Molecular Dynamics and Mechanistic Insights into the Ligand-Induced Conformational Stabilization of STAT3 and TNF-α

Abstract

The structural stabilization and conformational modulation of inflammatory targets such as TNF-α and STAT3 are of great significance in the discovery of novel anti-inflammatory agents and provide valuable insights into ligandprotein interaction mechanisms at the molecular level. In this study, we employed an integrated in silico framework, including ensemble molecular docking, long-timescale molecular dynamics (MD) simulations, and binding free energy calculations, to investigate the atomistic interaction mechanisms of two selected small molecules, isorhamnetin and 7-methoxy-2-methyl isoflavone, against TNF-α and STAT3. Initial ensemble docking identified energetically favorable candidate binding poses across different receptor conformations. Subsequent 500-ns MD simulations suggested that these ligands may contribute to stabilization of the proteinligand complexes and reduction of local structural fluctuations. Conformational dynamics analyses, including principal component analysis (PCA), free energy landscape (FEL), and dynamic cross-correlation matrix (DCCM), revealed ligand-associated changes in conformational convergence and altered intra-protein correlated motions. Furthermore, MM-PBSA calculations provided effective binding energy estimates (without entropic contributions), particularly for the STAT3–7-methoxy-2-methyl isoflavone complex (E effective = −167.44 kJ/mol). These computational findings provide atomistic insights into the possible ligand-associated conformational stabilization mechanisms, offering a computational structural basis for targeting the TNF–STAT3 inflammatory axis.