Summary The aging suppressor αKlotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion/vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex consisting of the shed extracellular domain of αKlotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, αKlotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23-FGFR1c proximity and conferring stability. The endocrine character of FGF23 notwithstanding, dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signaling. The structure of αKlotho is incompatible with its purported glycosidase activity. Thus, shed αKlotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signaling.

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Background and Purpose 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. Experimental Approach 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. Key Results Compound L6H21 inserted into the hydrophobic region of the MD‐2 pocket, forming hydrogen bonds with Arg90 and Tyr102 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 and Implications 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.

Endotoxin-induced acute inflammatory diseases such as sepsis, mediated by excessive production of various proinflammatory cytokines, remain the leading cause of mortality in critically ill patients. Lipopolysaccharide (LPS), the characteristic endotoxin found in the outer membrane of Gram-negative bacteria, can induce the innate immunity system and through the myeloid differentiation protein 2 (MD2) and Toll-like receptor 4 (TLR4) complex, increase the production of inflammatory mediators. Our previous studies have found that a curcumin analog, L48H37 [1-ethyl-3,5-bis(3,4,5-trimethoxybenzylidene)piperidin-4-one], was able to inhibit LPS-induced inflammation, particularly tumor necrosis factor a and interleukin 6 production and gene expression in mouse macrophages. In this study, a series of biochemical experiments demonstrate L48H37 specifically targets MD2 and inhibits the interaction and signaling transduction of LPS-TLR4/ MD2. L48H37 binds to the hydrophobic region of MD2 pocket and forms hydrogen bond interactions with Arg 90 and Tyr 102. Subsequently, L48H37 was shown to suppress LPS-induced mitogenactivated protein kinase phosphorylation and nuclear factor kB activation in macrophages; it also dose dependently inhibits the cytokine expression in macrophages and human peripheral blood mononuclear cells stimulated by LPS. In LPS-induced septic mice, both pretreatment and treatment with L48H37 significantly improved survival and protected lung injury. Taken together, this work identified a new MD2 specific inhibitor, L48H37, as a potential candidate in the treatment of sepsis.