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Article(id=1308455398621598463, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308455335962894804, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-0088, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1737561600000, receivedDateStr=2025-01-23, revisedDate=1743523200000, revisedDateStr=2025-04-02, acceptedDate=null, acceptedDateStr=null, onlineDate=1789889676957, onlineDateStr=2026-09-20, pubDate=1749657600000, pubDateStr=2025-06-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789889676957, onlineIssueDateStr=2026-09-20, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789889676957, creator=13701087609, updateTime=1789889676957, updator=13701087609, issue=Issue{id=1308455335962894804, tenantId=1146029695717560320, journalId=1189982191388893191, year='2025', volume='60', issue='6', pageStart='1573', pageEnd='1964', issueExtLink='null', onlineDate='null', pubDate='1749657600000', pubDateStr='2025-06-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789889662017, creator='13701087609', updateTime=1789893590327, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308471812564734358, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308455335962894804, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308471812564734359, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308455335962894804, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1721, endPage=1729, ext={EN=ArticleExt(id=1308455398864868096, articleId=1308455398621598463, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=High-throughput discovery of novel anti-colorectal cancer traditional Chinese active small molecules targeting TRAF2, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

In colorectal cancer, tumor necrosis factor receptor-associated factor 2 (TRAF2) is a positive regulator of the Wnt/β-catenin signaling pathway and is closely related to the occurrence and development of colorectal cancer. This study aims to discover novel anti-colorectal cancer small molecules targeting TRAF2 by establishing a high-throughput TRAF2 affinity screening system. The study established and optimized a high-throughput TRAF2 screening system based on Dianthus, and conducted a large-scale screening of 3 716 traditional Chinese medicine (TCM) small molecules from a TCM small molecule library. Microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) were further applied to verify the binding in both purified proteins and living cells. Among the candidates, the strongest binding compound, eupatilin (EUP), had an affinity of 7.87 μmol·L-1 with TRAF2. TOPFlash assays showed that EUP can inhibit the activity of the Wnt/β-catenin signaling pathway and the growth of colorectal cancer cells and mouse small intestinal adenoma organoids. After TRAF2 was knocked out using CRISPR/Cas9 technology, the inhibitory effect of EUP on the Wnt signaling pathway and colon cancer cells was significantly weakened, indicating that TRAF2 is the functional target protein of EUP. In summary, the novel high-throughput screening method based on Dianthus constructed in this study can be used for the screening of small molecule targeted drugs, and it has been confirmed that EUP is a TRAF2-targeted small molecule that inhibits the development of colorectal cancer, providing a theoretical basis for the development of targeted TRAF2 anti-tumor drugs.

, authors=Jie JIANG, Hui-hui LI, Ying XUAN, Min YANG, Zi-ye WANG, Shi-cheng ZHENG, Tian-yi ZHANG, Jia-yu XIONG, Xi-song KE, Rong YAN*, authorsList=Jie JIANG, Hui-hui LI, Ying XUAN, Min YANG, Zi-ye WANG, Shi-cheng ZHENG, Tian-yi ZHANG, Jia-yu XIONG, Xi-song KE, Rong YAN, authorCompany=null, correspAuthors=Rong YAN, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2025 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1308455401385644809, articleId=1308455398621598463, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=靶向TRAF2新型抗结直肠癌中药活性小分子的高通量发现, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

在结直肠癌中, 肿瘤坏死因子受体相关因子2 (tumor necrosis factor receptor-associated factor 2, TRAF2) 是Wnt/β-catenin信号通路的正性调控因子, 与结直肠癌发生发展密切相关。本研究旨在通过建立高通量TRAF2亲和力筛选系统, 发现新型靶向TRAF2的抗结直肠癌小分子。本研究建立并优化了Dianthus高通量TRAF2筛选系统, 大规模筛选中药小分子库中3 716个中药小分子; 应用微量热涌动技术(microscale thermophoresis, MST) 和活细胞热转移实验(cellular thermal shift assay, CETSA) 在纯化蛋白和活细胞中进一步验证结合, 其中结合能力最强的异泽兰黄素(eupatilin, EUP) 与TRAF2的亲和力为7.87 μmol·L-1。通过TOPFlash等实验表明, EUP能抑制Wnt/β-catenin信号通路的活性和结直肠癌细胞及小鼠小肠腺瘤类器官的生长。通过CRISPR/Cas9技术敲除TRAF2后显著减弱了EUP对Wnt信号通路和结肠癌细胞的抑制作用, 表明TRAF2是EUP的功能靶标蛋白。综上, 本研究构建的Dianthus高通量筛选新方法可以应用于小分子靶向药物的筛选, 且通过验证发现EUP是抑制结直肠癌发生发展的TRAF2靶向小分子, 为开发靶向TRAF2的抗肿瘤药物提供了理论依据。

