Article(id=1198652615572681503, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0553, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1682870400000, receivedDateStr=2023-05-01, revisedDate=1689177600000, revisedDateStr=2023-07-13, acceptedDate=null, acceptedDateStr=null, onlineDate=1763710653441, onlineDateStr=2025-11-21, pubDate=1691769600000, pubDateStr=2023-08-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763710653441, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763710653441, creator=13701087609, updateTime=1763710653441, updator=13701087609, issue=Issue{id=1198652605778985059, tenantId=1146029695717560320, journalId=1189982191388893191, year='2023', volume='58', issue='8', pageStart='0', pageEnd='2540', issueExtLink='null', onlineDate='null', pubDate='1691769600000', pubDateStr='2023-08-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763710651106, creator='13701087609', updateTime=1763710739504, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1198652976601596347, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1198652976601596348, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2193, endPage=2202, ext={EN=ArticleExt(id=1198652616088580921, articleId=1198652615572681503, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Research progress in controllable proteolysis targeting chimeras, columnId=null, journalTitle=Acta Pharmaceutica Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=

Proteolysis targeting chimeras (PROTACs) is an innovative technique in targeted protein degradation. PROTACs is a heterobifunctional molecule which can bind to the E3 ligase and target protein to form a ubiquitination complex, resulting in the ubiquitin-proteasome system dependent degradation of target protein. PROTACs has been regarded as the promising method in drug discovery campaign, for its high commonality, potent degradation activity and unique selectivity profile. However, the catalytic mechanism also induces the uncontrollable protein degradation risk. Controllable PROTACs contain the responsive element in the molecular entity. In certain conditions, the element can be triggered to activate or terminate the degradation event. In this review, we will briefly summarize the strategies in controllable PROTACs and describe the representative examples according to the responsive mechanism. We hope this review could provide some insight into the further development of controllable PROTACs.

