Leveraging High-Throughput Proteomics and AI-Based Protein Folding to Accelerate VAV1 Molecular Glue Discovery

Leveraging High-Throughput Proteomics and AI-Based Protein Folding to Accelerate VAV1 Molecular Glue Discovery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Leveraging High-Throughput Proteomics and AI-Based Protein Folding to Accelerate VAV1 Molecular Glue Discovery Jin Wang, Hanfeng Lin, Xin Yu, Haiyang Zheng, Ran Cheng, Min Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6712113/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract This study reports the discovery and characterization of novel CRBN molecular glues that selectively induce the proteasomal degradation of the hematopoietic-specific signaling protein VAV1, a key target in hematological malignancies and autoimmune diseases. Utilizing unbiased global proteomics, we identified phenyl-glutarimide derivatives NGT-201-12, as effective VAV1 degraders, with its C-terminal SH3 domain (SH3-2) being crucial for this interaction. A significant finding is the elucidation of a non-canonical RT-loop degron (RDxS motif, residues 796-799) within VAV1 SH3-2, distinct from previously characterized G-loop degrons. This discovery, supported by advanced computational modeling using the physics- and AI-driven GluePlex workflow and validated by site-directed mutagenesis, highlights versatility of CRBN in recognizing diverse neosubstrate motifs. Furthermore, we demonstrate that applying Free Energy Perturbation (FEP) calculations to these predicted ternary structures yields cooperativity metrics that correlate with experimental degradation potency, overcoming the limitations of standard molecular docking. This establishes a robust workflow where, once a ternary complex is predicted—even with initial weak binders—FEP can be utilized to prospectively rank analogs and optimize molecular glue potency. Additionally, we demonstrate that strategic chemical modifications, particularly conformational restriction via halogen substitution (e.g., NGT-201-18), markedly potentiate VAV1 degradation, a principle supported by density functional theory (DFT) calculations. Comprehensive structure-activity relationship (SAR) studies provided a roadmap for designing next-generation VAV1 degraders. Importantly, dose-response proteomics not only confirmed VAV1 as the primary target but also revealed LIMD1, possessing a canonical G-loop, as an off-target for some analogs, indicating a single molecular glue can engage disparate degron motifs. The identification of the VAV1 RT-loop degron prompted a proteome-wide search, revealing other SH3-containing proteins as potential targets or off-targets. In conclusion, this research unveils a novel non-canonical RT-loop degron in VAV1, demonstrates the utility of conformational restriction in enhancing degrader potency, and underscores the critical role of integrating global proteomics with advanced structural modeling and FEP calculations for understanding degrader potency and selectivity. These findings offer a promising therapeutic strategy for targeting VAV1 and significantly expand the landscape of CRBN neosubstrate recognition and the rational design of molecular glue degraders. Biological sciences/Drug discovery Biological sciences/Chemical biology/Proteomics Biological sciences/Computational biology and bioinformatics/Protein folding Full Text Additional Declarations Yes there is potential Competing Interest. J.W. is the co-founder of Chemical Biology Probes LLC. J.W. has stock ownership in CoRegen Inc and serves as a consultant for this company. J.W. and X.Y. are the co-founders of Fortitude Biomedicines, Inc. and hold equity interest in this company. J.W., H.L. and X.Y. are the coinventors of a patent application related to the disclosed VAV1 degraders. X.C. and S.Z. are the CEO and an employee of YDS Pharmatech, Inc., respectively. A.N. and S.Y. are the CEO and CSO of Receptor.AI, Inc., respectively. Y.Y. is an employee of ThermoFisher Scientific, Inc. Supplementary Files SupplementaryData1NGT20112CRBNVAV1GluePlexModel.txt Supplementary Data 1 SupplementaryData2CRBNLIMD1NGT20118dockingCK1aaligned.txt Supplementary Data 2 SupplementaryInformation.pdf Supplementary Information SupplementaryData3VAV1globalproteomics.pgmatrix.txt Supplementary Data 3A SupplementaryData3VAV1globalproteomics.prmatrix.txt Supplementary Data 3B vav1supplementaryfigures.pdf Supplementary figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6712113","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603028108,"identity":"73cc4b25-ef1c-4eef-b9a2-257f39bbe113","order_by":0,"name":"Jin 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Y.Y. is an employee of ThermoFisher Scientific, Inc.","formattedTitle":"Leveraging High-Throughput Proteomics and AI-Based Protein Folding to Accelerate VAV1 Molecular Glue Discovery","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6712113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6712113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study reports the discovery and characterization of novel CRBN molecular glues that selectively induce the proteasomal degradation of the hematopoietic-specific signaling protein VAV1, a key target in hematological malignancies and autoimmune diseases. Utilizing unbiased global proteomics, we identified phenyl-glutarimide derivatives NGT-201-12, as effective VAV1 degraders, with its C-terminal SH3 domain (SH3-2) being crucial for this interaction. A significant finding is the elucidation of a non-canonical RT-loop degron (RDxS motif, residues 796-799) within VAV1 SH3-2, distinct from previously characterized G-loop degrons. This discovery, supported by advanced computational modeling using the physics- and AI-driven GluePlex workflow and validated by site-directed mutagenesis, highlights versatility of CRBN in recognizing diverse neosubstrate motifs. Furthermore, we demonstrate that applying Free Energy Perturbation (FEP) calculations to these predicted ternary structures yields cooperativity metrics that correlate with experimental degradation potency, overcoming the limitations of standard molecular docking. This establishes a robust workflow where, once a ternary complex is predicted—even with initial weak binders—FEP can be utilized to prospectively rank analogs and optimize molecular glue potency. Additionally, we demonstrate that strategic chemical modifications, particularly conformational restriction via halogen substitution (e.g., NGT-201-18), markedly potentiate VAV1 degradation, a principle supported by density functional theory (DFT) calculations. Comprehensive structure-activity relationship (SAR) studies provided a roadmap for designing next-generation VAV1 degraders. Importantly, dose-response proteomics not only confirmed VAV1 as the primary target but also revealed LIMD1, possessing a canonical G-loop, as an off-target for some analogs, indicating a single molecular glue can engage disparate degron motifs. The identification of the VAV1 RT-loop degron prompted a proteome-wide search, revealing other SH3-containing proteins as potential targets or off-targets. In conclusion, this research unveils a novel non-canonical RT-loop degron in VAV1, demonstrates the utility of conformational restriction in enhancing degrader potency, and underscores the critical role of integrating global proteomics with advanced structural modeling and FEP calculations for understanding degrader potency and selectivity. These findings offer a promising therapeutic strategy for targeting VAV1 and significantly expand the landscape of CRBN neosubstrate recognition and the rational design of molecular glue degraders.\u003c/p\u003e","manuscriptTitle":"Leveraging High-Throughput Proteomics and AI-Based Protein Folding to Accelerate VAV1 Molecular Glue Discovery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-10 16:37:33","doi":"10.21203/rs.3.rs-6712113/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7d895e93-a2de-4043-a736-6cca21be5522","owner":[],"postedDate":"March 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64163671,"name":"Biological sciences/Drug discovery"},{"id":64163672,"name":"Biological sciences/Chemical biology/Proteomics"},{"id":64163673,"name":"Biological sciences/Computational biology and bioinformatics/Protein folding"}],"tags":[],"updatedAt":"2026-04-09T08:00:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-10 16:37:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6712113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6712113","identity":"rs-6712113","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}