, authors=姜婕, 李慧慧, 宣颖, 杨敏, 王子叶, 郑诗成, 张天艺, 熊嘉昱, 柯细松, 闫蓉*, authorsList=姜婕, 李慧慧, 宣颖, 杨敏, 王子叶, 郑诗成, 张天艺, 熊嘉昱, 柯细松, 闫蓉, authorCompany=null, correspAuthors=闫蓉, authorNote=

作者贡献:

闫蓉和姜婕负责课题总体设计; 姜婕、李慧慧、宣颖、杨敏、王子叶、郑诗成、张天艺、熊嘉昱负责完成实验的具体实施; 姜婕负责实验数据分析、文章撰写; 闫蓉和柯细松对课题进行及论文撰写进行指导。

, correspAuthorsNote=
闫蓉, Tel: 86-21-51323190, E-mail:
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Antioxidants (Basel), 2021, 10: 957-972., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1308497550232093063, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, xref=null, ext=[AuthorCompanyExt(id=1308497550240481672, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, companyId=1308497550232093063, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China), AuthorCompanyExt(id=1308497550248870281, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, companyId=1308497550232093063, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=上海中医药大学交叉科学研究院, 上海 201203)])], figs=[ArticleFig(id=1308497557546959303, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=EN, label=null, caption=null, figureFileSmall=kbk4gC4B5P5Ekl1zoCoXwg==, figureFileBig=AIFTfUC+bP0VELPJGaUeEA==, tableContent=null), ArticleFig(id=1308497557651816904, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=CN, label=Fig.1, caption=The establishment of high-throughput screening system. 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A: Microscale thermophoresis (MST) assay was used to detect the binding of purified TRAF2 and candidate compounds; B: Cellular thermal shift assay (CETSA) was used to detect the changes of TRAF2 protein levels in HCT116 cells upon the treatment with candidate compounds (50 μmol·L-1) for 1 h. Tm: Temperature; C: CETSA assay was used to detect the changes of TRAF2 protein levels in LS174T cells upon the treatment of candidate compounds (50 μmol·L-1) for 1 h; D: The chemical structures of EUP, BEN, and ISO, figureFileSmall=sFtg/+FjahM3iH+AX/FYuQ==, figureFileBig=h1dk0MXJkc98kORlD3QrpA==, tableContent=null), ArticleFig(id=1308497558104801742, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=EN, label=null, caption=null, figureFileSmall=ofRvsaTVpvBdr9I99Ha5mw==, figureFileBig=mQxXfQGLeJYJwmMzLhIoHg==, tableContent=null), ArticleFig(id=1308497558394208719, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=CN, label=Fig.3, caption=EUP inhibited the Wnt/β-catenin signaling pathway by targeting TRAF2. A: TOPFlash assay was performed in HCT116 cells upon the treatment of candidate compounds (10 μmol·L-1) for 12 h; B: The expression of Wnt target genes was analyzed by RT-PCR in SW480, SW620, and DLD1 cells upon the treatment of EUP (10 μmol·L-1) for 12 h; C: Immunofluorescence assay was conducted in HEK293T cells upon the treatment of EUP (10 μmol·L-1) for 24 h, and Wnt 3A conditioned medium for 3 h; D: Western blot analysis was performed to validate TRAF2 expression in WT and TRAF2-/- in HEK293T and HCT116 cells; E: TOPFlash assay was performed in HCT116 WT and TRAF2-/- cells upon the treatment of EUP with dose course (0, 10, 25, 50 μmol·L-1) for 12 h; F: Immunofluorescence assay was performed in HEK293T cells upon the treatment of EUP (10 μmol·L-1) for 24 h, and 6-bromoindirubin-3'-oxime (6BIO) for 3 h. n = 3, x±s. Scale bars: 5 and 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001. ns: No significance, figureFileSmall=ofRvsaTVpvBdr9I99Ha5mw==, figureFileBig=mQxXfQGLeJYJwmMzLhIoHg==, tableContent=null), ArticleFig(id=1308497558482289104, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=EN, label=null, caption=null, figureFileSmall=S/WvOB1UOKyPBC3RbLs9vA==, figureFileBig=burRoozJizEfRrKQDyQV5Q==, tableContent=null), ArticleFig(id=1308497558570369489, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=CN, label=Fig.4, caption=EUP inhibited the growth of colorectal cancer. A: CCK8 assay was performed in HCT116 cells after the treatment with candidate compounds at an increasing dose (0, 2, 5, 10, 20, 50 μmol·L-1) for 72 h (n = 3); B: CCK8 assay was conducted in HCT116 WT and TRAF2-/- cells upon the treatment of EUP with dose course (0, 2, 5, 10, 20, 50 μmol·L-1) for 72 h (n = 3); C: APCmin/+ organoid models were used to evaluate the efficacy of EUP (10 μmol·L-1) on tumor growth after three consecutive days of treatment (n = 12). Scale bars: 1 μm. x±s. *P < 0.05, **P < 0.01, ***P < 0.001, figureFileSmall=S/WvOB1UOKyPBC3RbLs9vA==, figureFileBig=burRoozJizEfRrKQDyQV5Q==, tableContent=null), ArticleFig(id=1308497558763307474, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
HitCategoryKd /µmol·L-1
Palmaturbine hydroxide (PAL)Binder42.6
Eupatilin (EUP)Binder56.96
Benzylideneacetone (BEN)Potential binder/
Isovitexin (ISO)Potential binder/
Natamycin (NATA)Potential binder/
Tricosanoic acid (TA)Weak binder128.66
(±)-Naringenin [(±)-NGN]Weak binder300.01
Panasenoside (PAN)Weak binder422.13
β-Caryophyllene (BCP)Weak binder465.66
), ArticleFig(id=1308497558842999251, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308455398621598463, language=CN, label=Tab.1, caption=

