6,7-Dihydroxy-4-Methylcoumarin Suppresses Adipogenesis via AMPK and MAPK Signaling with In Silico Analysis of Adipogenic Proteins

Background/Objectives: Coumarin-based compounds exhibit diverse pharmacological properties, and 4-methylcoumarin (4MC) has emerged as a promising scaffold for drug development. However, its anti-obesity mechanisms remain insufficiently understood. This study aimed to evaluate the anti-adipogenic potential of 4MC derivatives in 3T3-L1 preadipocytes and to elucidate their underlying molecular mechanisms. Methods: 3T3-L1 preadipocytes were treated with structurally diverse 4MC derivatives. Lipid accumulation was analyzed using Oil Red O staining, cell viability by MTT assay, and the expression of adipogenic proteins by Western blotting. Molecular docking and molecular dynamics simulations were performed to predict the interactions between lead compounds and key adipogenic regulators. Results: Among the tested derivatives, 6,7-dihydroxy-4-methylcoumarin (6,7DH-4MC) markedly inhibited lipid accumulation in a dose-dependent manner without cytotoxicity. It suppressed the expression of major adipogenic transcription factors (PPAR-γ, C/EBPα, SREBP-1c) and FABP4. Additionally, 6,7DH-4MC inhibited ERK1/2 and p38 MAPK phosphorylation while activating AMPK. It also reduced CREB phosphorylation, indicating suppression of early adipogenesis. Computational analyses revealed stable binding of 6,7DH-4MC within the active sites of multiple adipogenic regulators, supporting its pleiotropic mode of action. Conclusions: 6,7DH-4MC exerts potent anti-adipogenic effects by modulating key adipogenic signaling pathways and transcriptional networks. These findings highlight 6,7DH-4MC as a promising lead compound for anti-obesity drug development, warranting further in vivo studies.