Exploring the molecular interaction of celastrol and HMGB1 by multi-spectra analysis

As damage-associated molecular patterns (DAMPs), the high mobility group box 1 (HMGB1) mediates the transmission of intercellular damage, inflammatory signals and plays a key role in pathological processes such as aseptic inflammation, autoimmune diseases and cancer. Celastrol, a natural product extracted from <i>Tripterygium wilfordii</i> Hook.f, exerts a neuroprotective effect by binding to HMGB1 in cerebral ischemia-reperfusion injury. To explore the binding characteristics between celastrol and HMGB1, surface plasmon resonance (SPR), dynamic light scattering (DLS) and multi-spectral technology, including fluorescence spectroscopy and circular dichroism (CD) spectra, were applied. Molecular docking as well as molecular dynamic (MD) simulation were also performed to predict the binding poses of celastrol and HMGB1. The SPR results showed that the <i>K_D</i> value of celastrol and HMGB1 was 5.57 × 10^-5 M. In fluorescence spectroscopy, the binding of celastrol can dose-dependently quench the endogenous fluorescence of HMGB1, and the quenching type is static quenching. Moreover, celastrol can also reduce the content of α-helix and enhance the random coil content of HMGB1, which could increase its particle size. Molecular docking celastrol was engaged in interactions with the amino acids Lys95, Arg104 and Ala133, resulting in the formation of multiple hydrogen bonds within the length of 1.8-2.0 Å. The main forces involved were electrostatic interaction, hydrophobic interaction and hydrogen bonds. The MD simulation further showed that a stable complex was formed between HMGB1 and celastrol. The <i>in vitro</i> biological evaluation showed that celastrol could inhibit NO release in the HMGB1-induced RAW264.7 inflammatory cell model with an IC₅₀ value of 0.89 μM. Celastrol could bind to HMGB1 and slightly change its secondary structure and spatial conformation, subsequently affecting its pro-inflammatory function.