High-throughput screening results of small molecules binding to tumor necrosis factor receptor-associated factor 2 (TRAF2)

, figureFileSmall=null, figureFileBig=null, tableContent=
HitCategoryKd /µmol·L-1
Palmaturbine hydroxide (PAL)Binder42.6
Eupatilin (EUP)Binder56.96
Benzylideneacetone (BEN)Potential binder/
Isovitexin (ISO)Potential binder/
Natamycin (NATA)Potential binder/
Tricosanoic acid (TA)Weak binder128.66
(±)-Naringenin [(±)-NGN]Weak binder300.01
Panasenoside (PAN)Weak binder422.13
β-Caryophyllene (BCP)Weak binder465.66
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靶向TRAF2新型抗结直肠癌中药活性小分子的高通量发现
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姜婕 , 李慧慧 , 宣颖 , 杨敏 , 王子叶 , 郑诗成 , 张天艺 , 熊嘉昱 , 柯细松 , 闫蓉 *
药学学报 | 研究论文 2025,60(6): 1721-1729
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药学学报 |研究论文 2025 , 60 (6) : 1721 -1729
靶向TRAF2新型抗结直肠癌中药活性小分子的高通量发现
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姜婕, 李慧慧, 宣颖, 杨敏, 王子叶, 郑诗成, 张天艺, 熊嘉昱, 柯细松, 闫蓉*
作者信息
  • 上海中医药大学交叉科学研究院, 上海 201203
通讯作者:
闫蓉, Tel: 86-21-51323190, E-mail:
作者简介:

作者贡献:

闫蓉和姜婕负责课题总体设计; 姜婕、李慧慧、宣颖、杨敏、王子叶、郑诗成、张天艺、熊嘉昱负责完成实验的具体实施; 姜婕负责实验数据分析、文章撰写; 闫蓉和柯细松对课题进行及论文撰写进行指导。

High-throughput discovery of novel anti-colorectal cancer traditional Chinese active small molecules targeting TRAF2
Jie JIANG, Hui-hui LI, Ying XUAN, Min YANG, Zi-ye WANG, Shi-cheng ZHENG, Tian-yi ZHANG, Jia-yu XIONG, Xi-song KE, Rong YAN*
Affiliations
  • Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
出版时间: 2025-06-12 doi: 10.16438/j.0513-4870.2025-0088
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在结直肠癌中, 肿瘤坏死因子受体相关因子2 (tumor necrosis factor receptor-associated factor 2, TRAF2) 是Wnt/β-catenin信号通路的正性调控因子, 与结直肠癌发生发展密切相关。本研究旨在通过建立高通量TRAF2亲和力筛选系统, 发现新型靶向TRAF2的抗结直肠癌小分子。本研究建立并优化了Dianthus高通量TRAF2筛选系统, 大规模筛选中药小分子库中3 716个中药小分子; 应用微量热涌动技术(microscale thermophoresis, MST) 和活细胞热转移实验(cellular thermal shift assay, CETSA) 在纯化蛋白和活细胞中进一步验证结合, 其中结合能力最强的异泽兰黄素(eupatilin, EUP) 与TRAF2的亲和力为7.87 μmol·L-1。通过TOPFlash等实验表明, EUP能抑制Wnt/β-catenin信号通路的活性和结直肠癌细胞及小鼠小肠腺瘤类器官的生长。通过CRISPR/Cas9技术敲除TRAF2后显著减弱了EUP对Wnt信号通路和结肠癌细胞的抑制作用, 表明TRAF2是EUP的功能靶标蛋白。综上, 本研究构建的Dianthus高通量筛选新方法可以应用于小分子靶向药物的筛选, 且通过验证发现EUP是抑制结直肠癌发生发展的TRAF2靶向小分子, 为开发靶向TRAF2的抗肿瘤药物提供了理论依据。

