Deep Learning-Assisted Discovery of HIF-1α/VHL Protein-Protein Interaction Inhibitors for Hypoxic Cell Protection

Deep Learning-Assisted Discovery of HIF-1α/VHL Protein-Protein Interaction Inhibitors for Hypoxic Cell Protection | 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 Research Article Deep Learning-Assisted Discovery of HIF-1α/VHL Protein-Protein Interaction Inhibitors for Hypoxic Cell Protection Binglin Huang, Bijuan Lin, Xiao Chen, Yifan Zhu, Na Li, Bin Zheng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9415420/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract The HIF-1α/VHL protein-protein interaction regulates cellular hypoxic adaptation. Targeting this PPI offers therapeutic potential for ischemia, yet current VHL inhibitors lack structural diversity, are primarily developed as PROTAC ligands, and lack systematic investigation into their hypoxic cell protective activity. Here, we developed a deep learning-augmented virtual screening workflow to discover novel HIF-1α/VHL PPI inhibitors. Using DDMut-PPI, we analyzed interface residue binding free energy in the HIF-1α/VHL complex to identify auxiliary key residues for pharmacophore optimization. We virtually screened 24,893 molecules and validated hits through biophysical, computational, cellular, and functional assays. We identified four hits with VHL binding affinity (IC₅₀ < 10 μM). Cmpd16 exhibited the highest affinity (IC₅₀ = 0.41 μM). Molecular dynamics simulations and MM-GBSA confirmed a stable Cmpd16-VHL binding mode involving core and auxiliary residues. Cmpd16 was non-cytotoxic up to 200 μM and selectively stabilized HIF-1α and hydroxylated HIF-1α in VHL-expressing cells. In an oxygen-glucose deprivation/reoxygenation model, 10 μM Cmpd16 significantly restored human umbilical vein endothelial cell migration (68% increase) and tube formation (55% increase), demonstrating protective efficacy comparable to VH032. This strategy accelerates PPI inhibitor discovery, presenting Cmpd16 as a promising therapeutic candidate for ischemic diseases and expanding VHL ligand applications beyond PROTACs. HIF-1α/VHL Protein-protein interaction Deep learning Virtual screening Hypoxic cell protection Small molecule inhibitor Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 18 May, 2026 Reviews received at journal 17 May, 2026 Reviews received at journal 15 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor assigned by journal 17 Apr, 2026 Submission checks completed at journal 17 Apr, 2026 First submitted to journal 14 Apr, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9415420","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625180854,"identity":"02ed5d74-15c5-482f-9d4b-1dc1459e6eb2","order_by":0,"name":"Binglin Huang","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Binglin","middleName":"","lastName":"Huang","suffix":""},{"id":625180855,"identity":"de6f004b-3d6e-4c6d-841f-b08c2b5021b5","order_by":1,"name":"Bijuan Lin","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bijuan","middleName":"","lastName":"Lin","suffix":""},{"id":625180856,"identity":"4c28aa58-c7bb-4b5f-8044-761d69060981","order_by":2,"name":"Xiao Chen","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Chen","suffix":""},{"id":625180857,"identity":"ef2c8fa1-20e5-4f91-9e63-6ab8203bceb5","order_by":3,"name":"Yifan Zhu","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Zhu","suffix":""},{"id":625180858,"identity":"ba4e88f6-168b-40e3-baed-daa85578a461","order_by":4,"name":"Na Li","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Li","suffix":""},{"id":625180859,"identity":"3c9968e5-48b4-42c7-8ab4-d5b61f48fa1a","order_by":5,"name":"Bin Zheng","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zheng","suffix":""},{"id":625180860,"identity":"1d85dcbc-ebc2-4b83-bea8-b287ea8ed0c7","order_by":6,"name":"Weiwei Zheng","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Zheng","suffix":""},{"id":625180861,"identity":"fea592b5-73ee-4d9a-96b0-4b1850c117cd","order_by":7,"name":"Xin Xue","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Xue","suffix":""},{"id":625180862,"identity":"f88a6325-ca99-4df6-9e1d-1ee5a6d8f518","order_by":8,"name":"Maobai Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACPmYGBmYGg39ybOyNDwzAQgcIaGEDa6k4YMzHc9iASC0MIC1nDiTOk0iG6CCshZ3HdHNh253ENsnHDEU32xjk+G4kMH4uwOswHrPbM9ueGbdJJzMY57YxGEveSGCWnkFIC28bs2ybdP4BkJbEDTcSgIJEaGFskzwMtqWeOC08Zw4rtkkwg7UkGBDWwlZ2e0ZFmjEbD9AvOeckDGeeedgsjU8LP//hbbcLDGzk5NsPsxnnlNnI8x1PPvgZnxYUG4ERIwGkGRuI1ACM0gdEKx0Fo2AUjIIRBQDOb0PRz5whiAAAAABJRU5ErkJggg==","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":true,"prefix":"","firstName":"Maobai","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-04-14 