iCRT3

β-catenin-responsive transcription (CRT)
The target of iCRT3 is the β-catenin/T-cell factor (TCF) complex, specifically inhibiting the interaction between β-catenin and TCF1; [2]
The target of iCRT3 is the Wnt/β-catenin signaling pathway, acting as a small-molecule inhibitor of β-catenin-responsive transcription; [3]
The target of iCRT3 is the Wnt/β-catenin signaling pathway; [1]

iCRT3 抑制对 Wnt 和 β-catenin 敏感的转录。 iCRT3 显着降低了 TOP Flash 活动和 NTSR1 级别。 iCRT3可以显着抵消神经降压素（NTS）和Wnt3a的抗凋亡作用[1]。与 DMSO 对照相比，长期 iCRT3 维持的细胞表现出经典多能性的表达增加，但同时分化标记物和 T 细胞因子 (TCF) 靶基因的表达减少[2]。 iCRT3 治疗在 12.5、25、50 和 75 μM 剂量下，TNF-α 水平分别降低 14.7%、18.5%、44.9% 和 61.3%。与媒介物相比，iCRT3 疗法的 IκB 水平呈剂量依赖性上升[3]。

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1. 对胶质母细胞瘤细胞中Wnt/β-catenin信号通路的抑制作用：iCRT3通过药理抑制Wnt/β-catenin通路，降低胶质母细胞瘤细胞中NTSR1的mRNA和蛋白表达水平[1]
2. 对小鼠胚胎干细胞（mESCs）中β-catenin/TCF1相互作用的抑制作用：iCRT3特异性抑制β-catenin与TCF1的相互作用，不影响β-catenin与TCF3的结合。这种抑制作用降低了TCF依赖性靶基因（如Cdx1、Axin2）的转录活性，延缓了mESCs的分化。经iCRT3处理14天后，mESCs的自我更新能力增强，维持较高的Nanog和Rex1表达水平，自发分化减少。在视黄酸（RA）诱导的分化模型中，iCRT3使mESCs能够抵抗分化，提高Nanog-GFP阳性细胞比例，增强集落形成效率。此外，iCRT3还可增加β-catenin/Oct4复合物的形成，该复合物与基础状态多能性相关[2]
3. 对脂多糖（LPS）刺激的RAW264.7巨噬细胞炎症反应的调控作用：用不同浓度的iCRT3预处理（50分钟）RAW264.7巨噬细胞后，加入LPS（1 ng/ml）刺激。iCRT3呈剂量依赖性抑制LPS诱导的Wnt/β-catenin通路激活（通过TOP-TK-Luc报告基因活性检测）和肿瘤坏死因子-α（TNF-α）的产生（通过酶联免疫吸附试验（ELISA）检测）。iCRT3还呈剂量依赖性抑制LPS诱导的IκB降解（通过蛋白质印迹法（Western blotting）分析），且不影响细胞活力（通过MTS实验检测，与未处理细胞相比活力维持在100%）[3]

iCRT3 治疗可显着降低肿瘤生长速度。 iCRT3 的肿瘤抑制功能始终与增殖标志物 Ki67 指数的下降相关[1]。与载体组相比，10 mg/kg iCRT3 治疗组的 IL-6 水平降低了 82.9%。在假手术中，检测不到 IL-1β 水平；然而，在脓毒症小鼠中，当给予 5 和 10 mg/kg iCRT3 时，它们分别达到 371 pg/mL 和下降 30.2% 和 53.2%。用 5 和 10 mg/kg 剂量的 iCRT3 治疗的这些脓毒症小鼠的 AST 水平分别比用载体治疗的动物低 15.4% 和 44.2%。与媒介物组相比，用 10 mg/kg iCRT3 治疗后，肺形态得到改善，微观退化更少。将 iCRT3 治疗动物的肺组织与媒介物组进行比较时，凋亡细胞减少了 92.7%[3]。

