CBP/β-catenin interaction

体外活性：PRI-724 特异性结合 CBP，但不结合相关转录共激活因子 p300，从而破坏 CBP 与 β-catenin 的相互作用。 PRI-724 处理选择性诱导结肠癌细胞凋亡，但不诱导正常结肠上皮细胞凋亡，并减少结肠癌细胞的体外生长。激酶测定：双荧光素酶报告基因 (DLR) 测定系统提供了执行双报告基因测定的有效方法。在 DLRTM 检测中，从单个样品中依次测量萤火虫 (Photinuspyralis) 和海肾 (Renilla reniformis，也称为海三色堇) 荧光素酶的活性。首先通过添加荧光素酶检测试剂 II (LAR II) 来测量萤火虫荧光素酶报告基因，以产生“辉光型”发光信号。对萤火虫发光进行定量后，该反应被淬灭，并通过同时向同一管中添加 Stop & Glo® 试剂来启动海肾荧光素酶反应。 Stop & Glo® 试剂还会从海肾荧光素酶产生“发光型”信号，该信号在测量过程中缓慢衰减。在 DLRTM 检测系统中，两种报告基因均可产生具有亚阿托摩尔 (<10-18) 灵敏度的线性检测结果，并且在实验宿主细胞中任一报告基因均无内源活性。此外，DLRTM 检测的集成形式可对转染细胞或无细胞转录/翻译反应中的两种报告基因进行快速定量。细胞测定：用 ICG-001 或 IQ1 处理大鼠 EMC，并进行免疫共沉淀 (co-IP) 测定。用 DMSO、ICG-001 或 IQ1 处理细胞 24 小时。在 DMSO 对照处理的细胞中，基本上所有 β-连环蛋白都与 CBP 相关。 IQ1 治疗对 β-连环蛋白共激活剂的使用影响极小。然而，正如预期的那样，ICG-001 治疗减少了 β-连环蛋白/CBP 相互作用，同时增加了 β-连环蛋白/p300 相互作用。

PRI-724处理降低了sl4接种肝脏中β-catenin靶基因mRNA的表达[1]
PRI-724处理降低了sl4接种肝脏中β-catenin靶基因mRNA的表达[1]
PRI-724增加转移性肝肿瘤t淋巴细胞浸润 [1]
抗pd - l1 Ab和PRI-724联合治疗的抗肿瘤作用需要CD8+ t细胞。[1]

---

PRI-724 在小鼠结肠癌异种移植模型中表现出抗肿瘤活性。 PRI-724的I期临床试验初步结果已公开披露。该药物表现出可接受的毒性特征，只有一种剂量限制性毒性，即 3 级可逆性高胆红素血症。 PRI-724 针对晚期骨髓恶性肿瘤患者的开放标签剂量递增 I/II 期研究仍在进行中。

血清细胞因子和趋化因子的测定[1]
采用Wako转氨酶cii检测试剂盒检测血清ALT水平。血清细胞因子和趋化因子采用Luminex MILLIPLEX MAP小鼠细胞因子/趋化因子磁珠面板-免疫多重检测。这个程序是按照制造商的说明进行的。

小鼠ihl的分离[1]
在含有0.02%胶原酶IV和0.002% dna酶I的RPMI-1640中，37℃下消化40分钟，制备肝正中叶单细胞悬液。PBS将细胞覆盖在淋巴细胞- m上。密度分离后，用流式细胞仪（FACS）对分离的ils进行评价。

---

FACS分析[1]
使用抗cd3、抗cd4、抗cd8、抗nk1.1、抗cd69和抗foxp3抗体，用荧光染料偶联的抗体在冰上表面染色20 min。为了进行细胞内细胞因子染色，将ihl与SL4细胞（1 × 105个/孔）在含200 μL/孔rmi -1640培养基的96孔圆底板上，37℃共培养4 h。每孔加入50个单位的小鼠重组IL-2和0.2 μL的BD GolgiPlug蛋白转运抑制剂。孵育后，收获细胞，用含有1% FBS的PBS洗涤，用未标记的抗小鼠CD16/32 Ab在冰上孵育10分钟，以阻断fc - γ rii /III的结合。然后用指定的抗体在冰上表面染色20分钟。染色后，冲洗细胞以去除未结合的抗体，并使用Cytofix/Cytoperm Kit进行固定。然后用biolgend公司提供的试剂进行二次染色，除特别说明外，这些试剂包括：fitc偶联的CD107a、pe偶联的抗干扰素γ和抗il -10。

