Structure-guided design of a novel, stable, and soluble Cecropin A variant for antimicrobial therapeutic applications

Structure-guided design of a novel, stable, and soluble Cecropin A variant for antimicrobial therapeutic applications | 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 Structure-guided design of a novel, stable, and soluble Cecropin A variant for antimicrobial therapeutic applications Samaneh Hashemi, Armin Zarei, Mohammad Sadegh Taghizadeh, Cambyz Iraji, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6996527/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Cecropin A is a potent antimicrobial peptide with broad-spectrum activity; however, its clinical application is limited by poor stability, solubility, and bioavailability. In this study, an engineered analog of Cecropin A was developed by incorporating an M cell-targeting peptide (MTP) at the N-terminus, a self-assembling EAK16-II sequence at the C-terminus, and a rigid EAAAK linker to preserve domain integrity. This engineered peptide (Pep A) exhibited significantly improved physicochemical properties, including enhanced solubility, a higher extinction coefficient, and a more favorable net positive charge, all of which may support stronger interactions with microbial membranes and enhanced therapeutic potential. Despite a slightly elevated instability index, the peptide remained within the stable range. The engineered peptide was predicted to be non-toxic, in contrast to its native form, which was identified as toxic. Furthermore, its high solubility upon overexpression facilitates large-scale recombinant production in bacterial systems. Computational modeling using AlphaFold and Robetta confirmed the structural accuracy of the design, with Pep A outperforming other models in compactness and favorable folding. Molecular dynamics simulations further validated the improved stability of Pep A, demonstrating reduced flexibility, consistent hydrogen bonding, and lower solvent exposure. Point mutations at surface-accessible, flexible, and non-critical residues further enhanced its stability. Collectively, these modifications produced a multifunctional Cecropin A analog with improved structural stability, solubility, and bioavailability, establishing Pep A as a promising candidate for advanced antimicrobial and mucosal delivery applications. However, in vitro and in vivo validations are critical for confirming its final applications. Biological sciences/Biochemistry Biological sciences/Biotechnology Physical sciences/Chemistry Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Biological sciences/Microbiology Cecropin A Antimicrobial peptides Peptide engineering Computational studies Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Reviewers agreed at journal 17 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviewers invited by journal 17 Jul, 2025 Editor assigned by journal 16 Jul, 2025 Editor invited by journal 10 Jul, 2025 Submission checks completed at journal 02 Jul, 2025 First submitted to journal 02 Jul, 2025 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-6996527","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":486969284,"identity":"d4b6a4e0-d899-40ee-b5b5-5a30e7f578d3","order_by":0,"name":"Samaneh Hashemi","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Samaneh","middleName":"","lastName":"Hashemi","suffix":""},{"id":486969285,"identity":"ea12d5fb-e7f0-4140-9907-084f4c9d56e5","order_by":1,"name":"Armin Zarei","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Armin","middleName":"","lastName":"Zarei","suffix":""},{"id":486969286,"identity":"c8bc2e0d-4237-44ee-96d9-eb0773c2b375","order_by":2,"name":"Mohammad Sadegh Taghizadeh","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Sadegh","lastName":"Taghizadeh","suffix":""},{"id":486969287,"identity":"23f8670d-3e61-40c8-9e1b-9d0904208165","order_by":3,"name":"Cambyz Iraji","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Cambyz","middleName":"","lastName":"Iraji","suffix":""},{"id":486969291,"identity":"ff2691e3-1ba1-4d2d-8a52-0fd937d5008c","order_by":4,"name":"Navid Nezafat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIie3RsQrCMBCA4ZOALtGuKaK+wkmg4qC+SkPBqUN3BeOiS9BV8FUcKgW7+BAWoS4uIkgHETO4ia1uDvnhhgwfuRAAk+l/awJUJBDA19ktJhwoDX8mzNXkmzpQ3SbZGFsD+5Reg6AvZCU6QLL5TLqy5iHbYVvVfb5eoSckHSK46WeCIXUYlu8lTQihSIQEX78lzCWdzH3gQNnxUZOJkNa5kDiwnaFQDLgmkZCs6JaIcnu6QE/tfU5WGPMZSzHMJbFqX7Ib9uZzvVhwHzWWlpckWQ55/4uynjxgMplMpi96Ai/ZSPGvufvLAAAAAElFTkSuQmCC","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Navid","middleName":"","lastName":"Nezafat","suffix":""},{"id":486969292,"identity":"cd9463de-e31a-41f3-aa3c-189fc13b1601","order_by":5,"name":"Amir Savardashtaki","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Amir","middleName":"","lastName":"Savardashtaki","suffix":""}],"badges":[],"createdAt":"2025-06-28 08:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6996527/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6996527/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18067-x","type":"published","date":"2025-09-25T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92430466,"identity":"5bad0503-198b-4749-ba94-62cfd0f1a3ce","added_by":"auto","created_at":"2025-09-29 16:05:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":854298,"visible":true,"origin":"","legend":"","description":"","filename":"2FCManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6996527/v1_covered_89783a1d-307b-4b35-95ac-07527142ec05.pdf"},{"id":87214838,"identity":"a083259e-3ebb-4041-9d73-feece229eafc","added_by":"auto","created_at":"2025-07-21 15:18:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":38459,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6996527/v1/f1df496be7eeb1c8704f679d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structure-guided design of a novel, stable, and soluble Cecropin A variant for antimicrobial therapeutic applications","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Cecropin A, Antimicrobial peptides, Peptide engineering, Computational studies","lastPublishedDoi":"10.21203/rs.3.rs-6996527/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6996527/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCecropin A is a potent antimicrobial peptide with broad-spectrum activity; however, its clinical application is limited by poor stability, solubility, and bioavailability. In this study, an engineered analog of Cecropin A was developed by incorporating an M cell-targeting peptide (MTP) at the N-terminus, a self-assembling EAK16-II sequence at the C-terminus, and a rigid EAAAK linker to preserve domain integrity. This engineered peptide (Pep A) exhibited significantly improved physicochemical properties, including enhanced solubility, a higher extinction coefficient, and a more favorable net positive charge, all of which may support stronger interactions with microbial membranes and enhanced therapeutic potential. Despite a slightly elevated instability index, the peptide remained within the stable range. The engineered peptide was predicted to be non-toxic, in contrast to its native form, which was identified as toxic. Furthermore, its high solubility upon overexpression facilitates large-scale recombinant production in bacterial systems. Computational modeling using AlphaFold and Robetta confirmed the structural accuracy of the design, with Pep A outperforming other models in compactness and favorable folding. Molecular dynamics simulations further validated the improved stability of Pep A, demonstrating reduced flexibility, consistent hydrogen bonding, and lower solvent exposure. Point mutations at surface-accessible, flexible, and non-critical residues further enhanced its stability. Collectively, these modifications produced a multifunctional Cecropin A analog with improved structural stability, solubility, and bioavailability, establishing Pep A as a promising candidate for advanced antimicrobial and mucosal delivery applications. However, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e validations are critical for confirming its final applications.\u003c/p\u003e","manuscriptTitle":"Structure-guided design of a novel, stable, and soluble Cecropin A variant for antimicrobial therapeutic applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 15:10:01","doi":"10.21203/rs.3.rs-6996527/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"175875586505463632475560922309221703108","date":"2025-07-17T06:58:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145573024655489699536406247159751871760","date":"2025-07-17T06:33:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-17T05:47:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-17T02:28:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-10T09:44:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-03T03:45:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-02T09:13:18+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":"1b5ed79b-1fa9-4e3e-8001-7ba40ffe71d3","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51701554,"name":"Biological sciences/Biochemistry"},{"id":51701555,"name":"Biological sciences/Biotechnology"},{"id":51701556,"name":"Physical sciences/Chemistry"},{"id":51701557,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":51701558,"name":"Biological sciences/Drug discovery"},{"id":51701559,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-09-29T16:00:07+00:00","versionOfRecord":{"articleIdentity":"rs-6996527","link":"https://doi.org/10.1038/s41598-025-18067-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-25 15:57:25","publishedOnDateReadable":"September 25th, 2025"},"versionCreatedAt":"2025-07-21 15:10:01","video":"","vorDoi":"10.1038/s41598-025-18067-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18067-x","workflowStages":[]},"version":"v1","identity":"rs-6996527","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6996527","identity":"rs-6996527","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}