AbstractWRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1,2,3,4,5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins.

摘要WRN解旋酶是治疗微卫星不稳定性（MSI）癌症的有希望的靶标，因为它在解决具有错误错配修复机制的细胞中积累的有害非规范DNA结构中起着至关重要的作用1,2,3,4,5。目前还没有批准的直接靶向人类DNA或RNA解旋酶的药物，部分原因是开发针对这类蛋白质的有效和选择性化合物具有挑战性。

Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells.

在这里，我们描述了化学蛋白质组学能够发现WRN的临床阶段共价变构抑制剂VVD-133214。该化合物选择性地与位于解旋酶结构域区域的半胱氨酸（C727）结合，该区域在DNA解旋过程中会发生域间运动。VVD-133214与核苷酸协同结合WRN蛋白，并稳定缺乏正确解旋酶功能所需的动态灵活性的紧凑构象，导致MSI高（MSI-H）中广泛的双链DNA断裂，核肿胀和细胞死亡，但不在微卫星稳定的细胞中。

The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers..

该化合物在小鼠中具有良好的耐受性，并导致多种MSI-H结直肠癌细胞系和患者来源的异种移植模型中的肿瘤消退。我们的工作显示了一种抑制WRN功能的变构方法，该方法规避了癌细胞中内源性ATP辅因子的竞争，并将VVD-133214指定为MSI-H癌症患者的有希望的候选药物。。

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Fig. 1: Discovery of a covalent WRN inhibitor, VVD-133214, using chemoproteomics.Fig. 2: Overall architecture of the closed conformation of the WRN helicase domain.Fig. 3: VVD-133214 inhibits growth in MSI-H cells but not in MSS.Fig. 4: VVD-133214 induces DNA damage-and-repair response in MSI-H cells.Fig.

图1：使用化学蛋白质组学发现共价WRN抑制剂VVD-133214。图2:WRN解旋酶结构域闭合构象的整体结构。图3:VVD-133214抑制MSI-H细胞的生长，但不抑制MSS的生长。图4:VVD-133214在MSI-H细胞中诱导DNA损伤和修复反应。图。

5: Pharmacokinetics/pharmacodynamics and tumour growth inhibition in MSI-H colorectal cancer xenograft models.Fig. 6: VVD-133214 inhibits tumour growth in MSI-H PDX models..

5： MSI-H结直肠癌异种移植模型中的药代动力学/药效学和肿瘤生长抑制。图6:VVD-133214抑制MSI-H PDX模型中的肿瘤生长。。

Data availability

数据可用性

The structural data have been deposited in wwPDB under IDs 7GQS, 7GQT and 7GQU and the files can be accessed under group ID G_1002280 (and password pagodan) from a group deposition account (deposit@deposit.rcsb.org). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository38 with the dataset identifier PXD046214.

结构数据已以ID 7GQS、7GQT和7GQU存放在wwPDB中，可以通过组存放帐户以组ID G\U 1002280（和密码pagodan）访问文件(deposit@deposit.rcsb.org)。质谱蛋白质组学数据已通过PRIDE partner repository38保存到ProteomeXchange Consortium，数据集标识符为PXD046214。

These data can be accessed with the following username (reviewer_pxd046214@ebi.ac.uk) and password (5VPbwpt0). Proteomics analysis was performed using the Homo sapiens (2016) and Mus musculus (2017) UniProt Fasta databases. Source data are provided with this paper..

可以使用以下用户名访问这些数据(reviewer_pxd046214@ebi.ac.uk)和密码（5VPbwpt0）。使用智人（2016）和小家鼠（2017）UniProt Fasta数据库进行蛋白质组学分析。本文提供了源数据。。

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Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).Article

Karplus，P.A。和Diederichs，K。链接晶体学模型和数据质量。科学3361030-1033（2012）。文章

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Download referencesAcknowledgementsWe thank J. Bemis, R. Nishimura, S. Larsen and I. Stiller for programme administration; I. Mochalkin, E. Aitchison, H. Binch, D. Heer, E. Jochnowitz, A. L. Lambert, A. Rufer, R. Thoma, F. Schuler and M. Wittwer for valuable technical and scientific inputs; and B.

下载参考文献致谢我们感谢J.Bemis，R.Nishimura，S.Larsen和I.Stiller的项目管理；一、 Mochalkin，E。Aitchison，H。Binch，D。Heer，E。Jochnowitz，A。L。Lambert，A。Rufer，R。Thoma，F。Schuler和M。Wittwer为有价值的技术和科学投入；和B。

Cravatt for helpful discussions and review of the manuscript.Author informationAuthor notesRobert T. AbrahamPresent address: Odyssey Therapeutics, San Diego, CA, USAAuthors and AffiliationsVividion Therapeutics, San Diego, CA, USAKristen A. Baltgalvis, Kelsey N. Lamb, Kent T. Symons, Chu-Chiao Wu, Melissa A.

