{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T11:45:19Z","timestamp":1784893519374,"version":"3.55.0"},"reference-count":53,"publisher":"Public Library of Science (PLoS)","issue":"10","license":[{"start":{"date-parts":[[2023,10,24]],"date-time":"2023-10-24T00:00:00Z","timestamp":1698105600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004704","name":"National Research Council of Thailand","doi-asserted-by":"publisher","award":["N42A650316"],"award-info":[{"award-number":["N42A650316"]}],"id":[{"id":"10.13039\/501100004704","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004704","name":"National Research Council of Thailand","doi-asserted-by":"publisher","award":["N42A650231"],"award-info":[{"award-number":["N42A650231"]}],"id":[{"id":"10.13039\/501100004704","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The COVID-19 pandemic has created an urgent need for effective therapeutic and diagnostic strategies to manage the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the emergence of numerous variants of concern (VOCs) has made it challenging to develop targeted therapies that are broadly specific in neutralizing the virus. In this study, we aimed to develop neutralizing nanobodies (Nbs) using computational techniques that can effectively neutralize the receptor-binding domain (RBD) of SARS-CoV-2 VOCs. We evaluated the performance of different protein-protein docking programs and identified HDOCK as the most suitable program for Nb\/RBD docking with high accuracy. Using this approach, we designed 14 novel Nbs with high binding affinity to the VOC RBDs. The Nbs were engineered with mutated amino acids that interacted with key amino acids of the RBDs, resulting in higher binding affinity than human angiotensin-converting enzyme 2 (ACE2) and other viral RBDs or haemagglutinins (HAs). The successful development of these Nbs demonstrates the potential of molecular modeling as a low-cost and time-efficient method for engineering effective Nbs against SARS-CoV-2. The engineered Nbs have the potential to be employed in RBD-neutralizing assays, facilitating the identification of novel treatment, prevention, and diagnostic strategies against SARS-CoV-2.<\/jats:p>","DOI":"10.1371\/journal.pone.0293263","type":"journal-article","created":{"date-parts":[[2023,10,24]],"date-time":"2023-10-24T17:23:27Z","timestamp":1698168207000},"page":"e0293263","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":14,"title":["Computational design of novel nanobodies targeting the receptor binding domain of variants of concern of SARS-CoV-2"],"prefix":"10.1371","volume":"18","author":[{"given":"Phoomintara","family":"Longsompurana","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thanyada","family":"Rungrotmongkol","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nongluk","family":"Plongthongkum","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kittikhun","family":"Wangkanont","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Wolschann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2549-0187","authenticated-orcid":true,"given":"Rungtiva P.","family":"Poo-arporn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2023,10,24]]},"reference":[{"issue":"1","key":"pone.0293263.ref001","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1186\/s12967-020-02520-8","article-title":"Pathophysiology and treatment strategies for COVID-19","volume":"18","author":"M Kumar","year":"2020","journal-title":"J Transl Med"},{"issue":"7798","key":"pone.0293263.ref002","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/s41586-020-2008-3","article-title":"A new coronavirus associated with human respiratory disease in China","volume":"579","author":"F Wu","year":"2020","journal-title":"Nature"},{"issue":"5","key":"pone.0293263.ref003","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1037\/hea0000875","article-title":"The novel coronavirus (COVID-2019) outbreak: Amplification of public health consequences by media exposure","volume":"39","author":"DR Garfin","year":"2020","journal-title":"Health Psychology"},{"issue":"1","key":"pone.0293263.ref004","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1038\/s41580-021-00418-x","article-title":"Mechanisms of SARS-CoV-2 entry into cells","volume":"23","author":"CB Jackson","year":"2022","journal-title":"Nat Rev Mol Cell Biol"},{"key":"pone.0293263.ref005","doi-asserted-by":"crossref","first-page":"834098","DOI":"10.3389\/fimmu.2022.834098","article-title":"SARS-CoV-2\u2019s Variants of Concern: A Brief Characterization","volume":"13","author":"AM Scovino","year":"2022","journal-title":"Front Immunol"},{"key":"pone.0293263.ref006","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1016\/j.ijid.2022.06.039","article-title":"Classification of Omicron BA.1, BA.1.1, and BA.2 sublineages by TaqMan assay consistent with whole genome analysis data","volume":"122","author":"Y