12:24:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9415420/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9415420/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107708001,"identity":"cfc0342a-1212-4a30-8718-c648e697e886","added_by":"auto","created_at":"2026-04-24 09:21:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1616520,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptR1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9415420/v1_covered_3f2ac2c3-8422-43b9-b76e-f907af92620e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deep Learning-Assisted Discovery of HIF-1α/VHL Protein-Protein Interaction Inhibitors for Hypoxic Cell Protection","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HIF-1α/VHL, Protein-protein interaction, Deep learning, Virtual screening, Hypoxic cell protection, Small molecule inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-9415420/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9415420/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe HIF-1α/VHL protein-protein interaction regulates cellular hypoxic adaptation. Targeting this PPI offers therapeutic potential for ischemia, yet current VHL inhibitors lack structural diversity, are primarily developed as PROTAC ligands, and lack systematic investigation into their hypoxic cell protective activity. Here, we developed a deep learning-augmented virtual screening workflow to discover novel HIF-1α/VHL PPI inhibitors. Using DDMut-PPI, we analyzed interface residue binding free energy in the HIF-1α/VHL complex to identify auxiliary key residues for pharmacophore optimization. We virtually screened 24,893 molecules and validated hits through biophysical, computational, cellular, and functional assays. We identified four hits with VHL binding affinity (IC₅₀ \u0026lt; 10 μM). Cmpd16 exhibited the highest affinity (IC₅₀ = 0.41 μM). Molecular dynamics simulations and MM-GBSA confirmed a stable Cmpd16-VHL binding mode involving core and auxiliary residues. Cmpd16 was non-cytotoxic up to 200 μM and selectively stabilized HIF-1α and hydroxylated HIF-1α in VHL-expressing cells. In an oxygen-glucose deprivation/reoxygenation model, 10 μM Cmpd16 significantly restored human umbilical vein endothelial cell migration (68% increase) and tube formation (55% increase), demonstrating protective efficacy comparable to VH032. This strategy accelerates PPI inhibitor discovery, presenting Cmpd16 as a promising therapeutic candidate for ischemic diseases and expanding VHL ligand applications beyond PROTACs.\u003c/p\u003e","manuscriptTitle":"Deep Learning-Assisted Discovery of HIF-1α/VHL Protein-Protein Interaction Inhibitors for Hypoxic Cell Protection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 05:41:18","doi":"10.21203/rs.3.rs-9415420/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-18T10:51:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-17T13:20:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T09:35:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247219366005408349239026046037872633601","date":"2026-05-14T10:51:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320126656303628433199578282270490016790","date":"2026-04-29T11:09:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203340952485261971944573365680694367469","date":"2026-04-29T08:23:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204028293769362489191066306518703909785","date":"2026-04-17T19:52:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T11:37:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T11:03:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T07:12:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Diversity","date":"2026-04-14T12:18:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0c7f3be0-cc0d-4566-aa34-04ad984b670a","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-18T10:51:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-17T13:20:44+00:00","index":36,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T09:35:14+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"247219366005408349239026046037872633601","date":"2026-05-14T10:51:09+00:00","index":33,"fulltext":""},{"type":"reviewerAgreed","content":"320126656303628433199578282270490016790","date":"2026-04-29T11:09:40+00:00","index":26,"fulltext":""},{"type":"reviewerAgreed","content":"203340952485261971944573365680694367469","date":"2026-04-29T08:23:02+00:00","index":25,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T10:55:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 05:41:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9415420","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9415420","identity":"rs-9415420","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}