肿瘤坏死因子受体相关因子2  /  高通量亲和力筛选  /  中药小分子  /  异泽兰黄素  /  Wnt/β-catenin信号通路

In colorectal cancer, tumor necrosis factor receptor-associated factor 2 (TRAF2) is a positive regulator of the Wnt/β-catenin signaling pathway and is closely related to the occurrence and development of colorectal cancer. This study aims to discover novel anti-colorectal cancer small molecules targeting TRAF2 by establishing a high-throughput TRAF2 affinity screening system. The study established and optimized a high-throughput TRAF2 screening system based on Dianthus, and conducted a large-scale screening of 3 716 traditional Chinese medicine (TCM) small molecules from a TCM small molecule library. Microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) were further applied to verify the binding in both purified proteins and living cells. Among the candidates, the strongest binding compound, eupatilin (EUP), had an affinity of 7.87 μmol·L-1 with TRAF2. TOPFlash assays showed that EUP can inhibit the activity of the Wnt/β-catenin signaling pathway and the growth of colorectal cancer cells and mouse small intestinal adenoma organoids. After TRAF2 was knocked out using CRISPR/Cas9 technology, the inhibitory effect of EUP on the Wnt signaling pathway and colon cancer cells was significantly weakened, indicating that TRAF2 is the functional target protein of EUP. In summary, the novel high-throughput screening method based on Dianthus constructed in this study can be used for the screening of small molecule targeted drugs, and it has been confirmed that EUP is a TRAF2-targeted small molecule that inhibits the development of colorectal cancer, providing a theoretical basis for the development of targeted TRAF2 anti-tumor drugs.

