AbstractMolecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed ‘template-assisted covalent modification’.

。然而，开发分子胶仍然具有挑战性，需要创新的机械策略来稳定新蛋白界面并加速发现。在这里，我们揭示了一种反式标记共价分子胶机制，称为“模板辅助共价修饰”。

We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58.

我们确定了一系列新的BRD4分子胶降解剂，可将CUL4DCAF16连接酶募集到BRD4的第二个溴结构域（BRD4BD2）。通过全面的生化，结构和诱变分析，我们阐明了DCAF16和BRD4BD2之间预先存在的结构互补性如何作为模板，以最佳地定向降解物以共价修饰DCAF16Cys58。

This process stabilizes the formation of BRD4–degrader–DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK–BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes ‘template-assisted covalent modification’ as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology..

该过程稳定了BRD4降解剂-DCAF16三元复合物的形成，并促进了BRD4的降解。支持普遍性，我们发现降解物的一个子集还通过GAK的反式标记诱导GAK–BRD4BD2相互作用。总之，我们的工作建立了“模板辅助共价修饰”作为共价分子胶的机制，这为邻近驱动的药理学开辟了一条新途径。。

MainMolecular glue degraders have emerged as a powerful therapeutic modality, as demonstrated by the clinical successes of thalidomide analogs in the treatment of hematological malignancies1,2. These small-molecule degraders stabilize the protein–protein interface between ubiquitin ligases and disease-relevant neosubstrates, resulting in ubiquitination and proteasomal degradation of the targets3.

沙利度胺类似物在治疗血液系统恶性肿瘤方面的临床成功证明，主要分子胶降解剂已成为一种强大的治疗方式1,2。这些小分子降解物稳定了泛素连接酶和疾病相关新底物之间的蛋白质-蛋白质界面，导致靶标的泛素化和蛋白酶体降解3。

Unlike traditional occupancy-driven pharmacology of inhibitors, the event-driven pharmacology of degraders can result in more potent and sustained drug activity4. The elimination of target proteins by molecular glue degraders decreases both enzymatic and scaffold function of target proteins, leading to differentiated pharmacology and often superior inhibition of protein function5.

与抑制剂的传统占用驱动药理学不同，降解物的事件驱动药理学可以产生更有效和持续的药物活性4。通过分子胶降解物消除靶蛋白会降低靶蛋白的酶和支架功能，从而导致药理学的分化，并且通常对蛋白质功能具有更好的抑制作用5。

Moreover, molecular glue degraders hold the potential to target proteins that do not have ligandable pockets and are considered difficult to drug, including transcription factors6.The clinical efficacy of thalidomide-derived drugs, such as lenalidomide, and the broad utility of targeted protein degradation in research and drug discovery have inspired numerous efforts to explore proximity-driven pharmacology7,8,9.

此外，分子胶降解剂具有靶向不具有可连接口袋且被认为难以药物治疗的蛋白质的潜力，包括转录因子6。沙利度胺衍生药物（如来那度胺）的临床疗效以及靶向蛋白质降解在研究和药物发现中的广泛应用激发了许多探索邻近驱动药理学的努力7,8,9。

Although bifunctional molecules, such as PROTACs, can lead to rapid proof of concept and highly potent chemical probes, molecular glues are favorable for clinical development due to reduced size and overall chemical properties3,6. Despite these advantages, to date, only a small number of ubiquitin ligases have been exploited by molecular glue degraders, including CRBN10,11, DCAF15 (ref.

虽然双功能分子，如PROTACs，可以导致概念的快速验证和高效的化学探针，但分子胶由于尺寸减小和整体化学性质而有利于临床开发3,6。尽管有这些优点，但迄今为止，只有少数泛素连接酶被分子胶降解剂利用，包括CRBN10,11，DCAF15（参考文献）。

12) and DDB1 (refs. 13,14,15). Other proximity-driven approaches lack molecular glues. Covalency has the potential not only to aid the discovery of molecular glues but also to impart improved efficacy through .

