{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:27:53Z","timestamp":1760149673897,"version":"build-2065373602"},"reference-count":30,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2023,8,29]],"date-time":"2023-08-29T00:00:00Z","timestamp":1693267200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Thymalin is an immunomodulatory drug containing a polypeptide extract of thymus that has demonstrated efficacy in the therapy of acute respiratory distress syndrome and chronic obstructive pulmonary disease, as well as in complex therapy related to severe COVID-19 in middle-aged and elderly patients.. KE and EW dipeptides are active substances of Thymalin. There is evidence that KE stimulates cellular immunity and nonspecific resistance in organisms, exerting an activating effect on macrophages, blood lymphocytes, thymocytes, and neutrophils, while EW reduces angiotensin-induced vasoconstriction and preserves endothelium-dependent vascular relaxation by inhibiting ACE2, the target protein of SARS-CoV-2. However, the mechanism of the immunomodulatory action of Thymalin, KE, and EW during COVID-19 remains unclear. To identify the potential mechanism of action underlying the immunomodulatory activity of Thymalin and its active components, EW and KE dipeptides, we assessed inflammatory response in the context of COVID-19. Interactions between EW and KE dipeptides and double-stranded DNA (dsDNA) were investigated by molecular modeling and docking using ICM-Pro. Analysis of the possible effect of EW and KE dipeptides on gene expression and protein synthesis involved in the pathogenesis of COVID-19 was conducted through the use of bioinformatics methods, including a search for promoter sequences in the Eukaryotic Promoter Database, the determination of genes associated with the development of COVID-19 using the PathCards database of human biological pathways (pathway unification database), identification of the relationship between proteins through cluster analysis in the STRING database (\u2018Search Tool for Retrieval of Interacting Genes\/Proteins\u2019), and assessment of the functional enrichment of protein\u2013protein interaction (PPI) using the terms of gene ontology (GO) and the Markov cluster algorithm (MCL). After that, in vitro studying of a lipopolysaccharide (LPS)-induced model of inflammation using human peripheral blood mononuclear cells was performed. ELISA was applied to assess the level of cytokines (IL-1\u03b2, IL-6, TNF\u03b1) in the supernatant of cells with or without the impact of EW and KE peptides. Blood samples were obtained from four donors; for each cytokine, ELISA was performed 2\u20134 times, with two parallel experimental or control samples for each experiment (experiments to assess the effects of peptides on LPS-stimulated cells were repeated four times, while additional experiments with unstimulated cells were performed two times). Using molecular docking, GGAG was found to be the best dsDNA sequence in the classical B-form for binding the EW dipeptide, while GCGC is the preferred dsDNA sequence in the curved nucleosomal form for the KE dipeptide. Cluster analysis revealed that potential target genes for the EW and KE peptides encode the AKT1 and AKT2 proteins involved in the development of the cytokine storm. The specific targets for the EW peptide are the ACE2 and CYSLTR1 genes, and specific target for the KE peptide is the CHUK gene. Protein products of the ACE2, CYSLTR1, and CHUK genes are functionally associated with IL-1\u03b2, IL-6, TNF-\u03b1, IL-4, and IL-10 cytokines. An in vitro model of an inflammatory reaction demonstrated that Thymalin and EW and KE dipeptides reduced the synthesis of IL-1\u03b2, IL-6, and TNF-\u03b1 cytokines in human peripheral blood mononuclear cells by 1.4\u20136.0 times. The immunomodulatory effect of Thymalin under the inflammatory response conditions in COVID-19 is based on the potential ability of its active components, EW and KE dipeptides, to regulate protein synthesis involved in the development of the cytokine storm.