Hirotsu","year":"2022","journal-title":"International Journal of Infectious Diseases"},{"issue":"8","key":"pone.0293263.ref007","doi-asserted-by":"crossref","first-page":"104798","DOI":"10.1016\/j.isci.2022.104798","article-title":"Potent and broad neutralization of SARS-CoV-2 variants of concern (VOCs) including omicron sub-lineages BA.1 and BA.2 by biparatopic human VH domains","volume":"25","author":"C Chen","year":"2022","journal-title":"iScience"},{"key":"pone.0293263.ref008","article-title":"Potent Neutralization of Omicron and Other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains","author":"C Chen","year":"2022","journal-title":"Immunology"},{"key":"pone.0293263.ref009","article-title":"Nebulized Delivery of a Broadly Neutralizing SARS-CoV-2 RBD-Specific Nanobody Prevents Clinical, Virological and Pathological Disease in a Syrian Hamster Model of COVID-19","author":"TJ Esparza","year":"2021","journal-title":"Biochemistry"},{"key":"pone.0293263.ref010","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1016\/j.ijbiomac.2018.11.122","article-title":"Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody","volume":"123","author":"C Xu","year":"2019","journal-title":"International Journal of Biological Macromolecules"},{"issue":"1","key":"pone.0293263.ref011","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","article-title":"Nanobodies: Natural Single-Domain Antibodies","volume":"82","author":"S. Muyldermans","year":"2013","journal-title":"Annu Rev Biochem"},{"issue":"17","key":"pone.0293263.ref012","doi-asserted-by":"crossref","first-page":"1568","DOI":"10.1177\/1535370219881129","article-title":"Structure and development of single domain antibodies as modules for therapeutics and diagnostics","volume":"244","author":"RJ Hoey","year":"2019","journal-title":"Exp Biol Med (Maywood)"},{"issue":"5","key":"pone.0293263.ref013","doi-asserted-by":"crossref","first-page":"3675","DOI":"10.3390\/molecules16053675","article-title":"High Affinity, Developability and Functional Size: The Holy Grail of Combinatorial Antibody Library Generation","volume":"16","author":"D Ponsel","year":"2011","journal-title":"Molecules"},{"issue":"15","key":"pone.0293263.ref014","doi-asserted-by":"crossref","first-page":"6071","DOI":"10.1007\/s00253-017-8347-9","article-title":"Site-saturation mutagenesis library construction and screening for specific broad-spectrum single-domain antibodies against multiple Cry1 toxins","volume":"101","author":"L Jiao","year":"2017","journal-title":"Appl Microbiol Biotechnol"},{"issue":"1","key":"pone.0293263.ref015","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The Protein Data Bank","volume":"28","author":"HM Berman","year":"2000","journal-title":"Nucleic Acids Research"},{"issue":"W1","key":"pone.0293263.ref016","doi-asserted-by":"crossref","first-page":"W252","DOI":"10.1093\/nar\/gku340","article-title":"SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information","volume":"42","author":"M Biasini","year":"2014","journal-title":"Nucleic Acids Research"},{"issue":"2","key":"pone.0293263.ref017","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1002\/prot.21949","article-title":"An iterative knowledge-based scoring function for protein-protein recognition","volume":"72","author":"SY Huang","year":"2008","journal-title":"Proteins"},{"issue":"7","key":"pone.0293263.ref018","doi-asserted-by":"crossref","first-page":"e55","DOI":"10.1093\/nar\/gku077","article-title":"A knowledge-based scoring function for protein-RNA interactions derived from a statistical mechanics-based iterative method","volume":"42","author":"SY Huang","year":"2014","journal-title":"Nucleic Acids Research"},{"issue":"3","key":"pone.0293263.ref019","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1002\/prot.25234","article-title":"Addressing recent docking challenges: A hybrid strategy to integrate template-based and free protein-protein docking: Docking Tool for Predicting Protein-Protein Interactions","volume":"85","author":"Y Yan","year":"2017","journal-title":"Proteins"},{"issue":"W1","key":"pone.0293263.ref020","doi-asserted-by":"crossref","first-page":"W365","DOI":"10.1093\/nar\/gkx407","article-title":"HDOCK: a web server for protein\u2013protein and protein\u2013DNA\/RNA docking based on a hybrid strategy","volume":"45","author":"Y Yan","year":"2017","journal-title":"Nucleic Acids Research"},{"issue":"5","key":"pone.0293263.ref021","doi-asserted-by":"crossref","first-page":"1829","DOI":"10.1038\/s41596-020-0312-x","article-title":"The HDOCK server for integrated protein\u2013protein docking","volume":"15","author":"Y Yan","year":"2020","journal-title":"Nat Protoc"},{"issue":"3","key":"pone.0293263.ref022","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1016\/j.bpj.2014.12.015","article-title":"A Web Interface for Easy Flexible Protein-Protein Docking with ATTRACT","volume":"108","author":"SJ de