, authors=null, authorsList=Yi MOU, Shuai WEN, Yan WANG, Zheng-yu JIANG, authorCompany=null, correspAuthors=Zheng-yu JIANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2023 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=1198652623034347814, articleId=1198652615572681503, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=可控性蛋白降解靶向嵌合体(PROTACs) 技术研究进展, columnId=1190335349655180086, journalTitle=药学学报, columnName=综述, runingTitle=null, highlight=null, articleAbstract=

蛋白降解靶向嵌合体(PROTACs) 是一类新型蛋白降解技术, 能同时结合E3连接酶与疾病相关蛋白, 利用内源性泛素-蛋白酶体系降解靶蛋白。PROTACs具有较佳的通用性、较强的蛋白降解活性和较好的目的蛋白选择性, 成为靶向蛋白降解领域的热点技术, 但具有降解不受控的风险。可控PROTACs引入了特定的刺激响应触发元件, 在特定的响应条件下可被触发, 产生/终止后续的蛋白质降解效应。本文概述了可控PROTACs设计策略, 并按照相应机制不同对代表化合物和研究进展进行了介绍, 从而为可控PROTACs的研究提供借鉴和思路。

, authors=null, authorsList=牟伊, 文帅, 王燕, 姜正羽, authorCompany=null, correspAuthors=姜正羽, authorNote=null, correspAuthorsNote=
*姜正羽, Tel: 86-25-83271351, E-mail:
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Jiangsu Key Laboratory of Drug Design and Optimization, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China), AuthorCompanyExt(id=1198960101143966569, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, companyId=1198960101127189351, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.中国药科大学药学院, 江苏省药物分子设计与成药性优化重点实验室, 江苏 南京 210009)])], figs=[ArticleFig(id=1198960104742678659, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=EN, label=null, caption=null, figureFileSmall=wwBjki71VgH+nGy3ODR9eQ==, figureFileBig=CmevEigAtrqq+muAKk1lsA==, tableContent=null), ArticleFig(id=1198960104939810966, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=CN, label=Figure 1, caption= The mechanism of PROTACs. PROTACs consists of three parts: target protein ligand, E3 ligase ligand and "linker" structure that connects the two ligands. PROTACs binds to the target protein and E3 ligase respectively through ligands at both ends to form a stable terpolymer complex, which labels the target protein for ubiquitination, and the ubiquitinated target protein is then recognized and degraded by the proteasome in the cell. PROTACs: Proteolysis targeting chimeras , figureFileSmall=wwBjki71VgH+nGy3ODR9eQ==, figureFileBig=CmevEigAtrqq+muAKk1lsA==, tableContent=null), ArticleFig(id=1198960105266966696, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=EN, label=null, caption=null, figureFileSmall=SwYBX6HxiWE8kjbpZ+TG7Q==, figureFileBig=2DBjmHvOaPiOV8jgh6Vg8A==, tableContent=null), ArticleFig(id=1198960105426350257, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=CN, label=Figure 