tumor necrosis factor receptor-associated factor 2  /  high-throughput affinity screening  /  TCM small molecule  /  eupatilin  /  Wnt/β-catenin signaling pathway
姜婕, 李慧慧, 宣颖, 杨敏, 王子叶, 郑诗成, 张天艺, 熊嘉昱, 柯细松, 闫蓉. 靶向TRAF2新型抗结直肠癌中药活性小分子的高通量发现. 药学学报, 2025 , 60 (6) : 1721 -1729 . DOI: 10.16438/j.0513-4870.2025-0088
Jie JIANG, Hui-hui LI, Ying XUAN, Min YANG, Zi-ye WANG, Shi-cheng ZHENG, Tian-yi ZHANG, Jia-yu XIONG, Xi-song KE, Rong YAN. High-throughput discovery of novel anti-colorectal cancer traditional Chinese active small molecules targeting TRAF2[J]. Acta Pharmaceutica Sinica, 2025 , 60 (6) : 1721 -1729 . DOI: 10.16438/j.0513-4870.2025-0088
正文
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肿瘤坏死因子受体相关因子2 (tumor necrosis factor receptor-associated factor 2, TRAF2) 是典型的信号转接因子, 广泛参与肿瘤信号通路, 在结直肠癌、肺癌和乳腺癌等多种肿瘤中TRAF2有异常激活, 基因敲除或敲低TRAF2显著抑制肿瘤的生长和侵袭[1, 2]。课题组前期研究发现, TRAF2在结直肠癌中会与Wnt信号通路激活的关键蛋白β-catenin、TCF4和TNIK结合形成复合物, 且TRAF2是该复合物形成过程中的必要条件[3]。其次, 敲除TRAF2使结直肠癌细胞HCT116的克隆形成数量显著减少; 在裸鼠异种移植瘤模型中, TRAF2敲除的HCT116细胞形成的肿瘤生长速度显著减缓, 这表明TRAF2在肿瘤生长中起关键作用。此外, TCGA数据显示TRAF2在结直肠癌中的表达水平显著高于正常组织, 与结直肠癌患者的生存期呈负相关(http://gepia.cancer-pku.cn/), 进一步表明TRAF2是治疗结直肠癌的潜在靶点。
TRAF2的蛋白结构已被解析超过20年[4, 5], 唯一报道的靶向TRAF2的小分子是由本课题组基于表型筛选得到的中药路路通的活性成分之一路路通酸(liquidambaric acid, LDA)[3]。LDA在多种结直肠癌细胞模型中表现出显著的抗肿瘤活性, 且研究发现LDA通过破坏TRAF2/β-catenin和β-catenin/TCF4的相互作用抑制Wnt/β-catenin信号通路[3]。然而, LDA是一种五环三萜类化合物, 存在溶解度差、结构改造困难和生物利用度低等问题。
小分子的亲和力筛选是开发抗肿瘤药物的关键步骤, 它有助于识别和优化与靶标蛋白结合的药物候选小分子[6]。然而, 目前的药物亲和力筛选方法存在局限[7]。基于此, 本研究通过Dianthus仪器建立并优化了靶向TRAF2的亲和力筛选体系, 筛选得到了苗头化合物。该体系在40 min内可以同时完成384个样品的扫描, 极大增加了筛选通量, 缩短了检测的时间。该体系的建立为高效化开发靶向TRAF2的抗结直肠癌中药活性小分子奠定了基础, 同时为其他蛋白的靶向药物高通量发现提供了新思路。
材料与方法
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材料   Dianthus筛选仪器、Dianthus 384孔板、蛋白氨基标记试剂盒、16道移液器枪头购自德国Nanotemper科技有限公司。人结直肠癌细胞HCT116、LS174T、DLD1、SW480、SW620和人胚胎肾细胞HEK293T均购自中国科学院细胞库; Apcmin/+小鼠购自江苏集萃药康生物科技股份有限公司。小鼠小肠类器官Plus版试剂盒购自苏州伯桢生物科技公司(货号: K2001P-MI)。CCK8试剂购自美国APExBIO公司。TRAF2抗体购自美国Cell Signaling Technology公司(货号: 4724S); mouse-488荧光二抗及rabbit-568荧光二抗购自Cell Signaling Technology公司。中药小分子购自美国Med Chem Express生物科技公司; 蛋白酶抑制剂购自瑞士Roche公司; 二甲基亚砜DMSO购自德国Sigma公司; RPMI 1640培养基、DMEM培养基及胎牛血清购自美国Gibco Life Technologies生物试剂有限公司; 青霉素-链霉素溶液购自美国Invitrogen公司; Hoechst 33342、4%多聚甲醛溶液、Triton X-100、BSA购自上海碧云天生物技术有限公司。
细胞培养   所有细胞均在添加1%青霉素-链霉素和10%胎牛血清的培养基中生长。细胞均在含5% CO2的37 ℃培养箱中培养。
蛋白纯化   为了纯化人TRAF2的TRAF结构域(残基310~501), 采用pET24d载体过表达, 纯化了含有羧基末端多组氨酸标签的TRAF结构域[5]。重组蛋白经Ni-NTA亲和层析纯化通过尺寸排除。收集纯化蛋白, 浓缩于20 mmol·L-1 Tris (pH 7.5)、150 mmol·L-1 NaCl、2 mmol·L-1 TCEP和10%甘油中, 保存于-80 ℃。