12） 和DDB1（参考文献13,14,15）。其他接近驱动的方法缺乏分子胶。共价不仅有助于发现分子胶，而且有可能通过提高功效。

To determine the mechanism of DCAF16 recruitment, we sought to reconstitute the BRD4–DCAF16 interaction in a fully recombinant system. We developed a time-resolved fluorescence energy transfer (TR-FRET) assay (Extended Data Fig. 2b) and observed a tighter TMX1-induced interaction between DDB1–DCAF16 and BRD4BD2 compared to BRD4BD1, supporting the finding that the BD2 domain is the primary degron for TMX1-mediated degradation (Fig.

为了确定DCAF16募集的机制，我们试图在完全重组系统中重建BRD4-DCAF16相互作用。我们开发了一种时间分辨荧光能量转移（TR-FRET）分析（扩展数据图2b），并观察到与BRD4BD1相比，DDB1-DCAF16和BRD4BD2之间更紧密的TMX1诱导的相互作用，支持了BD2结构域是TMX1介导的降解的主要degron的发现（图2b）。

2a). We repeated a similar TR-FRET experiment with GNE11 and observed similar trends, but we found that the BRD4–DCAF16 interaction was much weaker compared to TMX1 (Extended Data Fig. 2c), consistent with the lower potency of GNE11 as a BRD4 degrader. These findings suggest that TMX1 functions as a molecular glue to recruit DCAF16 selectively to BRD4BD2, causing degradation of BRD4.Fig.

2a）。我们用GNE11重复了类似的TR-FRET实验，并观察到类似的趋势，但我们发现BRD4-DCAF16相互作用比TMX1弱得多（扩展数据图2c），这与GNE11作为BRD4降解剂的效力较低一致。这些发现表明，TMX1起着分子胶的作用，可将DCAF16选择性地募集到BRD4BD2，从而导致BRD4降解。

2: Template-assisted covalent modification of DCAF16 and degraders with optimized electrophilic warheads.a, TR-FRET signal for DDB1–DCAF16–BODIPY to BRD4BD1-terbium or BRD4BD2-terbium with increasing concentrations of TMX1 (n = 3). b, Intact protein mass spectra of DDB1–DCAF16 alone, DDB1–DCAF16 co-incubated with TMX1 at 4 °C for 16 h or DDB1–DCAF16 co-incubated with TMX1 and BRD4BD2 at 4 °C for 16 h.

2： 模板辅助共价修饰DCAF16和具有优化的亲电弹头的降解物。a，随着TMX1浓度的增加，DDB1–DCAF16–BODIPY对BRD4BD1铽或BRD4BD2铽的TR-FRET信号（n=3）。b、 DDB1-DCAF16单独的完整蛋白质质谱，DDB1-DCAF16与TMX1在4℃共孵育16小时或DDB1-DCAF16与TMX1和BRD4BD2在4℃共孵育16小时。

c, Chemical structures of MMH1, MMH2, MMH1-NR and MMH2-NR. d, Western blot of BRD4 degradation in K562 cells that were treated with DMSO or different concentrations of MMH1, MMH2, dBET6 or MZ1 for 6 h. e, TR-FRET signal for DDB1–DCAF16–BODIPY to BRD4BD2-terbium with increasing concentrations of JQ1, MMH1, MMH2, MMH1-NR and MMH2-NR (n = 3).

c、 MMH1，MMH2，MMH1-NR和MMH2-NR的化学结构。d，用DMSO或不同浓度的MMH1，MMH2，dBET6或MZ1处理6小时的K562细胞中BRD4降解的Western印迹，随着JQ1，MMH1，MMH2，MMH1-NR和MMH2-NR浓度的增加，DDB1-DCAF16-BODIPY对BRD4BD2铽的TR-FRET信号（n=3）。

f, Western blots of BRD4 degradation in K562 cells that were treated with DMSO or different concentrations of MMH1, MMH1-NR, MMH2 or MMH2-NR for 16 h. All western blot data are representative of two independent measurements.Sou.