2, caption= The mechanism of controlled PROTACs. The development strategy of controllable-PROTACs are mainly to block key binding sites in molecules with different stimulus response fragments (cage) to make the molecules lose degradation activity. Controllable-PROTACs are activated under specific response conditions to produce subsequent protein degradation effects , figureFileSmall=SwYBX6HxiWE8kjbpZ+TG7Q==, figureFileBig=2DBjmHvOaPiOV8jgh6Vg8A==, tableContent=null), ArticleFig(id=1198960105564762296, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=EN, label=null, caption=null, figureFileSmall=SyJFAr4LJJly4txMlUroMA==, figureFileBig=SJxjY5pjPujTBqrOLxmq+g==, tableContent=null), ArticleFig(id=1198960105694785730, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=CN, label=Figure 3, caption= Commonly used photocleavable protecting groups , figureFileSmall=SyJFAr4LJJly4txMlUroMA==, figureFileBig=SJxjY5pjPujTBqrOLxmq+g==, tableContent=null), ArticleFig(id=1198960105887723727, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=EN, label=null, caption=null, figureFileSmall=3uFHhSclSaOqYMGTOEORVA==, figureFileBig=uVfVHmtjAV9l7OLIlfWxIQ==, tableContent=null), ArticleFig(id=1198960106072273109, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=CN, label=Figure 4, caption= The chemical structures of pc-PROTACs and the release of pc-PROTAC1 , figureFileSmall=3uFHhSclSaOqYMGTOEORVA==, figureFileBig=uVfVHmtjAV9l7OLIlfWxIQ==, tableContent=null), ArticleFig(id=1198960106240045282, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=EN, label=null, caption=null, figureFileSmall=FmpZOBXGTrSFMOGOf9E6qw==, figureFileBig=K7UTZi5QXb/bWj0RcHwIHA==, tableContent=null), ArticleFig(id=1198960106416206058, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=CN, label=Figure 5, caption= The chemical structures of opto-PROTACs , figureFileSmall=FmpZOBXGTrSFMOGOf9E6qw==, figureFileBig=K7UTZi5QXb/bWj0RcHwIHA==, tableContent=null), ArticleFig(id=1198960106558812401, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=EN, label=null, caption=null, figureFileSmall=TZcLmiU4n564tLT8dfFkjg==, figureFileBig=aYlc8tfPVkcezjURPvCBDw==, tableContent=null), ArticleFig(id=1198960106672058618, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=CN, label=Figure 6, caption= The chemical structure of PROTAC3 , figureFileSmall=TZcLmiU4n564tLT8dfFkjg==, figureFileBig=aYlc8tfPVkcezjURPvCBDw==, tableContent=null), ArticleFig(id=1198960106785304835, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=EN, label=null, caption=null, figureFileSmall=VtbLss7e9cKvDIc2YR2ssg==, figureFileBig=UcODREpdXI9BxcfQ01e6+A==, tableContent=null), ArticleFig(id=1198960106948882701, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652615572681503, language=CN, 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可控性蛋白降解靶向嵌合体(PROTACs) 技术研究进展
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牟伊 1 , 文帅 1 , 王燕 1 , 姜正羽 2, *
药学学报 | 综述 2023,58(8): 2193-2202
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药学学报 |综述 2023 , 58 (8) : 2193 -2202
可控性蛋白降解靶向嵌合体(PROTACs) 技术研究进展
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牟伊1, 文帅1, 王燕1, 姜正羽2, *
作者信息
  • 1.泰州学院医药与化学化工学院, 江苏 泰州 225300
  • 2.中国药科大学药学院, 江苏省药物分子设计与成药性优化重点实验室, 江苏 南京 210009
通讯作者:
*姜正羽, Tel: 86-25-83271351, E-mail:
Research progress in controllable proteolysis targeting chimeras
Yi MOU1, Shuai WEN1, Yan WANG1, Zheng-yu JIANG2, *
Affiliations
  • 1. College of Pharmacy and Chemistry & Chemical Engineering, Taizhou University, Taizhou 225300, China
  • 2. Jiangsu Key Laboratory of Drug Design and Optimization, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
出版时间: 2023-08-12 doi: 10.16438/j.0513-4870.2023-0553
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蛋白降解靶向嵌合体(PROTACs) 是一类新型蛋白降解技术, 能同时结合E3连接酶与疾病相关蛋白, 利用内源性泛素-蛋白酶体系降解靶蛋白。PROTACs具有较佳的通用性、较强的蛋白降解活性和较好的目的蛋白选择性, 成为靶向蛋白降解领域的热点技术, 但具有降解不受控的风险。可控PROTACs引入了特定的刺激响应触发元件, 在特定的响应条件下可被触发, 产生/终止后续的蛋白质降解效应。本文概述了可控PROTACs设计策略, 并按照相应机制不同对代表化合物和研究进展进行了介绍, 从而为可控PROTACs的研究提供借鉴和思路。

蛋白降解靶向嵌合体  /  靶向蛋白降解  /  可控性蛋白降解靶向嵌合体

Proteolysis targeting chimeras (PROTACs) is an innovative technique in targeted protein degradation. PROTACs is a heterobifunctional molecule which can bind to the E3 ligase and target protein to form a ubiquitination complex, resulting in the ubiquitin-proteasome system dependent degradation of target protein. PROTACs has been regarded as the promising method in drug discovery campaign, for its high commonality, potent degradation activity and unique selectivity profile. However, the catalytic mechanism also induces the uncontrollable protein degradation risk. Controllable PROTACs contain the responsive element in the molecular entity. In certain conditions, the element can be triggered to activate or terminate the degradation event. In this review, we will briefly summarize the strategies in controllable PROTACs and describe the representative examples according to the responsive mechanism. We hope this review could provide some insight into the further development of controllable PROTACs.