Liver metastasis model [1]
Male wild-type C57BL/6J mice 8-weeks of age were obtained from Japan SLC. After making a small incision under anesthesia to expose the spleen, 0.1 mL of a viable cell suspension containing 5 × 106 cells/mL was injected into the spleen. We chose SL4 cells because the cells grow rapidly, even in the liver of wild-type mice. The animals were then each intraperitoneally injected with or without 0.4 mg PRI-724 and/or 200 μg of an anti-PD-L1 Ab (10F.9G2) three times per week. In addition, some mice treated with PRI-724 and the anti-PD-L1 Ab were administrated anti-mouse CD4 or CD8 Ab (250 μg/mouse) three times per week. After the course of treatment, the mice were anesthetized and humanely sacrificed by exsanguination 14 days post cell-inoculation. The livers of the animals were immediately removed, washed in ice-cold PBS, and weighed before a portion of the dissected liver tissue was frozen in liquid nitrogen. Additional animals were maintained and used for survival analysis.

---

Dissolved in PBS; 300 mg/kg; i.p. administration

C57BL/6 and Balb/c mice

[1]. Oncotarget.2019Apr 30;10(32):3013-3026;
[2]. Transl Respir Med.2014 Sep 11;2:8;
[3]. Am J Cancer Res.2015 Jul 15;5(8):2344-60.

PRI-724 is under investigation in clinical trial NCT03620474 (Safety and Effectiveness of PRI-724 for Hepatitis C or B Virus Derived Liver Cirrhosis).
Foscenvivint is a potent, specific inhibitor of the canonical Wnt signaling pathway in cancer stem cells with potential antineoplastic activity. Foscenvivint specifically inhibits the recruiting of beta-catenin with its coactivator CBP (the binding protein of the cAMP response element-binding protein CREB); together with other transcription factors beta-catenin/CBP binds to WRE (Wnt-responsive element) and activates transcription of a wide range of target genes of Wnt/beta-catenin signaling. Blocking the interaction of CBP and beta-catenin by this agent prevents gene expression of many proteins necessary for growth, thereby potentially suppressing cancer cell growth. The Wnt/beta-catenin signaling pathway regulates cell morphology, motility, and proliferation; aberrant regulation of this pathway leads to neoplastic proliferation.

---

Emami and colleagues have identified the small molecule PRI-724 (also named as ICG-001) that down-regulates the Wnt/β-catenin signaling by specifically binding to CBP. PRI-724 was shown to selectively induce apoptosis in colon carcinoma cells but not in normal colon cells, and exhibit antitumor activity in the mouse xenograft models of colon cancer. Interestingly, PRI-724 binds specifically to the co-activator CBP, but not to the closely related homologue p300.[3]

---

Immune checkpoint blockade with specific antibodies can accelerate anti-tumor immunity, resulting in clinical responses in patients with various types of cancer. However, these antibodies achieve only partial tumor regression. Thus, a wide variety of treatment combinations based on programmed death-ligand 1 (PD-L1) pathway inhibition are under development to enhance such therapeutic effects. In this study, the effects of combination treatment using PRI-724, a selective inhibitor of CBP/β-catenin, and an anti-PD-L1 antibody were examined in a mouse model of colon cancer liver metastasis. Mice were inoculated with SL4 colon cancer cells to produce metastatic liver tumors. The combination treatment resulted in regression of tumor growth, whereas monotherapy with each treatment individually failed to exhibit any anti-tumor activity. In addition, co-administration of the inhibitor and antibody induced CD8+CD44lowCD62Llow cells and interferon (IFN)-γ production in CD8+ T-cells in the liver compared with that in control mice. Administration of an anti-CD8 antibody mitigated the anti-tumor effects of the combined treatment of PRI-724 and anti-PD-L1 antibody. In conclusion, targeting CBP/β-catenin, combined with PD-1/PD-L1 immune checkpoint blockade, shows potential as a new therapeutic strategy for treating liver metastasis during colon cancer.[1]