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1. 对胶质母细胞瘤模型肿瘤生长的抑制作用：iCRT3通过药理抑制Wnt/β-catenin信号通路，在体内抑制胶质母细胞瘤的肿瘤生长[1]
2. 对C57BL/6小鼠脓毒症诱导的炎症反应和器官损伤的缓解作用：对雄性C57BL/6小鼠进行盲肠结扎穿孔（CLP）诱导脓毒症，在CLP后5小时腹腔注射iCRT3（5 mg/kg或10 mg/kg体重）。CLP后20小时，iCRT3呈剂量依赖性降低血浆中促炎细胞因子（IL-6、TNF-α、IL-1β）和器官损伤标志物（AST、ALT、LDH）的水平。组织学分析显示，iCRT3（10 mg/kg）改善了肺组织完整性，降低了肺损伤评分，减少了肺胶原沉积（通过Masson三色染色），并抑制了肺细胞凋亡（通过TUNEL染色检测）。此外，iCRT3下调了肺组织中IL-6、TNF-α、IL-1β、中性粒细胞趋化因子（MIP-2、KC）的mRNA表达，降低了肺髓过氧化物酶（MPO）活性，从而减轻了中性粒细胞浸润[3]

β-catenin-TCF报告活性测定[3]
RAW264.7细胞在转染前一天以1.24 × 105个细胞/ ml的密度接种。细胞与250 ng TOP-TK-Luc或TOP-TK-Luc和25 ng pRL-TK报告质粒短暂共转染，使用Lipofectamine 3000 Reagent，按照制造商的说明。转染24 h后，用iCRT3或对照物预处理细胞50 min，然后用LPS （1 ng/ml）刺激24 h。转染后48小时裂解细胞，根据制造商的说明，用双荧光素酶报告基因检测系统测定荧光素酶活性。TOP-TK-Luc包含最优位点，TOP-TK-Luc包含位于萤火虫荧光素酶报告基因上游的突变tcf结合位点。将TOP和FOP萤火虫荧光素酶活性归一化为来自共转染pRL-TK质粒的Renilla荧光素酶活性，作为转染效率的内部对照。所有实验都进行了至少两次的三次重复。

荧光素酶报告试验[1]
细胞在24孔板中以4 × 105个细胞/孔的大小进行镀膜，用Lipofectamine 2000 瞬时转染TopFlash（0.5µg）和Renilla报告基因（0.05µg）。A172或U87细胞中分别加入NTS、Wnt3a、SR48692和iCRT3处理24 h。收集细胞，转染2天后测定荧光素酶活性。采用双荧光素酶报告基因检测系统测定荧光素酶活性。

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细胞增殖和细胞凋亡试验[1]
将细胞接种到96孔板中，每孔密度为5 × 103个细胞，在指定处理的培养基中再孵育48小时。根据制造商的说明，分别使用Cell Counting kit-8和Caspase-Glo 3/7检测试剂盒进行细胞活力和细胞凋亡检测。

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对于长期培养，在DMSO或iCRT3的干条件下（血清加LIF），将细胞以有限的稀释度在6孔或96孔板中进行多次传代（14 d），每天更换培养基。每代进行AP染色以监测相对多能性水平。使用的小分子包括10µM iCRT3和1µM XAV939，用DMSO稀释。L- wnt3a和对照L细胞是R.T. Moon赠送的。［2］