proteolysis targeting chimeras  /  targeted protein degradation  /  controllable proteolysis targeting chimeras
牟伊, 文帅, 王燕, 姜正羽. 可控性蛋白降解靶向嵌合体(PROTACs) 技术研究进展. 药学学报, 2023 , 58 (8) : 2193 -2202 . DOI: 10.16438/j.0513-4870.2023-0553
Yi MOU, Shuai WEN, Yan WANG, Zheng-yu JIANG. Research progress in controllable proteolysis targeting chimeras[J]. Acta Pharmaceutica Sinica, 2023 , 58 (8) : 2193 -2202 . DOI: 10.16438/j.0513-4870.2023-0553
蛋白降解靶向嵌合体(proteolysis targeting chimeras, PROTACs) 是一种能够同时靶向结合E3连接酶与疾病相关蛋白, 利用内源性泛素-蛋白酶体系降解靶蛋白的化学降解蛋白技术[1, 2]。PROTACs技术的基本思路是设计含有双功能基的化合物分别结合泛素E3连接酶和目的蛋白, 形成稳定复合物, 进而通过E3连接酶实现对靶标蛋白的泛素化[3-5], 最终利用泛素-蛋白酶体系统降解目标蛋白。PROTACs可介导泛素E3连接酶对特定蛋白质的识别并完成泛素化, 其最大特点是可有效借助体内泛素-蛋白酶体系统实现对目的蛋白的高效泛素化降解[6-10]。这一作用机制具备较佳的通用性, 较强的蛋白降解活性和较好的目的蛋白选择性, 成为诱导蛋白降解领域的前沿和热点技术[11-15]
PROTACs技术通过催化循环机制, 可不断诱导泛素化循环直至靶标蛋白被彻底降解, 且常以非选择性的方式对细胞或者组织发挥降解蛋白质的功能[16-18], 有可能会产生脱靶毒性。为了解决这一问题, 研究人员提出可控PROTACs概念: 通过不同的响应方式条件性调控PROTACs分子的靶向降解活性, 由此提高PROTACs的时空分辨率, 改善PROTACs对正常细胞的潜在毒性[19, 20]。本文将总结近期可控性PROTACs的设计思路及其研究进展。
2001年Crew课题组[21]首次提出PROTACs概念, 借助泛素E3连接酶可特异性介导蛋白泛素化的特点, 实现对特定蛋白质的选择性泛素化, 最终催化蛋白质降解。PROTACs分子的一端是和目的蛋白结合的小分子配体, 负责对目的蛋白的识别; 另一端是和E3连接酶结合的小分子配体, 负责结合特定的E3连接酶; 通过PROTACs分子促进E3连接酶和目的蛋白形成复合物, 为泛素转移至目的蛋白建立了合适的空间环境, 使目的蛋白和活化的泛素处于接近的合适空间位置, 催化了泛素转移反应速率, 实现了目的蛋白的泛素化(图 1)。
PROTACs技术的作用机制使得其具备较佳的通用性、较强的蛋白降解活性和较好的目的蛋白选择性[22, 23]。较好的通用性是指该技术能够充分利用体内泛素化系统对各种不同目的蛋白实现泛素化调节。人体内含有600多种不同的泛素E3连接酶参与催化底物泛素化过程, 其为PROTACs分子的设计和发现提供了广阔的空间, 能够充分适配不同底物蛋白的需要。已有研究已经证实PROTACs技术能实现对多种不同蛋白的泛素化降解[24-26]。较强的蛋白降解活性是指PROTACs通过催化作用促进目的蛋白泛素化, 少量的PROTACs分子就能够实现对目的蛋白的循环降解, 实现对目的蛋白的高效抑制[27, 28]
较好的目的蛋白选择性是指PROTACs分子能够充分利用泛素化系统实现对目的蛋白的选择性。其选择性机制主要包含以下方面: 目的蛋白配体对目的蛋白的选择性, 即泛素化的前提是目的蛋白与PROTACs结合; E3连接酶催化作用选择性, 即E3连接酶处在合适的催化活性状态; E3连接酶和目的蛋白适配性, 即目的蛋白和E3连接酶必须相匹配, 能够通过PROTACs分子介导, 使目的蛋白的合适赖氨酸残基靠近E3连接酶的催化活性位点。上述的选择性机制保证PROTACs分子对目的蛋白有较好的选择性。现有研究表明, 即使非选择性的目的蛋白配体也能通过PROTACs技术提升对诱导蛋白降解的选择性[29-32]。PROTACs技术的上述特点使得其成为诱导蛋白降解领域最具潜力的新技术[33]
PROTACs分子克服了传统小分子抑制剂的占据驱动的局限性, 能够持续参与降解循环, 具备较强的蛋白降解活性, 只需催化剂量的PROTACs分子就能够快速且持续的降解靶蛋白[34, 35]。但这种催化降解的特性也是目前限制PROTACs技术发展的关键问题之一, 即降解过程的不可调控性。PROTACs分子事件驱动的特点导致降解过程一旦发生就无法停止, 并且PROTACs分子可循环参与靶蛋白的泛素化过程, 对下游蛋白产生持久的抑制效果, 即使是正常细胞或组织内存在少量的PROTACs分子也会产生较强的蛋白降解效应这可能会导致功能性靶蛋白的长时间的过度降解, 带来潜在的风险。
