Structure–Activity Relationship Evaluation of Melatonin and Its Derivatives for Wound-Healing Applications: A Combined Network Pharmacology, Molecular Docking, and Biological Validation Approach

Non-healing wounds remain a clinical challenge due to their complex pathophysiology and limited therapeutic options. These conditions are driven by complex molecular mechanisms, including inflammation, cell proliferation, and tissue remodeling. Melatonin (MLT) and its N1- and N2-substituted derivatives are known to exhibit potent antioxidant and anti-inflammatory properties; however, their specific therapeutic mechanisms in wound healing remain largely unexplored. Therefore, this study aimed to investigate the potential wound-healing properties of MLT and its six derivatives using an integrated computational and in vitro validation approach. Potential targets of MLT and its derivatives were screened using SwissTargetPrediction (version 2023 release) and SuperPred (version 3.0), yielding 491 candidate targets. These targets were cross-referenced with the GeneCards (version 5.24.0) database to map their involvement across the four phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. Network interaction models were constructed using Cytoscape (version 3.10.3) and GeneMANIA (version 3.6.0), and pathway enrichment was analyzed using the ShinyGO (version 0.85.1) platform. Enrichment analysis prioritized HIF-1-related signaling as a candidate regulatory axis associated with the predicted targets of melatonin derivatives across the inflammatory, proliferative, and remodeling phases of wound healing. In vitro validation using normal human dermal fibroblasts (NHDFs) demonstrated that all compounds, at non-toxic concentrations, significantly enhanced cell viability, as measured by the MTT assay. Furthermore, wound scratch assays revealed that the N2-bromobenzoyl-substituted derivative (EBMLT) accelerated cell migration, achieving complete wound gap closure within 24 h and outperforming the parent compound. Molecular docking simulations using AutoDock 4.2 predicted favorable binding interactions of the derivatives toward key wound healing-related targets (NF-κB, EGFR, VEGFR-1, MMP-1, and MMP-13). Aromatic-substituted derivatives (BMLT, BBMLT, and EBMLT) exhibited more favorable predicted binding interactions than the parent compound across all targets, whereas the aliphatic-substituted derivative (SMLT) showed weaker predicted interactions, particularly with VEGFR-1. These findings suggest that N1- and N2-aromatic substitutions are associated with more favorable binding interactions. Notably, the N2-bromobenzoyl derivative (EBMLT) exhibited the most potent wound-closure activity, highlighting it as a candidate compound for wound-healing applications.