Development of Potential CDK9 Inhibitors through Pharmacophore-Based Virtual Screening, 3D-QSAR, Molecular Docking, MD Simulation, and In Vitro Anticancer Evaluation

Development of Potential CDK9 Inhibitors through Pharmacophore-Based Virtual Screening, 3D-QSAR, Molecular Docking, MD Simulation, and In Vitro Anticancer Evaluation | 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 Development of Potential CDK9 Inhibitors through Pharmacophore-Based Virtual Screening, 3D-QSAR, Molecular Docking, MD Simulation, and In Vitro Anticancer Evaluation Rajkumar Reddy Rajula, Umadevi Etikyala, Dinesha P, Sampat P V, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9086560/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Cyclin-dependent kinase 9 (CDK9) is a transcription-regulating serine/threonine kinase, and the dysregulation drives tumour initiation, thereby establishing CDK9 inhibition as a mechanistically validated and therapeutically attractive strategy for the treatment of diverse malignancies. In this study, a comprehensive computational strategy was utilized to identify novel CDK9 inhibitors. A pharmacophore-based virtual screening was implemented in combination with atom-based 3D-QSAR, molecular docking, binding free energies, in silico ADME, and MD simulation studies. A statistically validated five-point pharmacophore model (ADHRR) was developed and demonstrated strong predictive performance (R 2 = 0.98, Q 2 = 0.84). This optimized model was used to screen chemical databases for potential CDK9 inhibitors. Structural insights gained from the resulting hits guided the rational design of indole-based biphenyl amide hybrids (IBA’s). Seven analogues ( D1 - D7 ) exhibited strong binding affinities comparable to or greater than those of the screened hits and the reference CDK9 inhibitor 23 . Additionally, molecular dynamics simulations and DFT analysis confirmed the stability of both D3 and D6 complexes. Subsequent synthesis and biological evaluation against a panel of cancer cell lines identified compounds D3 and D6 as the most potent. Collectively, these results identify D3 and D6 as promising lead candidates for further CDK9-focused medicinal chemistry optimization and mechanistic studies. Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Pharmacophore-Based Virtual Screening 3D-QSAR Molecular Docking Binding Free Energy and Anticancer activity Full Text Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx Cite Share Download PDF Status: Published Journal Publication published 28 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Apr, 2026 Reviews received at journal 02 Apr, 2026 Reviews received at journal 28 Mar, 2026 Reviews received at journal 24 Mar, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviewers agreed at journal 21 Mar, 2026 Reviewers agreed at journal 21 Mar, 2026 Reviewers invited by journal 20 Mar, 2026 Editor assigned by journal 19 Mar, 2026 Editor invited by journal 19 Mar, 2026 Submission checks completed at journal 17 Mar, 2026 First submitted to journal 17 Mar, 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. 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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-9086560","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":609866182,"identity":"79dc268c-6a6b-4d62-9e67-b0aa9e8ef24e","order_by":0,"name":"Rajkumar Reddy Rajula","email":"data:image/png;base64,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","orcid":"","institution":"Woxsen University","correspondingAuthor":true,"prefix":"","firstName":"Rajkumar","middleName":"Reddy","lastName":"Rajula","suffix":""},{"id":609866183,"identity":"da321147-81ea-42f0-85c4-bbc60de30b0c","order_by":1,"name":"Umadevi Etikyala","email":"","orcid":"","institution":"Osmania University","correspondingAuthor":false,"prefix":"","firstName":"Umadevi","middleName":"","lastName":"Etikyala","suffix":""},{"id":609866184,"identity":"67a1fa84-392c-42c3-9230-119ac8e5c761","order_by":2,"name":"Dinesha P","email":"","orcid":"","institution":"National Institute of Technology Karnataka","correspondingAuthor":false,"prefix":"","firstName":"Dinesha","middleName":"","lastName":"P","suffix":""},{"id":609866185,"identity":"a9f9266c-f26f-4f90-b31b-0276de1ca40d","order_by":3,"name":"Sampat P V","email":"","orcid":"","institution":"National Institute of Technology Karnataka","correspondingAuthor":false,"prefix":"","firstName":"Sampat","middleName":"P","lastName":"V","suffix":""},{"id":609866186,"identity":"76587427-ee39-4f36-a143-b8277bd87cc9","order_by":4,"name":"Shreeganesh