因此, 如果要避免PROTACs分子的潜在安全风险, 其只能运用于某些疾病特异蛋白和正常生理状态下无活性, 或者活性影响较小的蛋白质。如已经被广泛研究的肿瘤细胞内的融合蛋白或是突变的蛋白激酶。而众多的非酶蛋白, 如转录因子、骨架蛋白等在生理状态下也具有重要的生物学功能。但是现有的PROTACs技术对细胞是缺乏选择性的, 即其难以区分生理和病理状态下的目的蛋白, 特别是难以实现肿瘤细胞和正常细胞内的差异化活性。因此, 如何提高PROTACs分子的组织靶向性, 实现功能性靶蛋白的可控降解是PROTACs研究中的重要瓶颈问题。
为了实现PROTACs诱导的蛋白降解过程的可控, 研究人员提出了“可控PROTACs”的概念, 即在PROTACs分子中引入特定的刺激响应触发元件[36, 37]。可控蛋白水解靶向嵌合体开发策略主要是采用不同的刺激响应片段(cage) 封闭PROTACs分子中关键性的结合位点, 使分子失去降解活性; 在特定的响应条件下PROTACs被活化, 产生后续的蛋白质降解效应(图 2)。根据刺激的类型不同, 将可控PROTACs分为光笼保护的PROTACs、光开关的PROTACs分子、终止型PROTACs、酶催化响应型PROTACs、GSH选择性响应PROTACs、放射线响应的PROTACs等。
光可去除保护基团(photocleavable protecting groups, PPG) 是一类光敏基团, 在一定的光照条件下可发生不可逆光裂解反应从分子上脱除[38]。光笼型蛋白水解靶向嵌合体(pc-PROTACs, photocaged PROTACs) 是指在活性PROTACs分子中的E3连接酶配体、靶蛋白配体或连接子三部分中引入光可去除保护基团, 光可去除保护基团通过掩盖具有生物活性的药效团, 阻碍PROTACs分子与靶标的相互作用, 阻断PROTACs分子的靶向降解作用活性[39]。pc-PROTACs光照前为惰性分子, 光照后阻断基团从pc-PROTACs中去除, 可以快速释放有活性的PROTACs, 并使靶蛋白降解, 利用光照激活提高了PROTACs的时空分辨率。常见的光可去除保护基团有DMNB1、DMNB2、NPOM、DEACM等, 具体结构式如图 3
Xue等[40]基于BRD4降解剂dBET1设计了一系列光笼型PROTACs (pc-PROTACs), 将4, 5-二甲氧基-2-硝基苄基(DMNB) 分别与JQ1 (BRD4配体) 酰胺键上的氮原子以及沙利度胺(E3连接酶配体) 上的戊二酰亚胺氮原子连接得到pc-PROTAC1和pc-PROTAC2 (图 4)。其中, pc-PROTAC1在365 nm光线照射下能够脱除DMNB释放dBET1, 从而实现对BRD4蛋白降解的调控。实验结果显示, pc-PROTAC1对BRD4的亲和力较dBET1明显降低(IC50 = 7.6 μmol·L-1)。该团队还对pc-PROTAC1以斑马鱼为模型进行了体内活性评价, 365nm紫外线照射处理10 min后的pc-PROTAC1组出现了与dBET1组类似的蛋黄延伸表型减少的现象, 而未照射的pc-PROTAC1组出现了与空白对照组类似的结果。
2020年Liu等[41]基于dBET1和dALK设计了两类光笼型PROTACs (opto-PROTACs) (图 5)。这类化合物是通过将4, 5-二甲氧基-2-硝基苄基(DMNB) 与泊马度胺上的戊二酰亚胺氮原子连接而得。实验结果表明, 在365 nm光线照射5~15 min后, 这两类化合物均能够脱除DMNB释放dBET1和dALK, 从而实现对靶蛋白降解的调控。其中, opto-dBET1光照激活后呈现剂量依赖性的方式抑制HEK293FT、C4-2细胞的增殖; opto-dALK光照激活后呈现剂量依赖性的方式抑制SU-DHL-1细胞的增殖。
Kounde等[42]以BRD4靶蛋白配体JQ1和VHL E3连接酶配体为基础, 和光去除保护基团DMNB合成了一种光笼型PROTAC (PROTAC3) (图 6)。实验结果表明, 在50 μmol·L-1溶液中, 365 nm照射180 s后, PROTAC3可以完全脱除DMNB, 此外, 在黑暗条件下, PROTAC3在溶液中表现出良好的稳定性。BRD4靶蛋白降解实验表明, 365 nm光线照射60 s后, PROTAC3对BRD4蛋白呈现浓度依赖性的降解, 避光条件下, PROTAC3在细胞环境中表现出良好的稳定性。
Naro等[43]采用两种光去除保护基团DEACM和NPOM分别与VHL、CRBN E3连接酶配体连接, 设计了两种光笼型PROTACs (DEACM-caged ERRα PROTAC2, NPOM-caged BRD4 PROTAC4) (图 7)。实验结果显示, 在无光照条件下, 两种光笼型PROTACs对靶蛋白均无明显降解, 在365 nm光照3 min后, DEACM-caged ERRα PROTAC2和NPOM-caged BRD4 PROTAC4均可以脱除DEACM和NPOM, 从而实现对ERRα和BRD4靶蛋白降解的调控。该团队还对两种光笼型PROTACs进行了体内活性评价, 365 nm紫外线照射处理后的DEACM-caged ERRα PROTAC2和NPOM-caged BRD4 PROTAC4组出现了与空白对照组类似的结果。