Vries","year":"2015","journal-title":"Biophysical Journal"},{"issue":"13","key":"pone.0293263.ref023","doi-asserted-by":"crossref","first-page":"1698","DOI":"10.1093\/bioinformatics\/btt262","article-title":"pyDockWEB: a web server for rigid-body protein\u2013protein docking using electrostatics and desolvation scoring","volume":"29","author":"B Jim\u00e9nez-Garc\u00eda","year":"2013","journal-title":"Bioinformatics"},{"issue":"Web Server","key":"pone.0293263.ref024","doi-asserted-by":"crossref","first-page":"W310","DOI":"10.1093\/nar\/gkl206","article-title":"GRAMM-X public web server for protein-protein docking","volume":"34","author":"A Tovchigrechko","year":"2006","journal-title":"Nucleic Acids Research"},{"issue":"Web Server","key":"pone.0293263.ref025","doi-asserted-by":"crossref","first-page":"W363","DOI":"10.1093\/nar\/gki481","article-title":"PatchDock and SymmDock: servers for rigid and symmetric docking","volume":"33","author":"D Schneidman-Duhovny","year":"2005","journal-title":"Nucleic Acids Research"},{"key":"pone.0293263.ref026","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1007\/3-540-45784-4_14","volume-title":"Algorithms in Bioinformatics","author":"D Duhovny","year":"2002"},{"issue":"19","key":"pone.0293263.ref027","doi-asserted-by":"crossref","first-page":"2544","DOI":"10.1093\/bioinformatics\/btp447","article-title":"FRODOCK: a new approach for fast rotational protein\u2013protein docking","volume":"25","author":"JI Garzon","year":"2009","journal-title":"Bioinformatics"},{"issue":"15","key":"pone.0293263.ref028","doi-asserted-by":"crossref","first-page":"2386","DOI":"10.1093\/bioinformatics\/btw141","article-title":"FRODOCK 2.0: fast protein\u2013protein docking server","volume":"32","author":"E Ram\u00edrez-Aportela","year":"2016","journal-title":"Bioinformatics"},{"issue":"12","key":"pone.0293263.ref029","doi-asserted-by":"crossref","first-page":"1771","DOI":"10.1093\/bioinformatics\/btu097","article-title":"ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers","volume":"30","author":"BG Pierce","year":"2014","journal-title":"Bioinformatics"},{"issue":"1","key":"pone.0293263.ref030","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1186\/1471-2105-7-339","article-title":"Tools for integrated sequence-structure analysis with UCSF Chimera","volume":"7","author":"EC Meng","year":"2006","journal-title":"BMC Bioinformatics"},{"issue":"5","key":"pone.0293263.ref031","doi-asserted-by":"crossref","first-page":"1623","DOI":"10.1002\/prot.22987","article-title":"New benchmark metrics for protein-protein docking methods: Assessment of Protein Docking Models","volume":"79","author":"M Gao","year":"2011","journal-title":"Proteins"},{"issue":"W1","key":"pone.0293263.ref032","doi-asserted-by":"crossref","first-page":"W276","DOI":"10.1093\/nar\/gkac240","article-title":"Search and sequence analysis tools services from EMBL-EBI in 2022","volume":"50","author":"F Madeira","year":"2022","journal-title":"Nucleic Acids Research"},{"issue":"1","key":"pone.0293263.ref033","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1002\/pro.3289","article-title":"PDBsum: Structural summaries of PDB entries","volume":"27","author":"RA Laskowski","year":"2018","journal-title":"Protein Science"},{"key":"pone.0293263.ref034","doi-asserted-by":"crossref","first-page":"571","DOI":"10.1385\/1-59259-890-0:571","volume-title":"The Proteomics Protocols Handbook","author":"E Gasteiger","year":"2005"},{"issue":"19","key":"pone.0293263.ref035","doi-asserted-by":"crossref","first-page":"3098","DOI":"10.1093\/bioinformatics\/btx345","article-title":"Protein\u2013Sol: a web tool for predicting protein solubility from sequence","volume":"33","author":"M Hebditch","year":"2017","journal-title":"Bioinformatics"},{"issue":"1","key":"pone.0293263.ref036","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.1016\/j.bbrc.2017.11.073","article-title":"The effect of surface charge on the thermal stability and ice recrystallization inhibition activity of antifreeze protein III (AFP III)","volume":"495","author":"RC Deller","year":"2018","journal-title":"Biochemical and Biophysical Research Communications"},{"key":"pone.0293263.ref037","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/j.biochi.2020.11.004","article-title":"Molecular docking simulation reveals ACE2 polymorphisms that may increase the affinity of ACE2 with the SARS-CoV-2 Spike protein","volume":"180","author":"M Calcagnile","year":"2021","journal-title":"Biochimie"},{"issue":"3","key":"pone.0293263.ref038","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1007\/s40268-021-00357-0","article-title":"A Novel Therapeutic Peptide Blocks SARS-CoV-2 Spike Protein Binding with Host Cell ACE2 Receptor","volume":"21","author":"S Rajpoot","year":"2021","journal-title":"Drugs R D"},{"key":"pone.0293263.ref039","doi-asserted-by":"crossref","first-page":"105226","DOI":"10.1016\/j.compbiomed.2022.105226","article-title":"SARS-CoV-2 