Subraya Hedge","email":"","orcid":"","institution":"Dayananda Sagar University","correspondingAuthor":false,"prefix":"","firstName":"Shreeganesh","middleName":"Subraya","lastName":"Hedge","suffix":""},{"id":609866187,"identity":"cd4bbb80-7b8d-4d5d-b62f-994ba1dfa76a","order_by":5,"name":"Muzaffar-Ur-Rehman Mohammed","email":"","orcid":"","institution":"Birla Institute of Technology and Science, Pilani","correspondingAuthor":false,"prefix":"","firstName":"Muzaffar-Ur-Rehman","middleName":"","lastName":"Mohammed","suffix":""},{"id":609866188,"identity":"e661d496-5752-4728-a87b-f2ffc48aeff5","order_by":6,"name":"Chandra Sekhar Kondapalli Venkata Gowri","email":"","orcid":"","institution":"Birla Institute of Technology and Science - 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In this study, a comprehensive computational strategy was utilized to identify novel CDK9 inhibitors. A pharmacophore-based virtual screening was implemented in combination with atom-based 3D-QSAR, molecular docking, binding free energies, \u003cem\u003ein silico\u003c/em\u003e ADME, and MD simulation studies. A statistically validated five-point pharmacophore model (ADHRR) was developed and demonstrated strong predictive performance (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.98, Q\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.84). This optimized model was used to screen chemical databases for potential CDK9 inhibitors. Structural insights gained from the resulting hits guided the rational design of indole-based biphenyl amide hybrids (IBA\u0026rsquo;s). Seven analogues (\u003cb\u003eD1\u003c/b\u003e-\u003cb\u003eD7\u003c/b\u003e) exhibited strong binding affinities comparable to or greater than those of the screened hits and the reference CDK9 inhibitor \u003cb\u003e23\u003c/b\u003e. Additionally, molecular dynamics simulations and DFT analysis confirmed the stability of both \u003cb\u003eD3\u003c/b\u003e and \u003cb\u003eD6\u003c/b\u003e complexes. Subsequent synthesis and biological evaluation against a panel of cancer cell lines identified compounds \u003cb\u003eD3\u003c/b\u003e and \u003cb\u003eD6\u003c/b\u003e as the most potent. Collectively, these results identify \u003cb\u003eD3\u003c/b\u003e and \u003cb\u003eD6\u003c/b\u003e as promising lead candidates for further CDK9-focused medicinal chemistry optimization and mechanistic studies.\u003c/p\u003e","manuscriptTitle":"Development of Potential CDK9 Inhibitors through Pharmacophore-Based Virtual Screening, 3D-QSAR, Molecular Docking, MD Simulation, and In Vitro Anticancer Evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 13:01:26","doi":"10.21203/rs.3.rs-9086560/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-03T12:55:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T21:31:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-28T12:27:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-24T05:55:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223629282006488022493216774809524036104","date":"2026-03-24T05:38:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220181827614536399547553266384367444156","date":"2026-03-21T12:24:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81862786202667181316119748649707490906","date":"2026-03-21T11:56:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-20T05:56:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-19T13:45:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-19T13:34:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-17T16:02:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-17T12:09:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3a7b10b1-4992-4e37-b810-ff36c4acd80a","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":64899026,"name":"Biological sciences/Cancer"},{"id":64899027,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":64899028,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2026-05-04T16:02:57+00:00","versionOfRecord":{"articleIdentity":"rs-9086560","link":"https://doi.org/10.1038/s41598-026-50502-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-28 15:57:31","publishedOnDateReadable":"April 28th, 2026"},"versionCreatedAt":"2026-03-24 13:01:26","video":"","vorDoi":"10.1038/s41598-026-50502-5","vorDoiUrl":"https://doi.org/10.1038/s41598-026-50502-5","workflowStages":[]},"version":"v1","identity":"rs-9086560","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9086560","identity":"rs-9086560","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}