光开关配体(photoswitchable PROTACs) 是一类在光照条件下能够可逆改变构象的化学基团[44]。在PROTACs的连接臂部分引入光开关配体, 使其可在特定的光照条件下实现构象转换, 并且化合物的两种构象能影响蛋白泛素化复合物的形成, 以实现蛋白降解的“开”与“关”, 该类分子也被称为光开关型PROTACs。偶氮苯基团是一类双波长调节的可控光开关配体, 在不同的波长照射下可实现顺式和反式构象的转化, 具有结构简单、化学稳定性好、可逆性好以及易于制备等优势, 因此近年来广泛应用于光可逆型PROTACs的开发。在PROTACs分子设计中, 在连接臂部分引入偶氮苯基团, 可实现光切换调节PROTACs分子的降解活性以及预测PROTACs分子构象变化与降解活性变化的相关性[45]
2020年, Reynders等[46]基于dBET1设计了靶向BRD2-4的一系列PROTACs。其中PROTAC-I-3为性质最优化合物, 该化合物在390 nm下发生transcis的异构化, 异构化效率大于90%, 并在黑暗中逐渐变回trans构型, 半衰期为8.8 h。降解活性实验表明, PROTAC-I-3在黑暗中未观察到降解活性, 而在390 nm照射下观察到依赖浓度的BET家族蛋白的降解。此外该团队还设计了靶向FKBP12的一系列PROTACs, 其中PROTAC-Ⅱ-5和PROTAC-Ⅱ-6为性质最优化合物, PROTAC-Ⅱ-5脉冲照射后对FKBP12的水平有显著影响, PROTAC-Ⅱ-5和PROTAC-Ⅱ-6降解FKBP12的时间进程展现出了一定的相似性(图 8)。
Pfaff等[47]基于BRD配体和VHL配体设计了靶向BRD4的一系列PROTACs (图 9)。其中photoPROTAC-1在415 nm下发生cistrans的异构化, 异构化效率为95%, 在530 nm下发生transcis的异构化, 异构化效率为68%。降解活性实验表明, 在415 nm照射下trans-photoPROTAC-1在低纳摩尔浓度下孵育6.5 h显著诱导BRD2蛋白的降解, 而cis-photoPROTAC-1不会引起BRD2蛋白的降解。
Jin等[48]利用来那度胺-偶氮-达沙替尼三功能系统, 设计合成了一系列Azo-PROTACs (图 10)。该团队也验证了这类化合物的反式和顺式异构体在靶蛋白质降解活性上有显著差异, 通过改变这类化合物的构型, 可以选择性地控制ABL和BCR-ABL蛋白的降解。其中Azo-PROTAC-4C为性质最优化合物, 在361 nm下光照1 h可以实现transcis的异构化。细胞实验表明, Azo-PROTAC-4C对BCR-ABL介导的K562细胞具有较强的抑制作用(IC50 = 68 nmol·L-1, EC50 = 28 nmol·L-1)。此外, Azo-PROTAC-4C对BCR-ABL介导的K562细胞具有较强的选择性, 不影响任何非BCR-ABL介导的细胞系, 如A549肺癌细胞、HCT116结肠直肠癌细胞和MCF-7乳腺癌细胞等。此外, 该团队还验证了Azo-PROTAC-4C两种构型ABL和BCR-ABL蛋白的降解活性, 结果表明4C-trans在25 nmol·L-1浓度下可以观察到BCR-ABL融合蛋白的轻微降解, 在100 nmol·L-1时观察到ABL和BCR-ABL蛋白的显著降解, 而在同等条件下, 4C-cis对BCR-ABL融合蛋白未发生明显的降解作用。
尽管光响应型Pro-PROTACs分子通过光控条件改善了PROTACs脱靶毒性, 但是由于光组织穿透性不佳与潜在光毒性的问题, 影响了这类技术的体内应用。如何开发体内适用的光响应型PROTACs仍然具有一定的挑战性。
作为事件驱动型分子, PROTACs在该过程中并未被消耗, 而是能够催化性地诱导靶蛋白降解。瞄准蛋白降解终止的难点问题, 最近研究者提出了连接-终止(ligation to scavenging) 策略实现对蛋白降解的可靠终止[49]。该策略由四嗪标记的PROTACs分子(Tz-PROTACs) 和PAMAM-G5-TCO组成(图 11)。其中, PAMAM-G5-TCO以商品化的树枝状大分子PAMAM作为载体, 并用功能性的反式环辛烯(TCO) 分子进行修饰。含有四嗪的Tz-PROTACs和含反式环辛烯的PAMAM-G5-TCO可通过生物相容性好、效率高的逆电子需求狄尔斯-阿尔德反应(IEDDA) 实现快速的连接, 以吸收游离的Tz-PROTACs, 从而实现靶向蛋白降解的终止调控。
修饰后的PAMAM-G5-TCO呈球状结构, 具有一定的刚性和良好的生物相容性。树枝状分子的巨大表面和IEDDA的高反应性使得修饰后的PAMAM分子能够快速结合并清除四嗪片段标记的PROTACs分子。实验结果显示, 在细胞中修饰后的PAMAM分子能够透过细胞膜, 快速清除细胞内外游离的PROTACs分子。在PAMAM的作用下, 靶蛋白的降解过程被有效终止, 已下调的靶蛋白逐步恢复到正常水平。
与正常细胞和组织相比, 肿瘤细胞和组织具有快速增殖、代谢、迁移和转移的特点, 通常表现出更高的还原水平、更高的缺氧状态[50, 51]和部分功能性酶的过表达[52, 53] (如硝基还原酶、水解酶等), 基于上述肿瘤微环境的特异性, 研究者设计出了一系列刺激响应型PROTACs前药。肿瘤细胞中的部分功能性酶, 如脱氢酶、水解酶与组织蛋白酶等, 表达量较高, 并在癌症发展过程中发挥重要作用[54, 55]。因此酶响应型的PROTACs也被用于提高蛋白降解的组织选择性, 有利于实现肿瘤的精准治疗。
在肿瘤组织中过表达硝基还原酶(NTR) 的情况下, 硝基咪唑作为NTR酶底物能经由酶催化被选择性除去, 是大多数前药设计的重要化学基团[56, 57]。2021年, Cheng等[58]关注肿瘤组织的缺氧环境, 在招募靶蛋白的分子配体部分中引入缺氧条件响应分子基序, 开发缺氧条件激活的Ha-PROTACs分子(图 12), 实现惰性PROTACs分子在肿瘤微环境缺氧条件刺激时的功能性激活。