缓冲液配置   150 mmol·L-1 Tris-HCl、150 mmol·L-1 NaCl、10 mmol·L-1 MgCl2、0.1% pluronic F-127, 含2.5% DMSO, pH为7.4, 配置后抽滤。
Dianthus单点筛选   取出存放小分子的96孔板室温融化后离心。加入57 μL buffer将化合物稀释至500 μL, 离心, 封板, 使用微孔板振荡器进行混匀。使用16道移液器从储液槽中吸取48 nmol·L-1蛋白10 μL加入Dianthus 384孔板, 不封板离心。取10 μL稀释后的化合物加入Dianthus孔板中, 离心并封板, 混匀。去掉封膜, 离心, 重新封膜。上机检测。离心参数为1 000 ×g, 2 min, 混匀参数为2 500 r·min-1, 5 min。
Dianthus亲和力筛选   用含5% DMSO的缓冲溶液将初始浓度为1 mmol·L-1的小分子于Dianthus 384孔板内梯度稀释12组, 体积为10 μL。随即用16道移液器同时加入48 nmol·L-1蛋白10 μL, 离心并混匀, 最后放入Dianthus仪器检测。离心参数为1 000 ×g, 2 min, 混匀参数为2 500 r·min-1, 5 min。
微量热泳动(microscale thermophoresis, MST)   将TRAF2纯化蛋白按照一定比例稀释, 利用微量热涌动仪检测蛋白稀释后的荧光值, 选取荧光值在200~400的蛋白稀释倍数, 将蛋白按照比例稀释好以备用。用含10% DMSO的PBS溶液将小分子梯度稀释, 浓度从100 μmol·L-1梯度稀释16组, 每组留5 μL的小分子溶液, 随即与前面稀释好的蛋白同时孵育, 最后按照小分子梯度稀释顺序放入MST仪进行检测, 仪器设置条件: 25 ℃, 20% LED功率, 10% IR激光功率; 最后用Nano Temper软件计算Kd值。
细胞热转移实验(cellular thermal shift assay, CETSA)   收集细胞前, HCT116细胞用DMSO或小分子孵育1 h, 用含有蛋白酶抑制剂的预冷PBS进行裂解, 即刻每组平均分成7管, 在预设好的温度梯度上孵育3 min, 随后进行3次冻融循环。每个循环, 试管暴露在液氮中3 min, 然后在25 ℃下保持3 min。最后在4 ℃下以20 000 ×g离心20 min, 转移上清液进行变性, 用于Western blot实验。
TOPFlash实验   通过双荧光素酶报告系统检测Wnt通路转录活性方法, 可用于研究小分子对Wnt通路活性的作用[8]。TOPFlash是一个由β-catenin介导的转录激活的荧光素酶报告系统, 其启动子有TCF/LEF结合位, 可依据β-catenin的活性强弱, 调控下游萤火虫荧光素酶的表达量高低。同时, 该实验还使用一个不受β-catenin调控的renilla质粒作为对照检测荧光值, 以消除细胞数目、活力、转染效率等的影响。
Wnt 3A培养基制备   L-Wnt 3A细胞密度达100%汇合度时, 以1∶10比例进行细胞传代。细胞生长4天后, 收取细胞培养基并用无菌过滤器进行过滤, 即得到第一批的Wnt 3A培养基。再往细胞中加入新鲜培养基, 培养3天, 收取细胞培养基后用无菌过滤器进行过滤, 即得到第二批Wnt 3A培养基。将第一批和第二批培养基1∶1混合, 即为Wnt 3A的条件培养基(Wnt 3A培养基)。
免疫荧光(immunofluorescence, IF) 实验   将HEK293T细胞以合适密度接种到共聚焦皿中, 细胞贴壁后给药。细胞用4%多聚甲醛固定, 0.1% Triton X-100通透, 5% BSA溶液封闭。再用一抗和二抗依次孵育, 用Hoechst 33342染液染细胞核。使用共聚焦显微镜进行拍照。
CRISPR/Cas9敲除实验   sgRNA构建: 在TRAF2蛋白编码区侧翼设计两个小向导RNA (sgRNA1和2); sgRNA寡核苷酸序列为: TRAF2 sgRNA1-F: CACCGCAGGCGGAGCACAGGTACT; TRAF2 sgRNA1-R: CACCGCAGGCGGA GCACAGGTACT; TRAF2 sgRNA2-F: CACCGATGGCATCGTCCCGCACGT; TRAF2 sgRNA2-R: AAACACGTGCGGGACGATGCCATC。合成引物经磷酸化, 退火后线性化, 去磷酸化, 得到靶向引物与线性化载体的连接产物, 成功构建TRAF2敲除的载体质粒。随后将敲除TRAF2的质粒对共转染至HEK293T细胞和HCT116细胞中, 转染48 h后, 流式分选技术富集细胞, 并铺单克隆培养。通过DNA测序和Western blot实验进一步证实阳性克隆。
Western blot实验   用含有蛋白酶抑制剂的RIPA裂解液裂解细胞, 冰上裂解30 min后, 放入4 ℃的离心机, 14 000 r·min-1离心15 min。取蛋白上清液进行BSA定量。根据蛋白浓度取适量蛋白加入5× SDS上样缓冲液, 100 ℃金属浴变性10 min得到样品。每个样品以20 μg蛋白量用7.5% SDS凝胶进行电泳, 并转移到PVDF膜。5%脱脂奶粉封闭, 一抗和二抗依次孵育, 化学发光法检测条带。
