{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:26:28Z","timestamp":1787214388199,"version":"build-2736575974"},"reference-count":43,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Union and the Greek National Funds","award":["T2EDK 02056"],"award-info":[{"award-number":["T2EDK 02056"]}]},{"award":["T2EDK 02056"],"award-info":[{"award-number":["T2EDK 02056"]}],"id":[{"id":"https:\/\/ror.org\/019w4f821","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Gonadotropin-Releasing Hormone receptors (GnRHRs) are overexpressed in several hormone-dependent malignancies, making them attractive molecular targets for selective anticancer drug delivery. Peptide\u2013drug conjugates (PDCs) are a promising therapy for cancer and autoimmune diseases with high specificity and reduced toxicity. In this study, the three-dimensional structures of two previously synthesized mitoxantrone\u2013GnRH conjugates, con3 and con7, were elucidated using high-resolution NMR spectroscopy in combination with molecular dynamics (MD) simulations. Complete 1H and 13C resonance assignments were achieved in DMSO-d6 through two-dimensional NMR experiments. NOESY-derived distance restraints were subsequently used to refine the conformational ensembles obtained from MD simulations performed in water and DMSO. Both conjugates exhibited compact bent conformations with a U-shaped peptide backbone. The mitoxantrone moiety is positioned close to the peptide backbone in water simulations and NMR-refined structures, while it is positioned farther away in DMSO, without affecting the orientation of key residues involved in GnRH receptor binding. Importantly, His2, Trp3, and Arg8 remain solvent-exposed, whereas the disulfide bond is easily accessible to the solvent, consistent with the proposed drug release mechanism by the thioredoxin system. NMR-restrained molecular modeling confirmed the dominant conformational features predicted by the unconstrained theoretical simulations. Overall, these findings provide better structural understanding of the molecular organization of mitoxantrone\u2013GnRH conjugates, highlighting key receptor-recognition residues and supporting both the proposed thioredoxin-mediated drug release mechanism and their previously reported biological properties. These insights may facilitate the rational design and optimization of improved GnRH peptide\u2013drug conjugates for targeted therapy.<\/jats:p>","DOI":"10.3390\/ijms27167437","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:10:20Z","timestamp":1787213420000},"page":"7437","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["NMR Structural Elucidation of Mitoxantrone\u2013Gonadotropin-Releasing Hormone (GnRH) Conjugates Implicated in Hormone-Dependent Cancer"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-5789-8884","authenticated-orcid":false,"given":"Georgia","family":"Biniari","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Patras, 26504 Rion Patras, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1534-2762","authenticated-orcid":false,"given":"Haralambos","family":"Tzoupis","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Patras, 26504 Rion Patras, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7959-6681","authenticated-orcid":false,"given":"Uro\u0161","family":"Javornik","sequence":"additional","affiliation":[{"name":"Slovenian NMR Centre, National Institute of Chemistry, SI-1001 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3226-7434","authenticated-orcid":false,"given":"Nikitas","family":"Georgiou","sequence":"additional","affiliation":[{"name":"Department of Chemistry, National and Kapodistrian University of Athens, 10679 Athens, Greece"},{"name":"Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Safarik University in Kosice, Jesenna 5, 04001 Kosice, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2423-3135","authenticated-orcid":false,"given":"Georgios","family":"Liapakis","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Medicine, University of Crete, 71003 Heraklion, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5309-992X","authenticated-orcid":false,"given":"Thomas","family":"Mavromoustakos","sequence":"additional","affiliation":[{"name":"Department of Chemistry, National and Kapodistrian University of Athens, 10679 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1310-0591","authenticated-orcid":false,"given":"Theodore","family":"Tselios","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Patras, 26504 Rion Patras, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7096-4281","authenticated-orcid":false,"given":"Carmen","family":"Simal","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Patras, 26504 Rion Patras, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2254","DOI":"10.1016\/j.cell.2025.12.049","article-title":"Hallmarks of cancer\u2014Then and now, and beyond","volume":"189","author":"Hanahan","year":"2026","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Mustafa, M., Ahmad, R., Tantry, I.Q., Ahmad, W., Siddiqui, S., Alam, M., Abbas, K., Hassan, I., and Habib, S. 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