## 5 Docking Analysis of AMPK with Flavonoids
For the remaining six flavonoids, the mechanism of AMPK activation remains uncertain. Whether these flavonoids activate AMPK directly or indirectly has not yet been clarified in the existing research. To address this gap, docking studies were performed on these six flavonoids to assess whether they act as direct AMPK activators. Molecular docking analysis was used to evaluate their binding affinity and interactions with the \(\alpha\)1, \(\beta\)1, and \(\gamma\)1 subunits of AMPK. The goal was to determine whether these flavonoids directly bind to AMPK, thus providing deeper insights into their potential role as direct AMPK activators and their therapeutic applications in metabolic diseases. Ligand-protein docking studies offer significant advantages in elucidating how small molecules, such as flavonoids, interact with their protein targets, like AMPK. These studies not only predict the binding affinity and orientation of the ligands within the protein's active site but also provide valuable insights into the molecular basis of enzyme activation or inhibition. Moreover, docking studies can help identify potential binding sites and guide the design of more potent and selective AMPK activators, thereby accelerating the drug discovery process. By simulating ligand-protein interactions at the atomic level, docking studies contribute to a deeper understanding of the mechanisms underlying both direct and indirect AMPK activation, which is essential for developing targeted therapies for metabolic diseases. Molecular docking analysis was conducted using CB-Dock2 to assess the interactions between AMPK (comprising the \(\alpha\)1, \(\beta\)1, and \(\gamma\)1 subunits; PDB ID 4 QFG) and various flavonoids. The docking scores, binding chains, and contact residues were analyzed using Biovia Discovery Studio Visualizer (version 21.1.0.20298), providing a detailed understanding of the molecular interactions involved. Baicalein (Figure 3A) showed a strong binding affinity, with a docking score of \(-8.3\), primarily interacting with the \(\alpha\)1 subunit of AMPK. The key contact residues included LEU22, GLY25, VAL30, ALA43, IXS45, ILE77, MET93, GLU94, TYR95, VAL96, SER97, GLY98, GLU100, LEU146, ALA156, and ASP157. These interactions were mediated through a combination of hydrogen bonds and pi-sigma, pi-alkyl, and pi-sulfur interactions, suggesting that baicalein could potentially activate AMPK through a robust and multifaceted binding mode. Dihydromyricetin (Figure 3B) displayed a slightly lower docking score of \(-7.3\) but still showed significant binding affinity to the \(\alpha\)1 subunit of AMPK. The binding involved residues such as LEU22, GLY23, VAL30, ALA43, ILE77, MET93, GLU94, TYR95, VAL96, GLY99, GLU100, ASP103, LEU146, ALA156, and ASP157, indicating stable interactions through hydrogen bonds and pi-alkyl forces that could facilitate AMPK activation. Bavachin (Figure 4A) exhibited a moderate docking score of \(-4.4\), indicating a weaker interaction with the \(\alpha\)1 subunit of AMPK. The binding involved residues such as LEU22, VAL30, ALA43, LYS45, GLU64, LEU68, ILE77, MET93, GLU94, TYR95, and others. The interactions included hydrogen bonds and pi-alkyl contacts, suggesting that bavachin might have a less potent effect on AMPK activation. Figure 3: Molecular docking analysis of baelaein and dihydromyricetin with AMPK’s \(\alpha\)1, \(\beta\)1, and \(\gamma\)1 subunits (PDB ID 4 QFG). This figure illustrates the molecular docking interactions between AMPK (composed of \(\alpha\)1, \(\beta\)1, and \(\gamma\)1 subunits; PDB ID 4 QFG) and two flavonoids, baelaein (**A**) and dihydromyricetin (**B**), analyzed using CB-Dock2 and visualized with Biovia Discovery Studio Visualizer (version 21.1.0.20928). **(A)** Baelaein demonstrated a docking score of \(-8.3\), indicating strong binding affinity primarily to the \(\alpha\)1 subunit of AMPK. Key interacting residues include LEU22, GLY25, VAL30, ALA43, LY545, ILE77, MET93, GLU94, TYR95, VAL96, SER97, GLY98, GLU100, LEU146, ALA156, and ASP157.