f、 用DMSO或不同浓度的MMH1，MMH1-NR，MMH2或MMH2-NR处理16小时的K562细胞中BRD4降解的蛋白质印迹。所有蛋白质印迹数据均代表两次独立测量。苏。

Data availability

数据可用性

Cryo-EM maps and coordinates have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank, under accession codes EMD-29714 and 8G46, respectively. Raw data files of whole-cell proteome mass spectrometry, IP-MS and Biotin pulldown mass spectrometry in this study have been deposited in the PRIDE Archive, including PXD047137, PXD047138, PXD047141 and PXD051457.

低温电磁图和坐标已分别以登录号EMD-29714和8G46保存在电子显微镜数据库和蛋白质数据库中。本研究中全细胞蛋白质组质谱，IP-MS和生物素下拉质谱的原始数据文件已保存在PRIDE档案中，包括PXD047137，PXD047138，PXD047141和PXD051457。

Intact mass spectrometry raw data related to Figs. 2b and 4e and Extended Data Figs. 2d,e, 4e,f and 7f are available for free download at ftp://massive.ucsd.edu/MSV000093731. Synthetic procedures of JQ1-derived compounds, schematics of sorting strategies and deep sequencing results for DCAF16-knockout clones are provided in the Supplementary Information.

与图2b和4e相关的完整质谱原始数据以及扩展数据图2d，e，4e，f和7f可免费下载ftp://massive.ucsd.edu/MSV000093731.补充信息中提供了JQ1衍生化合物的合成程序，DCAF16敲除克隆的分选策略示意图和深度测序结果。

Coding sequences of the DNA constructs used in this study and mammalian cell line authentication results are provided as supplementary tables. Source data are provided with this paper..

本研究中使用的DNA构建体的编码序列和哺乳动物细胞系鉴定结果作为补充表提供。本文提供了源数据。。

Code availability

代码可用性

Codes used to identify hits and generate volcano plots in screens, as well as codes used to generate dose−response curves and correlation plots, are provided as Supplementary Code.

作为补充代码提供了用于识别命中并在屏幕中生成火山图的代码，以及用于生成剂量-反应曲线和相关图的代码。

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Download referencesAcknowledgementsWe thank the Broad Institute Walk-Up Sequencing team, the Broad Institute Genetic Perturbation Platform and the Broad Institute PRISM team for technical assistance. We thank the staff of the Harvard Cryo-Electron Microscopy Center for Structural Biology for their technical expertise and support during grid screening and data collection.

下载参考文献致谢我们感谢Broad Institute Walk Up测序团队，Broad Institute遗传扰动平台和Broad Institute PRISM团队的技术援助。我们感谢哈佛低温电子显微镜结构生物学中心的工作人员在网格筛选和数据收集过程中的技术专业知识和支持。

We acknowledge the SBGrid consortium for assistance with structural biology software packages. We thank members of the Eck laboratory for valuable structural discussions. We are grateful to all members of the Ebert, Fischer and Gray laboratories for discussions on many project-related topics.Y.-D.L.

我们感谢SBGrid联盟对结构生物学软件包的帮助。我们感谢Eck实验室的成员进行了宝贵的结构讨论。我们感谢Ebert，Fischer和Gray实验室的所有成员就许多项目相关主题进行的讨论。Y、 -D.L。

was supported by a Harvard Institutional Stipend and the Genevieve Castrodale Carpenter Graduate Financial Aid Fund. M.W.M. was supported by the Chleck Fellowship Foundation and the Fujifilm Fellowship. M.T. is a CPRIT Scholar in cancer research, and M.T. thanks the CPRIT for research funding support (RR220012).

