Discovery of potent MD2 inhibitors by hierarchical virtual screening strategy and bioactivity evaluation

Myeloid differentiation 2 (MD2), an accessory protein of Toll-like receptor 4 (TLR4), plays a crucial role in inflammation and represents a promising target for the development of anti-inflammatory drugs. This study used a hierarchical virtual screening strategy based on ligand and receptor pharmacophore models and docking to identify potential MD2 inhibitors. From an initial library of 257,706 compounds, 15 candidates were selected, with hit12 demonstrating the most potent anti-inflammatory activity. Hit12 inhibited the lipopolysaccharide (LPS)-induced formation of the TLR4/MD2 complex and blocked the NF-κB and MAPK signaling pathways. Molecular docking studies revealed hydrogen bonds between hit12 and MD2, particularly with Arg90. Cellular thermal shift analysis (CETSA) showed that hit12 enhanced the thermal stability of MD2, and molecular dynamics (MD) simulations and free energy calculations proposed the binding mode of hit12. In vivo experiments showed that hit12 significantly reduced foot swelling in the adjuvant-induced arthritis (AIA) model rats. These results highlight the potential of MD2 as a target for the development of anti-inflammatory therapies.