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1. 胶质母细胞瘤细胞实验：用iCRT3（浓度未明确）处理胶质母细胞瘤细胞以抑制Wnt/β-catenin通路。处理后提取细胞总RNA和蛋白，通过实时定量聚合酶链反应（qPCR）检测NTSR1的mRNA表达水平，通过Western blotting分析NTSR1的蛋白表达水平[1]
2. 小鼠胚胎干细胞（mESCs）实验：
- β-catenin/TCF转录活性实验：将mESCs（NG4-TOPluc）转染TOPFlash报告基因质粒和内参pRL-TK质粒。转染后，用iCRT3（浓度未明确）处理细胞，并在含血清和白血病抑制因子（S+LIF）或含血清和RA（S+RA）的培养基中培养，检测荧光素酶活性以评估对β-catenin/TCF依赖性转录活性的抑制作用[2]
- 流式细胞术分析：在S+LIF培养基中用iCRT3处理TNGA（Nanog报告基因）和Rex1-GFP mESCs 14天，通过流式细胞术检测Nanog-GFP和Rex1-GFP的表达水平，评估自我更新能力。在分化抵抗实验中，无LIF条件下用iCRT3处理mESCs 6天或RA诱导分化时处理48小时，通过流式细胞术分析Nanog-GFP阳性细胞比例[2]
- qPCR分析：收集经iCRT3处理四代或RA诱导分化过程中的mESCs，提取总RNA并逆转录为cDNA，以肌动蛋白（actin）为内参，通过qPCR检测多能性标志物（Nanog、Oct4、Sox2）、分化标志物及TCF靶基因（Cdx1、Axin2）的mRNA表达水平[2]
- 集落形成效率（CFE）实验：将mESCs用RA联合iCRT3或二甲基亚砜（DMSO）预处理48小时后，以有限稀释法接种到不含抑制剂的S+LIF培养基中，再培养48小时，量化集落数量、集落面积和碱性磷酸酶（AP）活性以评估集落形成效率[2]
- 免疫共沉淀（CoIP）实验：制备RA诱导分化过程中经iCRT3处理的mESCs总细胞裂解液，用抗β-catenin、TCF1和TCF3的抗体进行CoIP实验，检测β-catenin与TCF1/TCF3的相互作用，通过Western blotting进行检测和密度分析[2]
3. RAW264.7巨噬细胞实验：
- 报告基因实验：将RAW264.7细胞共转染β-catenin/TCF应答报告基因TOP-TK-Luc（或对照FOP-TK-Luc）和内参pRL-TK。转染后，用不同浓度的iCRT3预处理细胞50分钟，再用LPS（1 ng/ml）刺激24小时，检测荧光素酶活性以评估Wnt/β-catenin通路激活情况[3]
- TNF-α ELISA实验：用不同浓度的iCRT3预处理RAW264.7细胞50分钟，再用LPS（1 ng/ml）刺激4小时，收集细胞上清液，通过ELISA检测TNF-α水平[3]
- MTS细胞活力实验：用不同浓度的iCRT3处理RAW264.7细胞，通过MTS实验检测细胞活力，以未处理细胞的活力为100%[3]
- IκB Western blotting实验：用不同浓度的iCRT3预处理RAW264.7细胞50分钟，再用LPS（1 ng/ml）刺激15分钟，制备总细胞裂解液，用抗IκB和actin的抗体进行Western blotting，检测IκB降解情况[3]

Dissolved in 5% DMSO in saline; 5 and 10 mg/kg; i.p.
C57BL/6 mice A172 cells were used to establish a subcutaneous xenograft and to determine the anti-tumor effects of SR48692 and iCRT3. NOD-SCID BALB/c mice were inoculated subcutaneously in the right back with 2 × 106 A172 cells. The growth of the primary tumors was recorded every 4 days. SR48692 (10 mg/kg) and iCRT3 (5 mg/kg) was diluted in PBS i.p. triweekly when tumors grew to ∼200 mm3. The control mice were treated with blank PBS containing 5% (v/v) DMSO. Tumor volume was evaluated with the following formula: volume = tumor length × width2/2. The mice were sacrificed 24 days after pharmaceutical treatment. The tumors were resected and embedded in paraffin, and the Ki67 staining was analyzed by immunohistochemistry.[1]

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Mice were randomly allocated to three groups: sham (n = 5 mice), vehicle and treatment (n = 8 mice per group). iCRT3 was reconstituted with cell culture grade 100% DMSO as 50 mg/ml stock. 5 and 10 mg/kg body weight (BW) concentrations of iCRT3 were made by diluting stock in sterile normal saline with 5% DMSO. At 5 h after CLP, 5% DMSO in normal saline (vehicle) or iCRT3 at 5 or 10 mg/kg BW doses in 200 μl volume was delivered by intraperitoneal injection using 25 G × 7/8″ hypodermic needle. The investigator performing the animal experiments was blinded to the treatment assignment to eliminate any bias.[3]

