Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages

Abstract

Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/ Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 mM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-a, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88-and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-b-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-b knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 mM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-a and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 mM) inhibited LPS-induced prostaglandin E 2 production, cyclooxygenase-2 expression, and nuclear factor kB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.

Hepatoprotective Limonoids from Andiroba (Carapa guianensis)

“…Recently, it was reported that 1 suppressed the activation of macrophages through binding to myeloid differentiation protein 2 (MD-2), and not by affecting TLR4-mediated signaling. Their data supports our results of inhibitory effects of 1 on NO production in LPS-activated macrophages [ 60 ]. In addition, the structural requirements of limonoids ( 1 – 17 ) with regard to inhibition of LPS-induced NO production in mouse peritoneal macrophages and TNF-α-induced cytotoxicity in L929 cells were found to show different tendencies as mentioned above.…”

Toll-Like Receptors: Expression and Roles in Otitis Media

“…LPS binding thus promotes the dimerization of TLR4 and MD-2. TLR4 not only mediates responses to LPS, a major cellular component of Gram-negative bacteria, it is also involved in inflammatory responses to various other substances, such as ligands of harmful factors produced by Gram-positive bacteria and fusion protein (F protein) of respiratory syncytial virus [18,26,51]. In addition, individuals with a TLR4 D299G polymorphism are at increased risk of septic shock induced by Gram-negative bacteria infections.…”

Targeting myeloid differentiation protein 2 by the new chalcone L2H21 protects LPS‐induced acute lung injury

“…However, eritoran failed its phase III clinical trial because there was no obvious improvement with the compound over placebo . Some natural and synthetic chemicals, such as curcumin , JSH , gedunin and xanthohumol , and two new compounds, L6H21 and L48H37 , which we investigated in our laboratory, bind directly to the MD‐2 hydrophobic pocket and showed obvious anti‐inflammatory activity in that they displace LPS from MD‐2. In this study, we found that L2H21 is an inhibitor of MD‐2 (Figs and ).…”