克雷瓦特对稿件进行了有益的讨论和审查。作者信息作者注释Robert T.AbrahamPresent地址：Odyssey Therapeutics，San Diego，CA，USA作者和附属机构Vividion Therapeutics，San Diego，CA，USAKristen A.Baltgalvis，Kelsey N.Lamb，Kent T.Symons，Chu Chiao Wu，Melissa A。

Hoffman, Aaron N. Snead, Xiaodan Song, Thomas Glaza, Shota Kikuchi, Jason C. Green, Donald C. Rogness, Betty Lam, Maria E. Rodriguez-Aguirre, David R. Woody, Christie L. Eissler, Socorro Rodiles, Seth M. Negron, Steffen M. Bernard, Eileen Tran, Jonathan Pollock, Ali Tabatabaei, Victor Contreras, Heather N.

霍夫曼、亚伦·N·斯奈德、宋晓丹、托马斯·格拉扎、肖塔·菊池、杰森·C·格林、唐纳德·C·罗杰斯、贝蒂·林、玛丽亚·E·罗德里格斯·阿奎尔、大卫·R·伍迪、克里斯蒂·L·艾斯勒、索科罗·罗德里斯、塞思·M·内格罗、斯特芬·M·伯纳德、艾琳·陈、乔纳森·波洛克、阿里·塔巴塔贝、维克多·孔特雷拉斯、希瑟·N。

Williams, Martha K. Pastuszka, John J. Sigler, Laurence E. Burgess, Robert T. Abraham, David S. Weinstein, Gabriel M. Simon, Matthew P. Patricelli & Todd M. KinsellaPharma Research and Early Development pRED F. Hoffmann-La Roche, Ltd, Basel, SwitzerlandPiergiorgio Pettazzoni, Markus G. Rudolph, Moritz Classen, Doris Brugger, Christopher Claiborne & Jean-Marc PlancherVall d’Hebron Institute of Oncology, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, CIBERONC, Barcelona, SpainIsabel Cuartas & Joan SeoaneAuthorsKristen A.

威廉姆斯（Williams），玛莎·K·帕斯图兹卡（MarthaK.Pastuszka），约翰·J·西格勒（JohnJ.Sigler），劳伦斯·E·伯吉斯（LaurenceE.Burgess），罗伯特·T·亚伯拉罕（RobertT.Abraham），大卫·S·温斯坦（DavidS.Weinstein），加布里埃尔·M·西蒙（GabrielM.Simon），马修·P·帕特里切利（MatthewP.Patricelli）和托德·M·金塞拉制药研究与早期开发有限公司（pRED F.Hoffmann-La Roche），巴塞尔（Basel），瑞士比尔·乔治·佩塔佐尼（SwitzerlandPiergioPettazzoni），马库斯·G·鲁道夫（Markus G.Rudo巴塞罗那，锡伯龙，巴塞罗那，西班牙萨贝尔·卡塔斯和琼·塞奥阿奈·奥斯汀·克里斯汀A。

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PubMed Google ScholarContributionsG.M.S., M.P.P. and T.M.K. conceptualized the project. Investigation was carried out by K.A.B., K.N.L., K.T.S., C.-C.W., M.A.H., A.N.S., X.S., T.G., S.K., J.C.G., D.C.R., B.L., M.E.R.-A., D.R.W., C.L.E., S.R., S.M.N., S.M.B., E.T., V.C., H.N.W., M.K.P., J.J.S., M.G.R., M.C., D.B.

PubMed谷歌学术贡献。M、 S.，M.P.P.和T.M.K.将该项目概念化。调查由K.A.B.，K.N.L.，K.T.S.，C.C.W.，M.A.H.，A.N.S.，X.S.，T.G.，S.K.，J.C.G.，D.C.R.，B.L.，M.E.R.-A.，D.R.W.，C.L.E.，S.R.，S.M.N.，S.M.B.，E.T.，V.C.，H.N.W.，M.K.P.，J.S.，M.G.R.，M.C.，D.B。

and I.C. Resources were the responsibility of P.P., C.C. and J.-M.P. K.A.B. wrote the original draft. Writing, review and editing were undertaken by J.P., P.P., J.-M.P., G.M.S., M.P.P. and T.M.K. K.A.B., S.K., J.P., A.T., J.S., L.E.B., R.T.A., D.S.W., G.M.S., M.P.P. and T.M.K. supervised the project.Corresponding authorsCorrespondence to.

I.C.资源由P.P.负责，C.C.和J.-M.P.K.A.B.撰写了原始草案。写作，审查和编辑由J.P.，P.P.，J.-M.P.，G.M.S.，M.P.P.和T.M.K.K.A.B.，S.K.，J.P.，A.T.，J.S.，L.E.B.，R.T.A.，D.S.W.，G.M.S.，M.P.P.和T.M.K.负责监督该项目。通讯作者通讯。

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Competing interests

相互竞争的利益

K.N.L., K.T.S., M.A.H., A.N.S., X.S., T.G., S.K., J.C.G., D.C.R., B.L., M.E.R.-A., D.R.W., C.L.E., S.R., S.M.N., S.M.B., J.P., V.C., H.N.W., M.K.P., J.J.S., D.S.W., G.M.S., M.P.P. and T.M.K. are current employees of Vividion Therapeutics. K.A.B., A.T., L.E.B., R.T.A., E.T., S.R. and C.-C.W. are former employees of Vividion Therapeutics.