由哈佛大学奖学金和Genevieve Castrodale Carpenter研究生资助基金资助。M、 W.M.得到了Chleck Fellowship Foundation和Fujifilm Fellowship的支持。M、 T.是CPRIT癌症研究学者，M.T.感谢CPRIT的研究资助（RR220012）。

H.Y. was supported by the National Institutes of Health (NIH) grants (K00CA253754 and K99CA287069). This work was supported by NIH grants R01HL082945, P01CA066996, P50CA206963 and R35CA253125 (to B.L.E.); the Howard Hughes Medical Institute (to B.L.E.); NIH grants R01CA262188 and P01CA066996 (to E.S.F.); the Mark Foundation for Cancer Research 19-001-ELA (to E.S.F.); NIH High End Instrumentation grant (1S10OD028697-01) (to N.S.G.); departmental funds from Stanford Chemical and Systems Biology and Stanford Cancer Institute (to N.S.G.); NIH grants U24DK116204, R01CA219850, R01CA233800 and R21CA247671 (to J.A.M.); the Mark Foundation for Cancer Research; and the Massachusetts Life Science Center (to J.A.M.).Author informationAuthor notesThese authors contributed equally: Yen-Der Li, Mich.

H、 Y.得到了美国国立卫生研究院（NIH）的资助（K00CA253754和K99CA287069）。这项工作得到了NIH拨款R01HL082945，P01CA066996，P50CA206963和R35CA253125（授予B.L.E.）的支持；霍华德·休斯医学研究所（致B.L.E.）；NIH授予R01CA262188和P01CA066996（授予E.S.F.）；马克癌症研究基金会19-001-ELA（致E.S.F.）；NIH高端仪器拨款（1S10OD028697-01）（授予N.S.G.）；斯坦福化学与系统生物学和斯坦福癌症研究所（致N.S.G.）的部门资金；NIH授予U24DK116204，R01CA219850，R01CA233800和R21CA247671（授予J.A.M.）；马克癌症研究基金会；和马萨诸塞州生命科学中心（致J.A.M.）。作者信息作者注意到这些作者做出了同样的贡献：Yen Der Li，Mich。

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PubMed Google ScholarContributionsY.-D.L., B.L.E., E.S.F. and N.S.G. conceptualized and initiated the study. Y.-D.L. designed and performed functional genomics studies, mutagenesis screens and cellular validation experiments, with the help of J.C.R., B.S., S.X., W.S.B., I.Y., C.Z., J.M.T., P.M., C.P., H.Y., H.T.S.

PubMed谷歌学术贡献-D、 L.，B.L.E.，E.S.F.和N.S.G.概念化并启动了这项研究。Y、 -在J.C.R.，B.S.，S.X.，W.S.B.，I.Y.，C.Z.，J.M.T.，P.M.，C.P.，H.Y.，H.T.S.的帮助下，D.L.设计并进行了功能基因组学研究，诱变筛选和细胞验证实验。

and M.S. M.W.M. designed and carried out biochemical studies and structural analyses, with the help of M.H., K.P., C.Y.J. and R.P.N. M.M.H. and M.T. developed and synthesized covalent BRD4 molecular glue degraders, with the help of B.J.G. and F.C.M. R.J.L. and K.A.D. performed whole-cell proteomics and IP-MS experiments.

M.S.M.W.M.在M.H.，K.P.，C.Y.J.和R.P.N.的帮助下，设计并进行了生化研究和结构分析。M.M.H.和M.T.在B.J.G.和F.C.M.的帮助下，开发并合成了共价BRD4分子胶降解剂。R.J.L.和K.A.D.进行了全细胞蛋白质组学和IP-MS实验。

A.M.S. performed DCAF16, GAK intact mass spectrometry and GAK digested mass spectrometry experiments, with the help of H.C. S.B.F. performed DCAF16 intact mass spectrometry experiments, with the help of I.T. R.J.M. performed MMH2-Biotin pulldown mass spectrometry experiments. M.Y.W. performed AlphaScreen experiments, with the help of L.H.S.

