title: MD-2 as the target of a novel small molecule, L6H21, in the attenuation of LPS-induced inflammatory response and sepsis.
abstract: OBJECTIVE
  Myeloid differentiation 2 (MD-2) recognizes LPS, which is required for TLR4 activation, and represents an attractive therapeutic target for severe inflammatory disorders. We previously found that a chalcone derivative, L6H21, could inhibit LPS-induced overexpression of TNF-α and IL-6 in macrophages. Here, we performed a series of biochemical experiments to investigate whether L6H21 specifically targets MD-2 and inhibits the interaction and signalling transduction of LPS-TLR4/MD-2.
  
  
  METHODS
  The binding affinity of L6H21 to MD-2 protein was analysed using computer docking, surface plasmon resonance analysis, elisa, fluorescence measurements and flow cytometric analysis. The effects of L6H21 on MAPK and NF-κB signalling were determined using EMSA, fluorescence staining, Western blotting and immunoprecipitation. The anti-inflammatory effects of L6H21 were confirmed using elisa and RT-qPCR in vitro. The anti-inflammatory effects of L6H21 were also evaluated in septic C57BL/6 mice.
  
  
  RESULTS
  Compound L6H21 inserted into the hydrophobic region of the MD-2 pocket, forming hydrogen bonds with Arg(90) and Tyr(102) in the MD-2 pocket. In vitro, L6H21 subsequently suppressed MAPK phosphorylation, NF-κB activation and cytokine expression in macrophages stimulated by LPS. In vivo, L6H21 pretreatment improved survival, prevented lung injury, decreased serum and hepatic cytokine levels in mice subjected to LPS. In addition, mice with MD-2 gene knockout were universally protected from the effects of LPS-induced septic shock.
  
  
  CONCLUSIONS
  Overall, this work demonstrated that the new chalcone derivative, L6H21, is a potential candidate for the treatment of sepsis. More importantly, the data confirmed that MD-2 is an important therapeutic target for inflammatory disorders.
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Paediatrics, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Xiaoou
authors.initials: X
authors.lastname: Shan
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Gaozhi
authors.initials: G
authors.lastname: Chen
authors.affiliation: Department of Paediatrics, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Qilu
authors.initials: Q
authors.lastname: Fang
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Jingying
authors.initials: J
authors.lastname: Wang
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Yali
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Orbital and Oculoplastic Surgery, The Eye Hospital of Wenzhou Medical University, Wenzhou, China.
authors.forename: Wencan
authors.initials: W
authors.lastname: Wu
authors.affiliation: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Guang
authors.initials: G
authors.lastname: Liang
mesh_terms: D000818:Animals; D002460:Cell Line; D002599:Chalcone / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods*; D006801:Humans; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control; D018836:Inflammation Mediators / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018805:Sepsis / Q000378:metabolism* / Q000517:prevention & control*
chemical_list: D018836:Inflammation Mediators; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D002599:Chalcone