K、 N.L.，K.T.S.，M.A.H.，A.N.S.，X.S.，T.G.，S.K.，J.C.G.，D.C.R.，B.L.，M.E.R.-A.，D.R.W.，C.L.E.，S.R.，S.M.N.，S.M.B.，J.P.，V.C.，H.N.W.，M.K.P.，J.J.S.，D.S.W.，G.M.S.，M.P.和T.M.K.是Vividion Therapeutics的现任员工。K、 A.B.，A.T.，L.E.B.，R.T.A.，E.T.，S.R.和C.-C.W.是Vividion Therapeutics的前员工。

P.P., M.G.R., M.C., D.B., C.C. and J.-M.P. are current employees of F. Hoffmann-La Roche, Ltd..

P、 P.，M.G.R.，M.C.，D.B.，C.C.和J.-M.P.是F.Hoffmann-La Roche，Ltd.的现任员工。。

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Nature thanks Nicholas Larsen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

《自然》杂志感谢尼古拉斯·拉森（NicholasLarsen）和另一位匿名审稿人为这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended data figures and tablesExtended Data Fig. 1 Additional TE and helicase activity data.(a) Live cell and lysate TE50s for an off-target cysteine, TNFAIP3 C54 across all profiled compounds in WRN Type I (blue) and Type II (red) inhibitor series.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据图和表扩展数据图1其他TE和解旋酶活性数据。（a） WRN I型（蓝色）和II型（红色）抑制剂系列中所有分析化合物的脱靶半胱氨酸TNFAIP3 C54的活细胞和裂解物TE50。

(b) Ratios of hWRN519–1227 helicase activity without/with 0.2 mM ATP during compound preincubation period versus ratios of TE50s in lysates/live cells show that the ATP-cooperative compounds are the same ones that show enhanced potency in live cells. (c) Global TMT cysteine profiling of VVD-127101. (d) Dose-response curves of target engagement (WRN C727) by VVD-133214 in 4 cell lines: HCT-116 (gray circles), DLD-1 (white squares), SW480 (black triangles), and OCI-AML2 (circles).

（b） hWRN519-1227解旋酶活性与0.2的比值 化合物预孵育期间的mM ATP与裂解物/活细胞中TE50的比例表明，ATP协同化合物与在活细胞中显示增强效力的化合物相同。（c） VVD-127101的全球TMT半胱氨酸分析。（d） VVD-133214在4种细胞系中靶向参与（WRN C727）的剂量反应曲线：HCT-116（灰色圆圈），DLD-1（白色方块），SW480（黑色三角形）和OCI-AML2（圆圈）。

The comparison between both lysate (black circles) and live cells (white circles) is demonstrated in the screening line, OCI-AML2. (e) Evaluation of VVD-133214 for inhibition of mouse WRN helicase (fragment 486–1232) (mWRN486–1232) and (f) hBLM activity with- and without ATP during compound preincubation period.

在筛选系OCI-AML2中证明了裂解物（黑色圆圈）和活细胞（白色圆圈）之间的比较。（e） 在复合预孵育期间，评估VVD-133214对小鼠WRN解旋酶（片段486-1232）（mWRN486-1232）和（f）hBLM活性的抑制作用，无论有无ATP。

(g) Lack of WRN helicase inhibition activity by a non-covalent analog of VVD-133214, VVD-129448. (h) Reaction kinetics for VVD-133214 were determined using intact protein mass spectrometry with recombinant WRN. The rate of covalent modification of WRN was observed over time with varying concentrations of inhibitor.

（g） VVD-133214，VVD-129448的非共价类似物缺乏WRN解旋酶抑制活性。（h） 使用重组WRN的完整蛋白质质谱法测定VVD-133214的反应动力学。随着时间的推移，观察到不同浓度抑制剂对WRN的共价修饰速率。

Kobs/[I] was determined to be 4848 M-1s-1 (i) Forked DNA binding of hWRN519–1227 in presence of VVD-133214 with- and without ADP preincubation via HTRF. (j) Lack of time-dependent inhibition of WRN helicase activity by VVD-133214 after helicase reaction is initiated. (i) Eval.

Kobs/[I]被确定为4848 M-1s-1（i）在VVD-133214存在下，通过HTRF进行ADP预孵育和不进行ADP预孵育，hWRN519-1227的分叉DNA结合。（j） 解旋酶反应开始后，VVD-133214缺乏对WRN解旋酶活性的时间依赖性抑制。（i） 评估。

Nature (2024). https://doi.org/10.1038/s41586-024-07318-yDownload citationReceived: 19 October 2023Accepted: 14 March 2024Published: 24 April 2024DOI: https://doi.org/10.1038/s41586-024-07318-yShare 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.

《自然》（2024）。https://doi.org/10.1038/s41586-024-07318-yDownload收到引文日期：2023年10月19日接受日期：2024年3月14日发布日期：2024年4月24日OI：https://doi.org/10.1038/s41586-024-07318-yShare本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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