A、 。M、 Y.W.在L.H.S.的帮助下进行了AlphaScreen实验。

B.L.E., E.S.F., N.S.G., J.A.M. and J.Q. supervised the project. Y.-D.L., M.W.M., M.M.H., B.L.E., E.S.F. and N.S.G. wrote the manuscript, with input from all authors.Corresponding authorsCorrespondence to.

B、 L.E.，E.S.F.，N.S.G.，J.A.M.和J.Q.监督了该项目。Y、 -D.L.，M.W.M.，M.M.H.，B.L.E.，E.S.F.和N.S.G.在所有作者的投入下撰写了手稿。通讯作者通讯。

Nathanael S. Gray, Eric S. Fischer or Benjamin L. Ebert.Ethics declarations

Nathanel S.Gray，Eric S.Fischer或Benjamin L.Ebert。道德宣言

Competing interests

相互竞争的利益

B.L.E. has received research funding from Celgene, Deerfield, Novartis and Calico Life Sciences and consulting fees from AbbVie. He is a member of the scientific advisory board (SAB) for and a shareholder of Neomorph, Inc., TenSixteen Bio, Skyhawk Therapeutics and Exo Therapeutics. E.S.F. is a founder, SAB member and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics and Neomorph, Inc.

B、 。他是Neomorph，Inc.，TenSixteen Bio，Skyhawk Therapeutics和Exo Therapeutics的科学顾问委员会（SAB）成员和股东。E、 S.F.是麝香猫疗法、Lighthorse Therapeutics、邻近疗法和Neomorph，Inc.的创始人、SAB成员和股东。

(board of directors). He is an equity holder in and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Novartis, Sanofi, EcoR1 Capital and Deerfield. The Fischer laboratory receives or has received research funding from Deerfield, Novartis, Ajax, Interline and Astellas. N.S.G.

（董事会）。他是Avilar Therapeutics和Photys Therapeutics的股东和SAB成员，也是诺华、赛诺菲、EcoR1 Capital和迪尔菲尔德的顾问。菲舍尔实验室收到或已经收到迪尔菲尔德、诺华、阿贾克斯、Interline和Astellas的研究资金。N、 S.G。

is a founder, SAB member and equity holder in Syros, C4, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GlaxoSmithKline, Larkspur (board member), Shenandoah (board member) and Soltego (board member). The Gray laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, Springworks, Interline and Sanofi.

是Syros、C4、Allorion、Lighthorse、Voronoi、Inception、Matchpoint、CobroVentures、葛兰素史克、Larkspur（董事会成员）、Shenandoah（董事会成员）和Soltego（董事会成员）的创始人、SAB成员和股东。格雷实验室接受或已经接受了诺华、武田、阿斯特拉斯、太和、詹森、Kinogen、Arbella、迪尔菲尔德、Springworks、Interline和赛诺菲的研究资助。

M.S. has received research funding from Calico Life Sciences. K.A.D. receives or has received consulting fees from Kronos Bio and Neomorph, Inc. J.Q. is an equity holder of Epiphanes and Talus Bioscience and receives or has received research funding from Novartis. J.A.M. is a founder and equity holder of and advisor to Entact Bio, serves on the SAB of 908 Devices and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks, TUO Therapeutics and Bruker.

M、 美国已获得Calico Life Sciences的研究资助。K、 。J、 A.M.是Entact Bio的创始人、股东和顾问，在SAB服务908台设备，并获得或已经获得Vertex、AstraZeneca、Taiho、Springworks、TUO Therapeutics和Bruker的赞助研究资金。

Y.-D.L. is currently employed by Leerink Partners. M.W.M. is currently employed by Novartis Venture Fund. K.P. is currently employed by AbbVie. R.J.L. is currently employed by .

