

C₂₅H₂₈Cl₂N₄O₃

1699750-95-2

Room temperature in continental US; may vary elsewhere.

DMSO : 50 mg/mL (99.32 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。

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： 50% PEG300    50% saline

  Solubility: 10 mg/mL (19.86 mM); Suspended solution; Need ultrasonic
• 2.

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

  Solubility: ≥ 2.5 mg/mL (4.97 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.97 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 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

• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (4.97 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.97 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 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Potu H, et al. Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway. Oncotarget. 2014 Jul 30;5(14):5559-69.

  [2]. Peterson LF, et al. Targeting deubiquitinase activity with a novel small-molecule inhibitor as therapy for B-cell malignancies. Blood. 2015 Jun 4;125(23):3588-97.

  [3]. Pal A, et al. Usp9x Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth. Neoplasia. 2018 Feb;20(2):152-164.

UM-2, UM-6, UM-16, and UM-76 cells are seeded in a 96-well plate at 5000 per well in the presence of the indicated concentration of EOAI3402143 (1, 2, 3, 4, and 5 μM) for 3 days in a CO₂ incubator at 37°C. Twenty microliters of 5 g/L MTT solution are added to each well for 2 hours at 37°C. The cells are then lysed in 10% SDS buffer, and absorbance at 570 nm relative to a reference wavelength of 630 nm is determined with a microplate reader. To examine proliferation using the MTT assay, cells are plated in triplicates, and the samples are processed for MTT assay at day 0, 1, 2, 3, and 4^[3].

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.

Mice^[3]
NSG [NOD/SCID/IL2r-g (null)] mice are injected mid-dorsally with 2×10⁶ 8041 or 5×10⁶ MIAPACA2 cells in 0.1 mL of Matrigel/DMEM suspension. Tumors are allowed to establish to about 100 mm³, after which mice are tumor size matched and allocated to five per treatment group (vehicle or EOAI3402143) for 8041 tumor-bearing mice and four per treatment group for MIAPACA2 tumor-bearing mice. EOAI3402143 is administered in DMSO:PEG300 (1:1) by i.p injection every other day at 15 mg/kg. Tumor size is monitored by caliper measurements twice a week, and tumor volume is calculated^[3].

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]. Potu H, et al. Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway. Oncotarget. 2014 Jul 30;5(14):5559-69.

  [2]. Peterson LF, et al. Targeting deubiquitinase activity with a novel small-molecule inhibitor as therapy for B-cell malignancies. Blood. 2015 Jun 4;125(23):3588-97.

  [3]. Pal A, et al. Usp9x Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth. Neoplasia. 2018 Feb;20(2):152-164.