Computational and Cellular Evaluation of the Anti-Melanogenic Potential of Gancidin W: Integrating Network Pharmacology and Free-Energy Analyses.

Background/Objectives: Dysregulated melanogenesis underpins numerous hyperpigmentation disorders. Gancidin W, a naturally occurring diketopiperazine with diverse biological activities, has attracted interest as a potential bioactive compound; however, its role in melanogenesis remains largely unexplored. This study aimed to evaluate the anti-melanogenic potential of Gancidin W through an integrated experimental and computational approach. Methods: Network pharmacology analysis was performed to investigate the potential molecular mechanisms of Gancidin W against hyperpigmentation. The cellular effects of Gancidin W were evaluated in α-MSH-stimulated B16F10 melanoma cells by assessing intracellular tyrosinase activity, melanin content, and cytotoxicity. Furthermore, density functional theory (DFT) calculations, molecular dynamics (MD) simulations, MM/GBSA binding free-energy analysis, and relative binding free-energy (RBFE) calculations were conducted to investigate the molecular interactions of Gancidin W and its structurally related analogue Maculosin with human tyrosinase-related protein 1 (hTYRP1). Results: Network pharmacology analysis predicted potential targets and signaling pathways involved in melanogenesis regulation. Gancidin W reduced intracellular tyrosinase activity by 34.54% and melanin content by 25.09% without cytotoxic effects. Multiscale computational analyses demonstrated stable interactions of Gancidin W within the hTYRP1 active site and favorable binding energetics (ΔGbind = -28.77 ± 0.75 kcal/mol). Compared with Maculosin, Gancidin W exhibited a more favorable binding profile, as supported by MM/GBSA and RBFE analyses, consistent with the inhibitory effects observed in the cellular assays. Conclusions: Gancidin W exhibits anti-melanogenic potential, and multiscale computational analyses provide molecular insights into its interaction with hTYRP1. These findings support further investigation of Gancidin W as a potential natural depigmenting agent for hyperpigmentation-related disorders.