NMR Structural Elucidation of Mitoxantrone-Gonadotropin-Releasing Hormone (GnRH) Conjugates Implicated in...

Gonadotropin-Releasing Hormone receptors (GnRHRs) are overexpressed in several hormone-dependent malignancies, making them attractive molecular targets for selective anticancer drug delivery. Peptide-drug 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-GnRH 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-GnRH 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-drug conjugates for targeted therapy.