Akt; ERK

在几种癌细胞系中，TIC10 以不依赖于 p53 的方式诱导 TRAIL 蛋白在细胞表面定位，并以剂量依赖的方式增加 TRAIL mRNA。虽然 TIC10 在体外表现出广谱抗肿瘤活性，并导致 TRAIL 敏感的 HCT116 p53/p53 细胞表现出指示细胞死亡的亚 G1 DNA 含量增加，但正常成纤维细胞在同等剂量下不受 TIC10 影响。 TIC10 可以保护健康的成纤维细胞，同时减少癌细胞系的克隆存活。与 TRAIL 介导的细胞凋亡类似，TIC10 以不依赖 p53 且依赖 Bax 的方式增加癌细胞中亚 G1 DNA 的比例。 TIC10引起的TRAIL上调依赖于Foxo3a，Foxo3a也会上调TRAIL死亡受体DR5和其他靶标，可能使一些TRAIL耐药的肿瘤细胞变得敏感。 ERK 和 Akt 激酶被 TIC10 灭活，导致 Foxo3a 进入细胞核并与 TRAIL 启动子结合以激活基因转录。 Foxo3a 然后被转运到细胞核中。有效的抗肿瘤药物 TIC10 通过增加肿瘤细胞及其周围组织中天然存在的肿瘤抑制因子 TRAIL 的水平来发挥作用。 [1]

当TIC10和TRAIL以多剂量给药时，TIC10和TRAIL治疗导致HCT116 p53−/−异种移植物中的肿瘤消退到相当的程度。 TIC10 还诱导 MDA-MB-231 人三阴性乳腺癌异种移植物消退，而 TRAIL 治疗的肿瘤则进展。在 DLD-1 结肠癌异种移植物中，TIC10 在治疗一周后诱导肿瘤停滞，而 TRAIL 治疗的肿瘤在单剂量后进展。 SW480异种移植物在接受单剂量TIC10后也表现出持续的消退，无论该药物是口服还是腹膜内给药，都可以看到这种效果。这表明 TIC10 具有良好的口服生物利用度。 TIC10 通过 TRAIL 介导的直接效应和旁观者效应引起肿瘤特异性细胞死亡。 TIC10 是一种针对原位人多形性胶质母细胞瘤的有效抗肿瘤剂。 [1]

ChIP测定[1]
如前所述，用Foxo3a的ChIP级抗体或等效浓度的兔免疫球蛋白G作为非特异性对照，对TRAIL启动子进行ChIP测定。

用 10 μM ONC201 或 DMSO 处理细胞 24 小时。
菌落形成测定[1]
将所指示的细胞系以每孔500个细胞进行铺板并使其粘附，然后第二天在新鲜的完全培养基中处理。治疗后3天，用无药物培养基代替培养基，细胞繁殖10天，每3天给予一次新鲜培养基。在10天周期结束时，将细胞在PBS中洗涤，用甲醇固定，用考马斯蓝染色，漂洗，并干燥以进行定量

---

蛋白质印迹分析[1]
如前所述（41）用NuPAGE 4-12%双-三凝胶进行蛋白质印迹分析，并用SuperSignal West Femto和x射线胶片进行可视化。用NIH ImageJ进行密度测定。用细胞质裂解缓冲液（10mM Hepes、10mM KCl、2mM MgCl2、1mM二硫苏糖醇）制备细胞核和细胞质提取物，然后制备细胞核裂解缓冲液。对于所有裂解缓冲液，在使用前立即加入新鲜蛋白酶抑制剂和1mM原钒酸钠。

Female athymic nu/nu mice
25, 50, 100 mg/kg
Intraperitoneal/oral

---

All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee at the Pennsylvania State University College of Medicine. For subcutaneous xenografts, 4- to 6-week-old female athymic nu/nu mice (Charles River Laboratories) were inoculated with 1 × 106 cells (2.5 × 106 for T98G) of the indicated cell lines in each rear flank as a 200-μl suspension of 1:1 Matrigel (BD)/PBS. All subcutaneous tumors were allowed to establish for 1 to 4 weeks after injection until reaching a volume of ~125 mm3 before treatment initiation.[1]

[1]. Sci Transl Med. 2013 Feb 6;5(171):171ra17.

[2]. ACS Chem Biol. 2019 May 17;14(5):1020-1029.

Dordaviprone (ONC-201) is under investigation in clinical trial NCT03394027 (ONC201 in Recurrent/Refractory Metastatic Breast Cancer and Advanced Endometrial Carcinoma).
Dordaviprone is a water soluble, orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) and extracellular signal-regulated kinase (ERK), with potential antineoplastic activity. Upon administration, dordaviprone binds to and inhibits the activity of Akt and ERK, which may result in inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathway as well as the mitogen-activated protein kinase (MAPK)/ERK-mediated pathway. This may lead to the induction of tumor cell apoptosis mediated by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL death receptor type 5 (DR5) signaling in AKT/ERK-overexpressing tumor cells. The PI3K/Akt signaling pathway and MAPK/ERK pathway are upregulated in a variety of tumor cell types and play a key role in tumor cell proliferation, differentiation and survival by inhibiting apoptosis. In addition, ONC201 is able to cross the blood-brain barrier.

---

Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an antitumor protein that is in clinical trials as a potential anticancer therapy but suffers from drug properties that may limit efficacy such as short serum half-life, stability, cost, and biodistribution, particularly with respect to the brain. To overcome such limitations, we identified TRAIL-inducing compound 10 (TIC10), a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal-regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL. [1]

---

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds. [2]

C24H26N4O

386.49

386.21

C, 74.58; H, 6.78; N, 14.50; O, 4.14

1616632-77-9

41276-02-2 (isomer);1616632-77-9;1638178-82-1 (HCl);1777785-71-3 (HBr);2007141-57-1 (2HBr);

73777259

White to off-white solid powder

1.2±0.1 g/cm3

559.7±60.0 °C at 760 mmHg

292.3±32.9 °C

0.0±1.5 mmHg at 25°C

1.672

3.14

0

3

4

29

693

0

O=C1C2C([H])([H])N(C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])C([H])([H])C([H])([H])C=2N2C([H])([H])C([H])([H])N=C2N1C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H]

RSAQARAFWMUYLL-UHFFFAOYSA-N

InChI=1S/C24H26N4O/c1-18-7-5-6-10-20(18)16-28-22-11-13-26(15-19-8-3-2-4-9-19)17-21(22)23(29)27-14-12-25-24(27)28/h2-10H,11-17H2,1H3

7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(4H)-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。