CCK8实验   将HCT116细胞接种至96孔板中, 使每孔细胞数为1×104个, 细胞贴壁后给药, 药物终浓度为2、5、10、20、50 μmol·L-1, 对照组加入等量DMSO。72 h后, 每孔加入10 μL CCK8试剂, 轻轻混匀放回培养箱孵育1 h, 孵育完成后使用酶标仪在450 nm波长下测定各孔的吸光度(A) 值。通过公式“细胞活力(%) = (A实验组-A空白组)/(A对照组-A空白组) × 100%”计算细胞活力。
类器官药效评价   按照小鼠小肠类器官Plus版试剂盒流程提取APC突变C57BL/6J小鼠类器官。得到的小肠腺瘤类器官用小鼠小肠类器官完全培养基于含5% CO2的37 ℃恒温培养箱培养。待类器官长成后, 将合适密度的类器官铺在24孔板的两个孔, 两天后更换新鲜培养基并给药小分子EUP及溶剂对照DMSO, 分别在0、24、48及72 h时对类器官的生长情况进行拍摄记录。
统计学方法   所有数据统计分析均采用Image J软件和GraphPad Prism软件, 数据以x±s表示, P < 0.05被认为差异有统计学意义。
结果
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1　Dianthus高通量筛选体系的建立与应用
Dianthus的筛选原理是基于光谱位移(spectral shift) 和温度依赖的荧光强度变化(temperature-related intensity change, TRIC) 技术的双重检测。将目标蛋白标记上荧光基团, 荧光标记的蛋白具有特定的发射光谱。小分子配体与蛋白结合后会引起发射光谱出现微弱的蓝移或红移, 从而可以通过光谱位移的检测得出解离常数Kd值, 并通过TRIC技术监测样品聚集和质控。
将TRAF2标记为带有荧光的TRAF2-NHS-Red, 随后使用binding check定性判断阳性对照小分子路路通酸与TRAF2结合(图1A)。在过程中, 根据结合信号逐步优化缓冲液体系, 以防止或减少蛋白聚集, 避免假阳性曲线的产生, 最终确定缓冲液成分, 并在配置后通过抽滤和离心, 减少杂质对体系的干扰。其次, 本研究在每一列的第一个孔和最后一个孔分别设置阴性对照和阳性对照及化合物两个复孔, 以确保检测体系的状态稳定和质量均一。
设置TRAF2蛋白浓度为24 nmol·L-1, 阴性对照为2.5% DMSO, 阳性对照为同等DMSO浓度的250 μmol·L-1 LDA。Z-score选择区间为3~7, 越大越严格, 本研究设置Z-score为7, standard deviation为0.005 799 62, hit threshold为Δratio ≥ 0.041。最后, 建立了基于Dianthus的靶向TRAF2高通量筛选体系(图1B)。
本研究对3 716个天然产物小分子进行初步单点筛选。在筛选结果的标准判定中, Hit定义为响应值超过hit threshold, 且数据质控较好的小分子; auto-fluorescence为初始荧光强度比阴性对照高20%的小分子; inconclusive则是指两次重复响应值不一致, 数据质控异常或初始荧光强度比阴性对照低20%的小分子; no binder即信号响应值低于hit threshold, 且数据质控较好的小分子。其中, 筛选得到了128个Hit小分子, 并在后续进行亲和力筛选(图1C)。
为了进一步确认Hit小分子与TRAF2的结合, 本研究在128个Hit中挑选排名前40响应值较高的小分子进行Dianthus亲和力复筛。Binder为能拟合出化学平衡常数Kd值的小分子; weak binder为结合曲线饱和度小于50%, Kd值大于100的小分子; potential binder的初始荧光不均一或信噪比较低, 需要调整实验条件的潜在结合小分子。
复筛的结果如表1所示。共得到了结合TRAF2的9个候选化合物, 其中巴马亭红碱(palmaturbine hydroxide, PAL) 和异泽兰黄素(eupatilin, EUP) 与TRAF2有较强的结合; 苯苄叉酮(benzylideneacetone, BEN)、异牡荆黄素(isovitexin, ISO) 和纳他霉素(natamycin, NATA) 与TRAF2有潜在结合, 仍需后续验证; 二十三酸(tricosanoic acid, TA)、(±)-柚皮素[(±)-naringenin, (±)-NGN]、人参黄酮苷(panasenoside, PAN) 和β-石竹烯(β-caryophyllene, BCP) 与TRAF2的结合相对较弱。
2　候选化合物的细胞内外结合验证
利用MST技术将复筛得到的候选小分子与TRAF2纯化蛋白进行体外验证。结果表明, 小分子PAL、EUP、BEN、ISO和NATA能与TRAF2纯化蛋白直接结合(图2A), 其中EUP与TRAF2的结合亲和力最高, Kd值为7.87 µmol·L-1。
随后, 使用CETSA方法测定小分子与TRAF2在结直肠癌活细胞内的结合。在HCT116细胞中, 小分子EUP、BEN和ISO可以明显改变TRAF2的热稳定性(图2B、D)。为进一步确定这3个小分子的细胞内结合情况, 本研究继续在LS174T细胞上进行验证, 结果表明小分子EUP、BEN和ISO同样可以明显改变LS174T细胞中TRAF2的热稳定性(图2C)。上述实验结果表明, 小分子EUP、BEN、ISO不仅与TRAF2纯化蛋白存在体外结合, 且在活细胞内也与TRAF2存在结合。
3　异泽兰黄素通过靶向TRAF2抑制Wnt/β-catenin信号通路
Wnt/β-catenin信号级联在驱动结直肠癌的发生发展中具有关键作用, 且TRAF2是Wnt信号通路的正性调控因子[3]。鉴于此, 本研究考察了EUP、BEN和ISO对Wnt/β-catenin信号通路的作用。TOPFlash结果表明, EUP可以显著抑制HCT116细胞的Wnt活性(图3A)。随后, 分别在3种Wnt激活的APC突变细胞系DLD1、SW480、SW620上考察了EUP对Wnt/β-catenin下游靶基因转录的影响。RT-PCR实验表明, EUP能够显著抑制Wnt靶基因AXIN2和BMP4的转录(图3B)。
