{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T11:04:36Z","timestamp":1781780676696,"version":"3.54.5"},"reference-count":56,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2025,4,25]],"date-time":"2025-04-25T00:00:00Z","timestamp":1745539200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health","award":["R15AR083143"],"award-info":[{"award-number":["R15AR083143"]}]},{"name":"P20GM103646 (Center for Biomedical Research Excellence in Pathogen Host Interactions)","award":["R15AR083143"],"award-info":[{"award-number":["R15AR083143"]}]},{"name":"Judy and Bobby Shackouls Honors College at Mississippi State University","award":["R15AR083143"],"award-info":[{"award-number":["R15AR083143"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>ADAMTS-5 (aggrecanase-2) is a major metalloprotease involved in regulating the cartilage extracellular matrix. Due to its role in removing aggrecan in the progression of osteoarthritis (OA), ADAMTS-5 is often regarded as a potential therapeutic target for OA. Punicalagin (PCG), a polyphenolic ellagitannin found in pomegranate (Punica grunatum L.), and ellagic acid (EA), a hydrolytic metabolite of PCG, have been widely investigated as potential disease-modifying osteoarthritis drugs (DMOADs) due to their potent antioxidant and anti-inflammatory properties, but their interaction with ADAMTS-5 has yet to be determined. In this study, molecular docking simulations were used to predict enzyme\u2013inhibitor binding interactions. The results suggest that both compounds may be able to bind within the active site via the formation of H bonds and interactions between the ligand\u2019s aromatic rings and hydrophobic residue in the enzyme with inhibition constants of 183.3 \u00b5M and 1.13 \u00b5M for PCG and EA, respectively. Biochemical activity against recombinant human ADAMTS-5 was assessed using a dimethylmethylene blue-based assay to determine residual sulfated glycosaminoglycan (sGAG) in porcine articular cartilage. Although its loss could not be attributed to ADAMTS-5, sGAG was effectively persevered by PCG and EA. The potential conversion of PCG to EA by enzyme-catalyzed hydrolysis activity was then investigated using liquid chromatography\u2013mass spectroscopy to determine the potential for the use of PCG and EA as a prodrug\u2013proactive metabolite pair in the development of drug delivery systems to arthritic synovial joints.<\/jats:p>","DOI":"10.3390\/ijms26094093","type":"journal-article","created":{"date-parts":[[2025,4,25]],"date-time":"2025-04-25T08:57:47Z","timestamp":1745571467000},"page":"4093","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["In Silico Insights into the Inhibition of ADAMTS-5 by Punicalagin and Ellagic Acid for the Treatment of Osteoarthritis"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-8581-6566","authenticated-orcid":false,"given":"Austen N.","family":"Breland","sequence":"first","affiliation":[{"name":"Department of Agricultural & Biological Engineering, Mississippi State University, Starkville, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9167-8452","authenticated-orcid":false,"given":"Matthew K.","family":"Ross","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedical Sciences, Mississippi State University, Starkville, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8993-2140","authenticated-orcid":false,"given":"Nicholas C.","family":"Fitzkee","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Mississippi State University, Starkville, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0446-2136","authenticated-orcid":false,"given":"Steven H.","family":"Elder","sequence":"additional","affiliation":[{"name":"Department of Agricultural & Biological Engineering, Mississippi State University, Starkville, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1002\/art.42089","article-title":"Prevalence Trends of Site-Specific Osteoarthritis from 1990 to 2019: Findings from the Global Burden of Disease Study 2019","volume":"74","author":"Long","year":"2022","journal-title":"Arthritis Rheumatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"S245","DOI":"10.21037\/atm.2019.06.68","article-title":"Non-operative treatment options for knee osteoarthritis","volume":"7","author":"DeRogatis","year":"2019","journal-title":"Ann. Transl. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1397","DOI":"10.1016\/j.joca.2024.07.014","article-title":"Osteoarthritis year in review 2024: Epidemiology and therapy","volume":"32","author":"Courties","year":"2024","journal-title":"Osteoarthr. Cartil."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"636","DOI":"10.1016\/j.joca.2021.03.002","article-title":"Genetics of osteoarthritis","volume":"30","author":"Aubourg","year":"2022","journal-title":"Osteoarthr. Cartil."