---

Background: Wnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. The previously identified and characterized small molecule inhibitor IQ1 can pharmacologically decrease the interaction between β-catenin and its transcriptional coactivator p300, thereby enhancing the β-catenin/CBP interaction. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem cells and epicardial progenitor cells; however, whether differential coactivator usage by β-catenin plays a role in proximal-distal determination of lung epithelium is unknown. Methods: We examined the effects of inhibiting the β-catenin/p300 interaction with IQ1 on lung branching morphogenesis in mouse embryos in utero and mouse embryonic lung organ culture ex vivo. The phenotype of IQ1 treated lungs was analyzed by epithelial staining, histology, quantitative PCR and in situ hybridization. Results: Inhibition of the β-catenin/p300 interaction by IQ1 disrupted the distal branching of mouse lung epithelium both in utero and ex vivo. IQ1 proximalized lung epithelium with decreased expression of the genes Bmp4 and Fgf10, hallmarks of distal lung determination, and increased expression of the proximal genes Sox2 and Scgb1a1 (CC10) as shown by quantitative PCR and in situ hybridization. The disruption of branching was reversible ex vivo as branching was reinitiated after removal of IQ1 from the media. Conclusions: The results demonstrate that the β-catenin/p300 interaction plays a critical role in proximal-distal determination of the epithelium in mouse lung branching morphogenesis and β-catenin/p300 inhibition pharmacologically proximalizes lung epithelium.[2]

---

As described previously, small molecule PRI-724 can block interaction of CBP with β-catenin for Wnt signaling inhibition. The initial results of the Phase I clinic trial of PRI-724 has been disclosed publically. Overall, PRI-724 was given to 18 patients as a continuous infusion for 7 days, and the drug exhibited an acceptable toxicity profile with only one dose-limiting toxicity of grade 3 reversible hyperbilirubinaemia. An Open-Label dose-escalation phase I/II study of PRI-724 for patients with advanced myeloid malignancies is still ongoing. Additionally, clinical trials of combination of PRI-724 with other therapeutic agents are underway. For instance, a Phase I trial was initiated to treat patient with colorectal cancer by administering PRI-724 in combination with a modified regimen of FOLFOX6 (mFOLFOX 6). Furthermore, a current Phase I trial is testing continuous intravenous doses of PRI-724, in combination with Gemcitabine, to treat patients with advanced or metastatic pancreatic adenocarcinoma.[3]

C33H35N6O7P

658.6408

658.23

C, 60.18; H, 5.36; N, 12.76; O, 17.00; P, 4.70

1422253-38-0

1422253-38-0 (PRI-724);1198780-38-9 847591-62-2 (deleted);780757-88-2 (ICG001);

71509318

Solid powder

0.3

156

3

9

8

47

1170

3

P(=O)(O)(O)OC1C=CC(=CC=1)C[C@H]1C(N(CC2=CC=CC3C=CC=NC2=3)[C@@H](C)[C@]2([H])N(C(NCC3C=CC=CC=3)=O)N(C)CC(N21)=O)=O

VHOZWHQPEJGPCC-AZXNYEMZSA-N

InChI=1S/C33H35N6O7P/c1-22-31-38(29(40)21-36(2)39(31)33(42)35-19-24-8-4-3-5-9-24)28(18-23-13-15-27(16-14-23)46-47(43,44)45)32(41)37(22)20-26-11-6-10-25-12-7-17-34-30(25)26/h3-17,22,28,31H,18-21H2,1-2H3,(H,35,42)(H2,43,44,45)/t22-,28-,31-/m0/s1

(6S,9aS)-N-benzyl-6-(4-hydroxybenzyl)-8-(naphthalen-1-ylmethyl)-4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidine-1-carboxamide

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。