Probing the interactions of genistein with HMGB1 through multi-spectroscopic and in-silico approaches

Functional regulation of proteins by ligand–protein interactions plays a crucial role in understanding biological processes and identifying potential drugs. High mobility group box 1 (HMGB1) plays a pivotal role in sterile inflammation as a key immunomodulatory protein. Genistein, a well-known isoflavone compound, has been shown to have neuroprotective effects. In this study, we investigated the genistein-HMGB1 interactions using experimental and computational approaches. Our results revealed that genistein binds to HMGB1 with a KD value of 6.06 × 10⁻⁵ M. The addition of genistein significantly quenched the fluorescence of HMGB1. Thermodynamic analyses demonstrated that hydrogen bonds and hydrophobic forces are the primary forces during the binding process. Furthermore, the interaction between genistein and HMGB1 led to changes in the microenvironment of protein chromogenic amino acids and subtle alterations in the protein secondary structure. Molecular modeling results indicate that Pro95, Pro98, and Lys154 are the major amino acid residues for genistein binding to HMGB1. Meanwhile, at the cellular level, an inhibitory effect of genistein on HMGB1-induced NO release from microglia was observed, demonstrating an inhibition rate of 42.1 %. Our studies demonstrated that genistein could be applied in treating neurological diseases through its interaction with HMGB1.