Akt; ERK

ONC201/TIC10是目前正在研究的TRAIL基因的小分子诱导物，是一种新型抗癌剂。在这项研究中，我们确定了ONC201敏感性的关键分子决定因素，作为药效学或预测反应标志物提供了潜在的用途。通过筛选激酶siRNA文库与亚细胞毒性剂量的ONC201相结合，我们鉴定了几种能消除肿瘤细胞敏感性的激酶，包括MAPK通路诱导剂KSR1。出乎意料的是，在ONC201存在或不存在的情况下，KSR1沉默不影响MAPK信号传导，而是降低了抗凋亡蛋白FLIP、Mcl-1、Bcl-2、cIAP1、cIAP2和生存素的表达。在这项工作的同时，我们还进行了协同筛选，将ONC201与已批准的小分子抗癌药物相结合。在多个癌症细胞群体中，ONC201与多种药物类别协同作用，包括多激酶抑制剂索拉非尼。值得注意的是，ONC201和索拉非尼的联合导致TRAIL及其受体DR5的协同诱导以及细胞死亡的有效诱导[1]。

在肝细胞癌的小鼠异种移植物模型中，我们证明ONC201和索拉非尼协同安全地触发了肿瘤消退。总体而言，我们的研究结果建立了一组ONC201敏感性的决定因素，这些决定因素可以预测治疗反应，特别是在索拉非尼联合治疗以增强抗癌反应的情况下[1]。

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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。 [2]

siRNA激酶文库筛选[1]
在96孔板中以20000个细胞/孔的速度转染HCT116 p53−/−细胞。使用Optimem中的RNAiMax用Stealth RNAi人激酶文库转染细胞。Scramble和AllStars Hs细胞死亡对照siRNA分别用作阴性和阳性对照。转染进行过夜，第二天在去除转染培养基后将含有ONC201或DMSO的完整培养基加入到平板中。根据制造商的说明在指定的时间点进行CellTiterGlo分析。

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ChIP测定[2]
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。

小分子协同筛选[1]
使用Biomek 2000机器人使用针工具进行药物治疗。将细胞以5×104个细胞/mL的速度接种在96孔黑色壁板中，12小时后进行处理。最初通过计算观察到的与组合的活性和单体活性之和之间的差异来评估组合活性。

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基于细胞的测定[1]
如前所述进行Cell TiterGlo、Western印迹分析和细胞周期流式细胞术分析。在用4%多聚甲醛在PBS中固定20分钟后，在PBS内漂洗，与抗TRAIL或抗DR5抗体在1:200下孵育过夜，在PBS中漂洗，与荧光团缀合的第二抗体在1:250下孵育30分钟，并通过流式细胞术分析细胞表面TRAIL和DR5。用p-T202/Y204（细胞信号传导）抗体评估pERK，用pT308抗体评估pAkt。

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菌落形成测定[2]
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。

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蛋白质印迹分析[2]
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。

6-week-old athymic nude mice were obtained from Charles River Laboratories. 107 HepG2 cells in 200 µL (1:1, PBS: Matrigel) were injected into each rear flank. Measurable tumors were assessed 1 week later. Treatment was then initiated as indicated. Sorafenib and ONC201 were administered as 100 µL solutions by oral gavage. ONC201 was administered 12 hours following sorafenib treatment. Tumor volume was assessed with digital calipers and calculated as a spheroid. Tumor dimensions and body weights were assessed twice a week. IHC (Vector Labs) and TUNEL (Millipore) analyses were performed as previously described [1].

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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.[2]

[1]. Cancer Res.2015 Apr 15;75(8):1668-74;
[2]. Sci Transl Med.2013 Feb 6;5(171):171ra17.

NC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses. [1]

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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. [2]

C24H28CL2N4O

459.411323547363

458.164

C, 62.75; H, 6.14; Cl, 15.43; N, 12.20; O, 3.48

1638178-82-1

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

121596510

Solid powder

39.2Ų

2

3

4

31

693

0

Cl.Cl.O=C1C2CN(CC3C=CC=CC=3)CCC=2N2CCN=C2N1CC1C=CC=CC=1C

HKBXPCQCBQFDML-UHFFFAOYSA-N

InChI=1S/C24H26N4O.2ClH/c1-18-7-5-6-10-20(18)16-28-23(29)21-17-26(15-19-8-3-2-4-9-19)13-11-22(21)27-14-12-25-24(27)28/h2-10H,11-17H2,1H32*1H

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

ONC-201 Dihydrochloride, ONC-201 HCl, ONC201; ONC 201 ONC-201; NSC350625; NSC-350625; NSC 350625; ONC-201 Dihydrochloride; 1638178-82-1; ONC201 HCl; 53VG71J90J; 7-Benzyl-4-(2-methylbenzyl)-2,4,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one dihydrochloride; 11-benzyl-7-[(2-methylphenyl)methyl]-2,5,7,11-tetrazatricyclo[7.4.0.02,6]trideca-1(9),5-dien-8-one;dihydrochloride; Imidazo(1,2-a)pyrido(3,4-E)pyrimidin-5(1H)-one, 2,4,6,7,8,9-hexahydro-4-((2-methylphenyl)methyl)-7-(phenylmethyl)-, hydrochloride (1:2); Imidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one, 2,4,6,7,8,9-hexahydro-4-[(2-methylphenyl)methyl]-7-(phenylmethyl)-, hydrochloride (1:2); imipridone; TIC10; TIC 10; TIC-10 TRAIL inducing compound 10.

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)

DMSO; > 10 mM

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。

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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。

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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)

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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。

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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

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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。

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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。