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Nedunchezhiyan, U., Varughese, I., Sun, A.R.J., Wu, X., Crawford, R., and Prasadam, I. (2022). Obesity, Inflammation, and Immune System in Osteoarthritis. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.907750"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1080\/13697137.2017.1421926","article-title":"Female hormonal factors and osteoarthritis of the knee, hip and hand: A narrative review","volume":"21","author":"Hussain","year":"2018","journal-title":"Climacteric"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1007\/s10067-019-04811-y","article-title":"Total joint replacement improves pain, functional quality of life, and health utilities in patients with late-stage knee and hip osteoarthritis for up to 5 years","volume":"39","author":"Neuprez","year":"2020","journal-title":"Clin. Rheumatol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"100749","DOI":"10.1016\/j.asmr.2023.05.006","article-title":"Radiofrequency Chondroplasty of the Knee Yields Excellent Clinical Outcomes and Minimal Complications: A Systematic Review","volume":"5","author":"Tuthill","year":"2023","journal-title":"Arthrosc. Sports Med. Rehabil."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"812","DOI":"10.1177\/03635465211053594","article-title":"Isolated Osteochondral Autograft Versus Allograft Transplantation for the Treatment of Symptomatic Cartilage Lesions of the Knee: A Systematic Review and Meta-analysis","volume":"51","author":"Trofa","year":"2023","journal-title":"Am. J. Sports Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"876","DOI":"10.1016\/j.joca.2023.03.008","article-title":"Osteoarthritis year in review 2022: Epidemiology & therapy","volume":"31","author":"Foster","year":"2023","journal-title":"Osteoarthr. Cartil."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1001\/jama.2020.22171","article-title":"Diagnosis and Treatment of Hip and Knee Osteoarthritis: A Review","volume":"325","author":"Katz","year":"2021","journal-title":"Jama"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1002\/jbm.b.34747","article-title":"Effects of short-duration treatment of cartilage with punicalagin and genipin and the implications for treatment of osteoarthritis","volume":"109","author":"Elder","year":"2021","journal-title":"J. Biomed. Mater. Res. Part B Appl. Biomater."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"123842","DOI":"10.1016\/j.ijpharm.2024.123842","article-title":"Development of in situ forming implants for controlled delivery of punicalagin","volume":"652","author":"Elder","year":"2024","journal-title":"Int. J. Pharm."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Xu, J., Cao, K., Liu, X., Zhao, L., Feng, Z., and Liu, J. (2021). Punicalagin Regulates Signaling Pathways in Inflammation-Associated Chronic Diseases. Antioxidants, 11.","DOI":"10.3390\/antiox11010029"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Cao, Y., Chen, J., Ren, G., Zhang, Y., Tan, X., and Yang, L. (2019). Punicalagin Prevents Inflammation in LPS- Induced RAW264.7 Macrophages by Inhibiting FoxO3a\/Autophagy Signaling Pathway. Nutrients, 11.","DOI":"10.3390\/nu11112794"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1016\/j.jff.2017.12.026","article-title":"Inhibitory effects of punicalagin from Punica granatum against type II collagenase-induced osteoarthritis","volume":"41","author":"Lee","year":"2018","journal-title":"J. Funct. Foods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"116070","DOI":"10.1016\/j.bone.2021.116070","article-title":"Punicalagin attenuates osteoarthritis progression via regulating Foxo1\/Prg4\/HIF3\u03b1 axis","volume":"152","author":"Liu","year":"2021","journal-title":"Bone"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"148","DOI":"10.22270\/jddt.v12i2.5377","article-title":"Pharmacological Activities of Punicalagin: A Review","volume":"12","author":"Rozadi","year":"2022","journal-title":"J. Drug Deliv. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1007\/s10753-014-9816-2","article-title":"Punicalagin inhibits inflammation in LPS-induced RAW264.7 macrophages via the suppression of TLR4-mediated MAPKs and NF-\u03baB activation","volume":"37","author":"Xu","year":"2014","journal-title":"Inflammation"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5082","DOI":"10.4049\/jimmunol.181.7.5082","article-title":"Prostaglandin E2 Exerts Catabolic Effects in Osteoarthritis Cartilage: Evidence for Signaling via the EP4 Receptor","volume":"181","author":"Attur","year":"2008","journal-title":"J. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.cbi.2013.06.018","article-title":"Inhibitory effects of polyphenol punicalagin on type-II collagen degradation in vitro and inflammation in vivo","volume":"205","author":"Li","year":"2013","journal-title":"Chem. Interact."