<\/jats:p>","DOI":"10.3390\/ijms241713377","type":"journal-article","created":{"date-parts":[[2023,8,29]],"date-time":"2023-08-29T08:26:52Z","timestamp":1693297612000},"page":"13377","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["The Influence of KE and EW Dipeptides in the Composition of the Thymalin Drug on Gene Expression and Protein Synthesis Involved in the Pathogenesis of COVID-19"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5156-5421","authenticated-orcid":false,"given":"Natalia","family":"Linkova","sequence":"first","affiliation":[{"name":"Saint Petersburg Institute of Bioregulation and Gerontology, 197110 Saint Petersburg, Russia"},{"name":"Saint Petersburg Research Institute of Phthisiopulmonology, 191036 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vladimir","family":"Khavinson","sequence":"additional","affiliation":[{"name":"Saint Petersburg Institute of Bioregulation and Gerontology, 197110 Saint Petersburg, Russia"},{"name":"Pavlov Institute of Physiology of Russian Academy of Sciences, 199034 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anastasiia","family":"Diatlova","sequence":"additional","affiliation":[{"name":"Saint Petersburg Institute of Bioregulation and Gerontology, 197110 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3771-1343","authenticated-orcid":false,"given":"Michael","family":"Petukhov","sequence":"additional","affiliation":[{"name":"Petersburg Nuclear Physics Institute Named after B.P. Konstantinov, NRC \u201cKurchatov Institute\u201d, 188300 Gatchina, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizaveta","family":"Vladimirova","sequence":"additional","affiliation":[{"name":"FSBSI Institute of Experimental Medicine, 197022 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Sukhareva","sequence":"additional","affiliation":[{"name":"FSBSI Institute of Experimental Medicine, 197022 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7112-1534","authenticated-orcid":false,"given":"Anastasiia","family":"Ilina","sequence":"additional","affiliation":[{"name":"Saint Petersburg Institute of Bioregulation and Gerontology, 197110 Saint Petersburg, Russia"},{"name":"FSBSI Institute of Experimental Medicine, 197022 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,29]]},"reference":[{"key":"ref_1","unstructured":"(2023, June 11). WHO Director-General\u2019s Opening Remarks at the Media Briefing\u20145 May 2023. Available online: https:\/\/www.who.int\/director-general\/speeches\/detail\/who-director-general-s-opening-remarks-at-the-media-briefing---5-may-2023."},{"key":"ref_2","unstructured":"(2023, June 11). Post COVID-19 Condition (Long COVID). Available online: https:\/\/www.who.int\/europe\/news-room\/fact-sheets\/item\/post-covid-19-condition."},{"key":"ref_3","unstructured":"Chippa, V., Aleem, A., and Anjum, F. (2023). StatPearls, StatPearls Publishing."},{"key":"ref_4","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":"Jackson","year":"2022","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Khavinson, V., Linkova, N., Dyatlova, A., Kuznik, B., and Umnov, R. (2020). Peptides: Prospects for Use in the Treatment of COVID-19. Molecules, 25.","DOI":"10.3390\/molecules25194389"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Jarczak, D., and Nierhaus, A. (2022). Cytokine Storm-Definition, Causes, and Implications. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231911740"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1038\/s41392-020-00243-2","article-title":"COVID-19: Immunopathogenesis and Immunotherapeutics","volume":"5","author":"Yang","year":"2020","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"102763","DOI":"10.1016\/j.ebiom.2020.102763","article-title":"Longitudinal Characteristics of Lymphocyte Responses and Cytokine Profiles in the Peripheral Blood of SARS-CoV-2 Infected Patients","volume":"55","author":"Liu","year":"2020","journal-title":"EBioMedicine"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1134\/S2079057021040068","article-title":"Peptide Drug Thymalin Regulates Immune Status in Severe COVID-19 Older Patients","volume":"11","author":"Kuznik","year":"2021","journal-title":"Adv. Gerontol."},{"key":"ref_10","first-page":"368","article-title":"Morphological compound and indicators of the blood clotting system in severe COVID-19 patients of middle aged and elderly during treatment of Tocilizumab and Thymalin","volume":"35","author":"Kuznik","year":"2022","journal-title":"Adv. Gerontol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Coleman, M.J., Zimmerly, K.M., and Yang, X.O. (2021). Accumulation of CD28null Senescent T-Cells Is Associated with Poorer Outcomes in COVID19 Patients. Biomolecules, 11.","DOI":"10.3390\/biom11101425"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e137799","DOI":"10.1172\/jci.insight.137799","article-title":"The Laboratory Tests and Host Immunity of COVID-19 Patients with Different Severity of Illness","volume":"5","author":"Wang","year":"2020","journal-title":"JCI Insight"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1007\/s00394-016-1374-y","article-title":"Effects of Tryptophan-Containing Peptides on Angiotensin-Converting Enzyme Activity and Vessel Tone Ex Vivo and in Vivo","volume":"57","author":"Khedr","year":"2018","journal-title":"Eur. J. Nutr."