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1. Glioblastoma in vivo experiment: Specific details such as animal model establishment (e.g., cell inoculation method, number of cells), iCRT3 dosage, administration route, and frequency were not explicitly stated. Pharmacological inhibition of the Wnt/β-catenin pathway by iCRT3 was used to observe the inhibitory effect on tumor growth [1]
2. Sepsis model experiment in C57BL/6 mice:
- Animal preparation: Male C57BL/6 mice were selected as experimental animals and randomly divided into sham-operated group, CLP + vehicle group, and CLP + iCRT3 treatment group (5 mg/kg and 10 mg/kg body weight, n = 5–8 mice per group) [3]
- Sepsis model establishment: Mice in the CLP groups were subjected to cecal ligation and puncture to induce sepsis, while the sham-operated group only underwent laparotomy without cecal ligation and puncture [3]
- Drug administration: At 5 h after CLP, mice in the treatment groups were intraperitoneally injected with iCRT3 dissolved in 5% DMSO in normal saline at the specified doses. Mice in the vehicle group were injected with the same volume of 5% DMSO in normal saline [3]
- Sample collection and detection: At 20 h after CLP, blood samples were collected from mice to detect plasma cytokine levels and organ injury markers. Lung tissues were harvested for histological analysis (H&E staining, Masson’s Trichrome staining), TUNEL staining, qPCR analysis of cytokine and chemokine mRNA expression, and MPO activity detection [3]

[1]. A Novel Positive Feedback Loop Between NTSR1 and Wnt/β-Catenin Contributes to Tumor Growth of Glioblastoma. Cell Physiol Biochem. 2017 Oct 24;43(5):2133-2142.

[2]. Inhibition of β-catenin-TCF1 interaction delays differentiation of mouse embryonic stem cells. J Cell Biol. 2015 Oct 12;211(1):39-51.

[3]. Mitigation of sepsis-induced inflammatory responses and organ injury through targeting Wnt/β-catenin signaling. doi: 10.1038/s41598-017-08711-6.

Background/aims: Neurotensin (NTS), an intestinal hormone, is profoundly implicated in cancer progression through binding its primary receptor NTSR1. The conserved Wnt/β-Catenin pathway regulates cell proliferation and differentiation via activation of the β-catenin/T-cell factor (TCF) complex and subsequent modulation of a set of target genes. In this study, we aimed to uncover the potential connection between NTS/NTSR1 signaling and Wnt/β-Catenin pathway.\n\nMethods: Genetic silencing, pharmacological inhibition and gain-of-function studies as well as bioinformatic analysis were performed to uncover the link between NTS/ NTSR1 signaling and Wnt/β-Catenin pathway. Two inhibitors were used in vivo to evaluate the efficiency of targeting NTS/NTSR1 signaling or Wnt/β-Catenin pathway.\n\nResults: We found that NTS/NTSR1 induced the activation of mitogen-activated protein kinase (MAPK) and the NF-κB pathway, which further promoted the expression of Wnt proteins, including Wnt1, Wnt3a and Wnt5a. Meanwhile, the mRNA and protein expression levels of NTSR1 were increased by the Wnt pathway activator Wnt3a and decreased by the Wnt inhibitor iCRT3 in glioblastoma cells. Furthermore, pharmacological inhibition of NTS/NTSR1 or Wnt/β-Catenin signaling suppressed tumor growth in vitro and in vivo.\n\nConclusion: These results reveal a positive feedback loop between NTS/NTSR1 and Wnt/β-Catenin signaling in glioblastoma cells that might be important for tumor development and provide potential therapeutic targets for glioblastoma.[1]

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\nThe ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/β-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how β-catenin's function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of β-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that β-catenin's function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein-protein interactions drive stem cell fate.\n\n[2]