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.nut.2016.08.004","article-title":"Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis","volume":"33","author":"Akhtar","year":"2017","journal-title":"Nutrition"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1901","DOI":"10.2147\/JIR.S302929","article-title":"Punicalagin inhibited inflammation and migration of fibroblast-like synoviocytes through nf-\u03bab pathway in the experimental study of rheumatoid arthritis","volume":"14","author":"Huang","year":"2021","journal-title":"J. Inflamm. Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1491","DOI":"10.1021\/jf0626720","article-title":"In vitro studies on the binding, antioxidant, and cytotoxic action of punicalagin","volume":"55","author":"Kulkarni","year":"2007","journal-title":"J. Agric. Food Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3493","DOI":"10.1021\/jf020842c","article-title":"Repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for 37 days is not toxic","volume":"51","year":"2003","journal-title":"J. Agric. Food Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.bbapap.2011.06.020","article-title":"Proteases involved in cartilage matrix degradation in osteoarthritis","volume":"1824","author":"Troeberg","year":"2012","journal-title":"Biochim. Biophys. Acta (BBA)-Proteins Proteom."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Li, T., Peng, J., Li, Q., Shu, Y., Zhu, P., and Hao, L. (2022). The Mechanism and Role of ADAMTS Protein Family in Osteoarthritis. Biomolecules, 12.","DOI":"10.3390\/biom12070959"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1038\/sj.cr.7290106","article-title":"Structure and function of aggrecan","volume":"12","author":"Kiani","year":"2002","journal-title":"Cell Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1038\/nature03369","article-title":"Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis","volume":"434","author":"Glasson","year":"2005","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Hering, N.A., Luettig, J., Jebautzke, B., Schulzke, J.D., and Rosenthal, R. (2021). The Punicalagin Metabolites Ellagic Acid and Urolithin A Exert Different Strengthening and Anti-Inflammatory Effects on Tight Junction-Mediated Intestinal Barrier Function In Vitro. Front. Pharmacol., 12.","DOI":"10.3389\/fphar.2021.610164"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"16273","DOI":"10.1021\/acs.jafc.2c06585","article-title":"Biodegradation of Punicalagin into Ellagic Acid by Selected Probiotic Bacteria: A Study of the Underlying Mechanisms by MS-Based Proteomics","volume":"70","author":"Caballero","year":"2022","journal-title":"J. Agric. Food Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"104713","DOI":"10.1016\/j.jddst.2023.104713","article-title":"Montmorillonite an efficient oral nanocarrier for punicalagin-rich pomegranate peel extract: An in vitro study","volume":"86","author":"Sabzevari","year":"2023","journal-title":"J Drug Deliv Sci Technol, J. Drug Deliv. Sci. Technol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1007\/s00253-007-1276-2","article-title":"Microbial production of ellagic acid and biodegradation of ellagitannins","volume":"78","author":"Augur","year":"2008","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Klein, P., Johe, P., Wagner, A., Jung, S., K\u00fchlborn, J., Barthels, F., Tenzer, S., Distler, U., Waigel, W., and Engels, B. (2020). New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain. Molecules, 25.","DOI":"10.3390\/molecules25061451"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2854","DOI":"10.1021\/acssensors.9b01783","article-title":"Real-Time Label-Free Kinetics Monitoring of Trypsin-Catalyzed Ester Hydrolysis by a Nanopore Sensor","volume":"4","author":"Li","year":"2019","journal-title":"ACS Sens."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.mrgentox.2011.12.022","article-title":"Enhanced activity of punicalagin delivered via polymeric implants against benzo[a]pyrene-induced DNA adducts","volume":"743","author":"Aqil","year":"2012","journal-title":"Mutat. Res. Toxicol. Environ. Mutagen."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1055\/a-0633-9492","article-title":"A Pharmacological Update of Ellagic Acid","volume":"84","author":"Giner","year":"2018","journal-title":"Planta Med."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Lin, Z., Lin, C., Fu, C., Lu, H., Jin, H., Chen, Q., and Pan, J. (2020). The protective effect of Ellagic acid (EA) in osteoarthritis: An in vitro and in vivo study. Biomed. Pharmacother., 125.","DOI":"10.1016\/j.biopha.2020.109845"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2937","DOI":"10.1021\/acs.jmedchem.0c02008","article-title":"Discovery of GLPG1972\/S201086, a Potent, Selective, and Orally Bioavailable ADAMTS-5 Inhibitor for the Treatment of Osteoarthritis","volume":"64","author":"Brebion","year":"2021","journal-title":"J. Med. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2020\/8293921","article-title":"The Role of Inflammation in the Pathogenesis of Osteoarthritis","volume":"2020","author":"Chow","year":"2020","journal-title":"Mediat. Inflamm."},{"key":"ref_41","unstructured":"(2024, May 27). The PyMOL Molecular Graphics System, Version 2.6 Schr\u00f6dinger, LLC. Available online: https:\/\/pymol.org\/."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1002\/jor.24457","article-title":"Role of Inflammation and the Immune System in the Progression of Osteoarthritis","volume":"38","author":"Sommerfeld","year":"2020","journal-title":"J. Orthop. Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"13505","DOI":"10.1021\/acs.jmedchem.2c01177","article-title":"Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition","volume":"65","author":"Cuffaro","year":"2022","journal-title":"J. Med. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"822","DOI":"10.1016\/j.drudis.2011.06.001","article-title":"Getting physical in drug discovery II: The impact of chromatographic hydrophobicity measurements and aromaticity","volume":"16","author":"Young","year":"2011","journal-title":"Drug Discov. Today"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"4421","DOI":"10.1021\/acs.jmedchem.8b00096","article-title":"Development of Thioaryl-Based Matrix Metalloproteinase-12 Inhibitors with Alternative Zinc-Binding Groups: Synthesis, Potentiometric, NMR, and Crystallographic Studies","volume":"61","author":"Nuti","year":"2018","journal-title":"J. Med. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1039\/C4MD00401A","article-title":"Discovery of a new class of histone deacetylase inhibitors with a novel zinc binding group","volume":"6","author":"Li","year":"2015","journal-title":"MedChemComm"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Michel, M., Homan, E.J., Wiita, E., Pedersen, K., Alml\u00f6f, I., Gustavsson, A.-L., Lundb\u00e4ck, T., Helleday, T., and Berglund, U.W. (2020). In silico Druggability Assessment of the NUDIX Hydrolase Protein Family as a Workflow for Target Prioritization. Front. Chem., 8.","DOI":"10.3389\/fchem.2020.00443"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Santamaria, S., Cuffaro, D., Nuti, E., Ciccone, L., Tuccinardi, T., Liva, F., D\u2019andrea, F., de Groot, R., Rossello, A., and Ahnstr\u00f6m, J. (2021). Exosite inhibition of ADAMTS-5 by a glycoconjugated arylsulfonamide. Sci. Rep., 11.","DOI":"10.1038\/s41598-020-80294-1"},{"key":"ref_49","first-page":"65","article-title":"Molecular docking: Current advances and challenges","volume":"21","author":"Arciniega","year":"2018","journal-title":"TIP Rev. Espec. En. Cienc. Qu\u00edmico Biol\u00f3gicas"},{"key":"ref_50","first-page":"1824","article-title":"Molecular docking analysis of major active compounds of pomegranate peel extract (Punica granatum L.) in inhibiting cyclooxygenase enzyme","volume":"20","author":"Iqbal","year":"2023","journal-title":"J. Adv. Res. Rev."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1002\/pro.606","article-title":"Structure analysis reveals the flexibility of the ADAMTS-5 active site","volume":"20","author":"Shieh","year":"2011","journal-title":"Protein Sci."},{"key":"ref_52","unstructured":"(2024, May 27). Zentrum f\u00fcr Bioinformatik: Universit\u00e4t Hamburg-Proteins Plus Server. Available online: https:\/\/proteins.plus\/."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1021\/ci200454v","article-title":"Combining global and local measures for structure-based druggability predictions","volume":"52","author":"Volkamer","year":"2012","journal-title":"J. Chem. Inf. Model."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/S0006-291X(67)80055-X","article-title":"On the size of the active site in proteases","volume":"27","author":"Schechter","year":"1967","journal-title":"I. Papain. Biochem. Biophys, Res. Commun."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"903","DOI":"10.1093\/protein\/4.8.903","article-title":"Electrostatic effects in proteins: Comparison of dielectric and charge models","volume":"4","author":"Mehler","year":"1991","journal-title":"Protein Eng."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2778","DOI":"10.1021\/ci200227u","article-title":"LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery","volume":"51","author":"Laskowski","year":"2011","journal-title":"J. Chem. Inf. Model."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/9\/4093\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:21:38Z","timestamp":1760030498000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/9\/4093"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,25]]},"references-count":56,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2025,5]]}},"alternative-id":["ijms26094093"],"URL":"https:\/\/doi.org\/10.3390\/ijms26094093","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,25]]}}}