β-catenin核转位是Wnt信号通路调控细胞行为的关键环节[9]。当Wnt通路被激活时, 细胞质中的β-catenin积累并转移到细胞核内, 与TCF/LEF家族的转录因子结合, 启动下游靶基因的表达[10]。在HEK293T细胞上使用Wnt 3A条件培养基处理后, 细胞核中的β-catenin有明显积累, EUP处理则部分抑制了Wnt激活引起的β-catenin入核(图3C), 且EUP对β-catenin在细胞上的分布、荧光强度无明显影响。
为了确定EUP是否是通过TRAF2介导Wnt/β-catenin信号通路, 本研究利用CRISPR/Cas9技术在HEK293T和HCT116细胞上构建了TRAF2敲除细胞(图3D)。通过TOPFlash实验, 观察到在HCT116细胞中EUP对Wnt抑制作用呈现出明显的浓度依赖性; 当TRAF2被敲除后, EUP对Wnt通路的抑制作用被显著减弱(图3E)。值得注意的是, TRAF2敲除本身也会对Wnt通路的激活产生抑制效果, 这也进一步说明了TRAF2在Wnt信号通路调控中的重要作用。
6-Bromoindirubin-3'-oxime (6BIO) 是GSK3β的小分子抑制剂, 对Wnt通路有明确的激活作用[11]。免疫荧光实验表明, EUP也能够抑制由6BIO激活的β-catenin核转位, 同时敲除TRAF2后, EUP的抑制作用被明显阻断(图3F)。以上结果表明, EUP抑制Wnt通路的作用依赖于其靶标蛋白TRAF2。
4　异泽兰黄素具有抗结直肠癌活性
为了探究EUP抗结直肠癌的作用, 本研究在细胞水平采用CCK8实验对小分子作用于HCT116的细胞增殖情况进行了观察(图4A)。EUP在20 μmol·L-1能够显著抑制HCT116的活性, 而BEN和ISO未见抑制效果。在敲除TRAF2的HCT116细胞上, EUP的抑制作用被阻断(图4B), 这表明EUP抑制结直肠癌细胞增殖的作用依赖TRAF2。
类器官是一种体外的三维立体细胞簇, 其空间结构与真实器官相近, 能够保留肿瘤组织的病理特征, 精准模拟人体器官的生理及病理状态, 是研究抗肿瘤药效的理想实验模型[12-14]。本研究通过构建APCmin/+小鼠小肠腺瘤类器官模型评价EUP的抗肿瘤活性, EUP 10 μmol·L-1处理类器官, 并在不同时间点观测小分子对类器官生长的作用。结果表明, EUP在24 h即可抑制小肠腺瘤类器官的生长, 且在后续时间点的监测中, EUP组的类器官一直处于生长抑制状态(图4C)。综上所述, EUP有较好的抗结直肠癌作用。
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目前, 已有研究借助大规模基因组测序和全基因敲除技术发现了500多个肿瘤驱动因子和潜在治疗靶标, 但约90%是“不可成药”或“难以成药”蛋白[15-17]。于1999年被鉴定出来的TRAF2便属于缺乏典型小分子结合口袋的“不可成药”蛋白[5, 15]。
EUP是从艾草(Artemisia argyi) 中提取的具有生物活性的黄酮类化合物, 具有多种药理作用。EUP通过抑制丝裂原活化蛋白激酶(MAPK) 和核因子κB (NF-κB), 抑制H2O2诱导的细胞凋亡, 发挥抗氧化作用[18]; 通过抑制炎症和氧化应激, 减轻急性肺损伤等[19]。此外, 已有研究报道EUP具有抑制Wnt/β-catenin信号通路的作用, 以缓解慢性鼻窦炎伴鼻息肉的炎症和上皮-间质转化[20]。在抗肿瘤方面, 有研究报道EUP通过降低线粒体膜电位和增加活性氧水平, 诱导结直肠癌细胞凋亡从而达到抗结直肠癌效果, 但未指出具体的药效靶点[21]。
Dianthus是近几年面世的第一台集检测及分析于一体的高通量分子互作亲和力检测仪器, 目前尚未有报道Dianthus筛选技术的应用情况, 也未有成体系的针对TRAF2的筛选方法。本研究通过构建基于Dianthus的靶向“不可成药”蛋白高通量筛选新方法, 成功发现并验证了中药活性小分子EUP通过TRAF2发挥抗结直肠癌作用。该方法的建立不仅为靶向TRAF2相关药物的研发和改造奠定了有力基础, 也为其他“不可成药”蛋白的小分子药物开发提供了新途径。

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2025年第60卷第6期
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doi: 10.16438/j.0513-4870.2025-0088
  • 接收时间：2025-01-23
  • 首发时间：2026-09-20
  • 出版时间：2025-06-12
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  • 收稿日期：2025-01-23
  • 修回日期：2025-04-02
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    上海中医药大学交叉科学研究院, 上海 201203

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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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