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10553","DOI":"10.1093\/nar\/gkz850","article-title":"Systematic Search for Structural Motifs of Peptide Binding to Double-Stranded DNA","volume":"47","author":"Kolchina","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"109920","DOI":"10.1016\/j.mehy.2020.109920","article-title":"Diacerein: A Potential Multi-Target Therapeutic Drug for COVID-19","volume":"144","author":"Termini","year":"2020","journal-title":"Med. Hypotheses"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1162","DOI":"10.1378\/chest.117.4.1162","article-title":"Anti-Inflammatory Cytokines","volume":"117","author":"Opal","year":"2000","journal-title":"Chest"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2255","DOI":"10.1056\/NEJMra2026131","article-title":"Cytokine Storm","volume":"383","author":"Fajgenbaum","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1007\/s12026-022-09268-x","article-title":"PI3K\/Akt\/MTOR Pathway: A Potential Target for Anti-SARS-CoV-2 Therapy","volume":"70","author":"Fattahi","year":"2022","journal-title":"Immunol. Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"105357","DOI":"10.1016\/j.meegid.2022.105357","article-title":"Epigenetic Mechanisms and Host Factors Impact ACE2 Gene Expression: Implications in COVID-19 Susceptibility","volume":"104","author":"Daniel","year":"2022","journal-title":"Infect. Genet. Evol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"6778","DOI":"10.4049\/jimmunol.0901800","article-title":"Cysteinyl-Leukotriene Receptor Type 1 Expression and Function Is down-Regulated during Monocyte-Derived Dendritic Cell Maturation with Zymosan: Involvement of IL-10 and Prostaglandins","volume":"183","author":"Thivierge","year":"2009","journal-title":"J. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/j.bj.2022.01.013","article-title":"Inherited Immunodeficiencies Associated with Proximal and Distal Defects in T Cell Receptor Signaling and Co-Signaling","volume":"45","author":"Latour","year":"2022","journal-title":"Biomed. J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"983","DOI":"10.1006\/jmbi.1994.1052","article-title":"Biased Probability Monte Carlo Conformational Searches and Electrostatic Calculations for Peptides and Proteins","volume":"235","author":"Abagyan","year":"1994","journal-title":"J. Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"D607","DOI":"10.1093\/nar\/gky1131","article-title":"STRING V11: Protein-Protein Association Networks with Increased Coverage, Supporting Functional Discovery in Genome-Wide Experimental Datasets","volume":"47","author":"Szklarczyk","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1002\/prot.22896","article-title":"Development of a New Physics-Based Internal Coordinate Mechanics Force Field and Its Application to Protein Loop Modeling","volume":"79","author":"Arnautova","year":"2011","journal-title":"Proteins"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1093\/nar\/28.1.302","article-title":"The Eukaryotic Promoter Database (EPD)","volume":"28","author":"Praz","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"bav006","DOI":"10.1093\/database\/bav006","article-title":"PathCards: Multi-Source Consolidation of Human Biological Pathways","volume":"2015","author":"Belinky","year":"2015","journal-title":"Database"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"586602","DOI":"10.3389\/fgene.2020.586602","article-title":"Multi-Approach Bioinformatics Analysis of Curated Omics Data Provides a Gene Expression Panorama for Multiple Cancer Types","volume":"11","author":"Feltes","year":"2020","journal-title":"Front. Genet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1097\/SHK.0000000000000470","article-title":"The Role of Lipopolysaccharide Structure in Monocyte Activation and Cytokine Secretion","volume":"45","author":"Plevin","year":"2016","journal-title":"Shock"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"62","DOI":"10.22141\/2224-0586.18.7.2022.1534","article-title":"Effectiveness of the Immunomodulator Thymalin in Experimental Coronavirus Infection","volume":"18","author":"Kozlovsky","year":"2022","journal-title":"Emerg. Med."},{"key":"ref_30","first-page":"PA3667","article-title":"Thymalin as an Immunomodulation Option in Severe COVID-19","volume":"58","author":"Lukyanov","year":"2021","journal-title":"Eur. Respir. J."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/17\/13377\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:41:36Z","timestamp":1760128896000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/17\/13377"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,29]]},"references-count":30,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2023,9]]}},"alternative-id":["ijms241713377"],"URL":"https:\/\/doi.org\/10.3390\/ijms241713377","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2023,8,29]]}}}