Omicron spike glycoprotein receptor binding domain exhibits super-binder ability with ACE2 but not convalescent monoclonal antibody","volume":"142","author":"O Omotuyi","year":"2022","journal-title":"Computers in Biology and Medicine"},{"key":"pone.0293263.ref040","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1016\/j.ijbiomac.2020.02.180","article-title":"Nanobody affinity improvement: Directed evolution of the anti-ochratoxin A single domain antibody","volume":"151","author":"X Wang","year":"2020","journal-title":"International Journal of Biological Macromolecules"},{"issue":"7807","key":"pone.0293263.ref041","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1038\/s41586-020-2180-5","article-title":"Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor","volume":"581","author":"J Lan","year":"2020","journal-title":"Nature"},{"issue":"25","key":"pone.0293263.ref042","doi-asserted-by":"crossref","first-page":"13967","DOI":"10.1073\/pnas.2008209117","article-title":"Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions","volume":"117","author":"Y Wang","year":"2020","journal-title":"Proc Natl Acad Sci USA"},{"issue":"5","key":"pone.0293263.ref043","doi-asserted-by":"crossref","first-page":"3273","DOI":"10.1074\/jbc.M806889200","article-title":"General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold","volume":"284","author":"C Vincke","year":"2009","journal-title":"Journal of Biological Chemistry"},{"issue":"1","key":"pone.0293263.ref044","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/1475-2859-10-60","article-title":"Post-production protein stability: trouble beyond the cell factory","volume":"10","author":"E Vazquez","year":"2011","journal-title":"Microb Cell Fact"},{"issue":"2","key":"pone.0293263.ref045","doi-asserted-by":"crossref","DOI":"10.15761\/IMM.1000408","article-title":"NL63: A Better Surrogate Virus for studying SARS-CoV-2","volume":"8","author":"A Chakraborty","year":"2020","journal-title":"Integr Mol Med"},{"key":"pone.0293263.ref046","first-page":"428","volume-title":"Encyclopedia of Virology","author":"DX Liu","year":"2021"},{"key":"pone.0293263.ref047","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.2147\/IDR.S357193","article-title":"Clinical Study of Human Coronavirus NL63, OC43, 229E, HKU1 Infentions in Hospitalized Children from 2015 to 2020","volume":"15","author":"C Wen","year":"2022","journal-title":"IDR"},{"issue":"8","key":"pone.0293263.ref048","doi-asserted-by":"crossref","first-page":"1840","DOI":"10.1080\/21645515.2018.1462639","article-title":"H3N2 influenza viruses in humans: Viral mechanisms, evolution, and evaluation","volume":"14","author":"JD Allen","year":"2018","journal-title":"Human Vaccines & Immunotherapeutics"},{"issue":"22","key":"pone.0293263.ref049","doi-asserted-by":"crossref","first-page":"12097","DOI":"10.3390\/ijms222212097","article-title":"Antiviral Gene Expression in Young and Aged Murine Lung during H1N1 and H3N2","volume":"22","author":"R Harris","year":"2021","journal-title":"IJMS"},{"issue":"15","key":"pone.0293263.ref050","doi-asserted-by":"crossref","first-page":"4657","DOI":"10.1111\/febs.16376","article-title":"Novel engineered nanobodies specific for N-terminal region of alpha-synuclein recognize Lewy-body pathology and inhibit in-vitro seeded aggregation and toxicity","volume":"289","author":"I Hmila","year":"2022","journal-title":"The FEBS Journal"},{"issue":"1","key":"pone.0293263.ref051","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/cbdd.13847","article-title":"Computational design and modeling of nanobodies toward SARS-CoV-2 receptor binding domain","volume":"98","author":"J Yang","year":"2021","journal-title":"Chem Biol Drug Des"},{"issue":"1","key":"pone.0293263.ref052","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1038\/s41392-022-00912-4","article-title":"An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants","volume":"7","author":"X Chi","year":"2022","journal-title":"Sig Transduct Target Ther"},{"key":"pone.0293263.ref053","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.bbrc.2021.07.054","article-title":"Positive charge in the complementarity-determining regions of synthetic nanobody prevents aggregation","volume":"572","author":"Z Zhong","year":"2021","journal-title":"Biochem. Biophys. Res. Commun"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0293263","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,24]],"date-time":"2023-10-24T17:24:01Z","timestamp":1698168241000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0293263"}},"subtitle":[],"editor":[{"given":"Sheikh Arslan","family":"Sehgal","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2023,10,24]]},"references-count":53,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2023,10,24]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0293263","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,10,24]]}}}