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\n\nThe Wnt/β-catenin pathway has been involved in regulating inflammation in various infectious and inflammatory diseases. Sepsis is a life-threatening condition caused by dysregulated inflammatory response to infection with no effective therapy available. Recently elevated Wnt/β-catenin signaling has been detected in sepsis. However, its contribution to sepsis-associated inflammatory response remains to be explored. In this study, we show that inhibition of Wnt/β-catenin signaling reduces inflammation and mitigates sepsis-induced organ injury. Using in vitro LPS-stimulated RAW264.7 macrophages, we demonstrate that a small-molecule inhibitor of β-catenin responsive transcription, iCRT3, significantly reduces the LPS-induced Wnt/β-catenin activity and also inhibits TNF-α production and IκB degradation in a dose-dependent manner. Intraperitoneal administration of iCRT3 to C57BL/6 mice, subjected to cecal ligation and puncture-induced sepsis, decreases the plasma levels of proinflammatory cytokines and organ injury markers in a dose-dependent manner. The histological integrity of the lungs is improved with iCRT3 treatment, along with reduced lung collagen deposition and apoptosis. In addition, iCRT3 treatment also decreases the expression of the cytokines, neutrophil chemoattractants, as well as the MPO activity in the lungs of septic mice. Based on these findings we conclude that targeting the Wnt/β-Catenin pathway may provide a potential therapeutic approach for treatment of sepsis.[3]\n

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1. Background: Neurotensin (NTS) binds to its primary receptor NTSR1 to promote cancer progression. The Wnt/β-catenin pathway regulates cell proliferation and differentiation by activating the β-catenin/TCF complex. There is a positive feedback loop between NTS/NTSR1 and Wnt/β-catenin signaling in glioblastoma cells, which is involved in tumor development [1]
2. Mechanism of action: iCRT3 inhibits the Wnt/β-catenin signaling pathway, thereby reducing the expression of NTSR1 and suppressing glioblastoma tumor growth [1]
3. Background: The Wnt/β-catenin pathway regulates the self-renewal and differentiation of mESCs. β-catenin alleviates TCF3-mediated repression of the pluripotency transcriptional network (PTN) to promote pluripotency, while its interaction with TCF1 promotes mESC differentiation [2]
4. Mechanism of action: iCRT3 specifically inhibits the interaction between β-catenin and TCF1, reducing the transcriptional activity of differentiation-promoting target genes. It enhances mESC self-renewal, delays differentiation, and improves functional pluripotency without affecting the interaction between β-catenin and TCF3 [2]
5. Background: The Wnt/β-catenin pathway is involved in regulating inflammation in infectious and inflammatory diseases. Sepsis is caused by a dysregulated inflammatory response to infection, and elevated Wnt/β-catenin signaling has been detected in sepsis [3]
6. Mechanism of action: iCRT3 blocks the Wnt/β-catenin signaling pathway, thereby inhibiting the NF-κB pathway, reducing the production of proinflammatory cytokines and chemokines, alleviating lung collagen deposition, apoptosis, and neutrophil infiltration, and ultimately mitigating sepsis-induced inflammatory responses and organ injury [3]
7. Therapeutic potential: iCRT3 has potential therapeutic value for the treatment of glioblastoma by targeting the Wnt/β-catenin pathway [1]
8. Therapeutic potential: iCRT3 provides a tool for studying the role of β-catenin/TCF1 interaction in mESC fate regulation and may have potential applications in stem cell research [2]
9. Therapeutic potential: Targeting the Wnt/β-catenin pathway with iCRT3 may be a potential therapeutic approach for the treatment of sepsis [3]

C23H26N2O2S

394.53

394.171

C, 70.02; H, 6.64; N, 7.10; O, 8.11; S, 8.13

901751-47-1

6622273

White to off-white solid powder

5.195

80.43

1

4

9

28

462

0

QTDYVSIBWGVBKU-UHFFFAOYSA-N

InChI=1S/C23H26N2O2S/c1-3-18-9-11-20(12-10-18)23-25-21(17(2)27-23)15-28-16-22(26)24-14-13-19-7-5-4-6-8-19/h4-12H,3,13-16H2,1-2H3,(H,24,26)

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.34 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.34 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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