Fig. 2. 7w and 7x are identified as new MD2 inhibitors.
a, b Molecular dockings of 7w and 7x with rhMD2 (PDB: 2E56) were analyzed with the Tripos molecular modeling software. c, d The direct interactions of 7w and 7x with rhMD2 were shown by SPR assay. Ka binding constant, Kd dissociation constant, KD equilibrium dissociation constant. e, f The effects of 7w (5 and 10 μM) and 7x (5 and 10 μM) on binding fluorescent bis-ANS (5 μM) to rhMD2. g As determined by ELISA, 7w and 7x dose-dependently inhibited the binding of biotin-LPS to rhMD2. h, i MPMs were pretreated with 7w or 7x at 10 μM for 30 min and then exposed to LPS for 15 min. The complex of MD2-TLR4 were detected by immunoprecipitation (upper two panels). 293T cells were co-transfected with HA-TLR4 and Flag-TLR4 plasmids. Then, cells were treated with LPS for 15 min with 7w or 7x at 10 μM. Cell extracts were co-immunoprecipitated and were detected by anti-Flag and anti-HA antibodies (lower two panels).