醌氧化还原酶(NQO1) 的过表达与癌症的发生发展密切相关, 该酶在自然生理环境中能够催化醌类物质的双电子还原反应, 是哺乳动物的重要解毒途径。Liang等[59]以癌细胞过表达的NQO1的催化活性设计了两种酶促响应脱笼的Pro-PROTACs分子(NQO1-PROTAC与ROS-PROTAC) (图 13), 解决PROTACs分子细胞选择性不高的科学问题。作者选择了肿瘤微环境中的特征酶响应化学条件性激活惰性的Pro-PROTACs分子, 利用癌症细胞中过表达的催化型蛋白酶NQO1内源性调节PROTACs的活性。
氧化还原因子如谷胱甘肽(GSH) 的上调, 肿瘤细胞表现出高还原活性[60]。肿瘤细胞中的GSH浓度约为10 mmol·L-1, 为正常细胞的两倍[61]。因此利用二硫键作为GSH的底物来构建GSH响应型PROTACs是一个有前景的策略。基于前期研发的ALK蛋白降解剂, Zhang等[62]报道了一种GSH响应型PROTACs分子FA-S2-POMA, 该分子具有笼化作用和靶向递送PROTACs的双重作用(图 14)。该团队用二硫键将叶酸受体结合配体连接到泊马度胺的戊二酰亚胺的氮原子上, 阻碍了泊马度胺与CRBN的相互作用。FA-S2-POMA能够与癌细胞上高表达的叶酸受体α (folate receptor α, FOLR1) 特异性结合, 在叶酸引导下进入癌细胞, 通过谷胱甘肽介导的二硫键断裂释放ALK蛋白降解剂用于靶蛋白的降解。实验结果显示, 该化合物对SU-DHL-1细胞中的ALK蛋白表现出浓度和时间依赖性的降解, 在3 μmol·L-1和2 h内基本降解完全, 并且通过实验验证该化合物对ALK蛋白的降解依赖叶酸受体和过表达的GSH。
X射线是重要的癌症放射性治疗策略, 局部的X射线治疗可导致DNA损伤、细胞凋亡以及肿瘤坏死[63, 64]。除此以外, X射线也能产生氢自由基、羟基自由基等化学反应产物, 已被用于设计化疗药物的前药激活策略。2022年, Yang等[65]将X射线响应的叠氮苯基笼状基团引入PROTACs分子的设计中, 首次合成了X射线响应的RT-PROTACs。由于X射线的高精度时空分辨率与高组织穿透能力, RT-PROTACs在X射线辐射前保持惰性状态, 经由X射线辐射后能够在特定肿瘤组织中被激活, 在体内协同抑制肿瘤生长(图 15)。
光响应型PROTACs的设计具有良好的时空分辨率以及高效的释放率, 但受限于光照的组织穿透能力不足与光毒性, 如何实现该技术的体内应用具有较大挑战。终止型PROTACs技术为有效停止蛋白降解提供了一种可靠的途径, 如何将终止型PROTACs技术整合到蛋白降解药物的开发中需要进一步的研究。
酶催化以及微环境响应型PROTACs的设计主要利用了癌细胞微环境与正常细胞的生理差异, 一定程度上实现了肿瘤细胞靶标的蛋白降解; 放射线响应型PROTACs的设计能够与放射线治疗协同抑制癌细胞的生长, 但这些策略改造后的PROTACs分子的分子量增加且结构进一步复杂化, 通常存在低细胞渗透性和口服生物利用度低的问题, 如何进一步优化、保证这类PROTACs分子的类药理化性质是该研发方向要解决的主要问题。
可控性PROTACs分子虽然在一定程度上增强了组织靶向性, 实现功能性靶蛋白的可控降解, 但是目前该类分子的研究还主要处于细胞水平, 应用于组织机体时的可控性尚需进一步验证。可控性PROTACs分子的设计进一步增加了药物分子的质量, 是否会进一步影响其吸收分布还需要进一步验证。相信随着该研究领域技术的不断突破, 将会有更多的安全有效的PROTACs分子被开发设计出来。
作者贡献: 牟伊负责文献的收集整理以及主要内容的撰写; 文帅负责文章图片和结构式的绘制和部分案例整理; 王燕负责PROTACs部分的文献检索和内容修改; 姜正羽负责综述选题与框架设计、稿件修改等工作。
利益冲突: 本文的研究内容无任何利益冲突。
  • 江苏省高校自然科学基金(21KJB350008)
  • 泰州市社会发展项目(TN202135)
  • 江苏省“六大人才高峰”资助(YY-110)
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2023年第58卷第8期
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doi: 10.16438/j.0513-4870.2023-0553
  • 接收时间:2023-05-01
  • 首发时间:2025-11-21
  • 出版时间:2023-08-12
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  • 收稿日期:2023-05-01
  • 修回日期:2023-07-13
基金
江苏省高校自然科学基金(21KJB350008)
泰州市社会发展项目(TN202135)
江苏省“六大人才高峰”资助(YY-110)
作者信息
    1.泰州学院医药与化学化工学院, 江苏 泰州 225300
    2.中国药科大学药学院, 江苏省药物分子设计与成药性优化重点实验室, 江苏 南京 210009

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*姜正羽, Tel: 86-25-83271351, E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
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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