Y、 -D.L.目前受雇于Leerink Partners。M、 W.M.目前受雇于诺华风险基金。K、 P.目前受雇于AbbVie。R、 J.L.目前受雇于。

Peer review

同行评审

Peer review information

同行评审信息

Nature Chemical Biology thanks Craig Crews and the other, anonymous reviewers for their contribution to the peer review of this work.

《自然化学生物学》感谢克雷格·克鲁斯（CraigCrews）和其他匿名审稿人对这项工作的同行评议做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Degradation characterization of JQ1-derived compounds.a. The domain structure of BRD4. b. Schematic of BRD4BD stability reporter.

。。b、 BRD4BD稳定性报告器的示意图。

IRES, internal ribosome entry site. c. Flow cytometry analysis of BRD4BD1-eGFP and BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of GNE11 for 16 h (n = 3). d. Western blots of BRD4 degradation in K562 cells that were treated with JQ1, TMX1 or GNE11 at 1 μM for increasing time points.

IRES，内部核糖体进入位点。c、 用增加浓度的GNE11处理16小时（n=3）的K562细胞中BRD4BD1-eGFP和BRD4BD2-eGFP降解的流式细胞术分析。d、 用JQ1，TMX1或GNE11以1μM处理K562细胞以增加时间点的BRD4降解的蛋白质印迹。

e. Flow cytometry analysis of BRD4BD1-eGFP and BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of TMX1 for 16 h (n = 3). f. Quantitative whole proteome analysis of K562 cells after treatment with JQ1 at 0.5 μM (n = 1) or DMSO (n = 3) for 5 h. Statistical analysis was performed using a two-sided moderated t-test as implemented in the limma package.

e、 用增加浓度的TMX1处理16小时（n=3）的K562细胞中BRD4BD1-eGFP和BRD4BD2-eGFP降解的流式细胞术分析。f、 用JQ1以0.5μM（n=1）或DMSO（n=3）处理K562细胞5小时后，对K562细胞进行定量全蛋白质组分析。使用limma软件包中实施的双侧缓和t检验进行统计分析。

g. Western blots of BRD4 degradation in K562 cells that were treated with DMSO, TMX1 at 1 μM, GNE11 at 1 μM, MG132 at 10 μM, MLN7243 at 1 μM, and MLN4924 at 1 μM for 16 h. All western blot data are representative of two independent measurements.Source dataExtended Data Fig. 2 Covalent recruitment of DCAF16 is facilitated by BRD4BD2.a.

g、 用DMSO，1μM的TMX1，1μM的GNE11，10μM的MG132，1μM的MLN7243和1μM的MLN4924处理16小时的K562细胞中BRD4降解的蛋白质印迹。所有蛋白质印迹数据代表两个独立的测量。源数据扩展数据图2 BRD4BD2促进了DCAF16的共价募集。

Flag immunoprecipitation (IP) followed by mass spectrometry in 293 T cells overexpressing BRD4BD2-Flag of cells treated with either MLN4924 plus GNE11 both at 1 μM (n = 4), or MLN4924 at 1 μM only (n = 4). Fold enrichment and p-values were calculated by comparing GNE11/MLN4924 treated samples to MLN4924 only control samples.

。通过比较GNE11/MLN4924处理的样品与仅MLN4924的对照样品来计算倍数富集和p值。

Statistical analysis was performed using a two-sided moderated t-test as implemented in the limma .

使用limma中实施的双侧调节t检验进行统计分析。

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Reprints and permissionsAbout this articleCite this articleLi, YD., Ma, M.W., Hassan, M.M. et al. Template-assisted covalent modification underlies activity of covalent molecular glues.

转载和许可本文引用本文Li，YD.，Ma，M.W.，Hassan，M.M.等人。模板辅助共价修饰是共价分子胶活性的基础。

Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01668-4Download citationReceived: 27 February 2024Accepted: 05 June 2024Published: 29 July 2024DOI: https://doi.org/10.1038/s41589-024-01668-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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