Discovery Of Innate Immune Receptor Inhibitor With Anti-hyperuricemic Activity And Research On Its Mechanism In Treatment Of Gout

Backgound:Gout is a common crystal-induced arthritis attributed to the deposition of monosodium uric acid crystals?MSU?in the joints resulted from persistent hyperuricemia.Combination of urate-lowering and anti-inflammatory therapy is considered to be available stratege for treatment of gout,nevertheless,there are lack of ideal drugs.Xanthine oxidase,which is the rate-limiting enzyme involved in uric acid production,as well as innate immune receptors NOD-like receptor protein3?NLRP3?and Toll-like receptor 4?TLR4?have been regarded as potential targets of gout therapy,however,lead compounds and drugs with multiple inhibitory effects on XOD,NLRP3 and TLR4 are deficient.Purpose:In the present study,we plan to synthesize a series of deoxybenzoin derivatives based on the framework of deoxybenzoin compounds,and screen a lead compound with anti-hyperuricemic and anti-inflammatory activities which could inhibit XOD,NLRP3 and TLR4 simultaneously.Then,in vitro and in vivo models would be established to evaluate molecular machanisms bu which the screened lead compound improves hyperuricemia and acute gouty arthritis.Methods:?1?Based on the framework of deoxybenzoin compounds,combining with computer-aided drug design,a series of deoxybenzoin derivatives were sythesized.After screening sythesized compounds with XOD enzymatic system,THP-1 cell model and HEK-Blue^TM-hTLR4 cell model,a lead compound with inhibitory effects on XOD,NLRP3 and TLR4 was identified;?2?Molecular docking detection was performed to confirm the binding of the lead compound to target molecules.The effects of the lead compound on XOD activity were investigated using XOD enzymatic system,while the effects of the lead compound on protein expressions of components of NLRP3 inflammasome and key factors involved in TLR4 signaling pathways were investigated in MSU-stimulated THP-1 cells,in order to preliminary affirm the mechanisms of the lead compound in treating gout in vitro;?3?The effects of the lead compound on biochemical index and hepatic XOD activities were evaluated in potassium oxonate-induced hyperuicemic mice.Moreover,the effects of the lead compound on joint swelling,synovial inflammatory reactions,activation of synovial NLRP3 inflammasome and TLR4 signaling pathway were assessed in acute gouty arthritis rats induced by intra-articular injection of MSU.Results:?1?After screening,we obtained a lead compound with inhibitory effectsofXODNLPR3andTLR4,called4-?2-?4-chlorophenyl?-1-??4-chlorophenyl?amino?ethyl?benzene-1,3-diol?CBED?.The purity of the product was 96.5%by optimizing the synthesis method.?2?Molecular docking detection exhibited that CBED could well bind to active sites of XOD,NLRP3 and TLR4.In XOD enzymatic system,CBED significantly inhibit XOD activity with a IC₅₀0 of 3.87?M.In MSU-sitmulated THP-1 cells,CBED not only obviously down-regulated expressions of NLRP3 inflammasome components,but apparently decreased protein levels of key factors involved in TLR4 signaling pathways,subsequently inhibit release of Interleukin-1??IL-1??and tumor necrosis factor alpha?TNF-??;?3?In potassium oxonate-induced hyperuricemic mice,CBED lowered serum uric acid levels and hepatic XOD activities with a dose-related manner.On the other hand,in acute gouty arthritis rats,CBED significantly attenuated MSU-induced joint swelling and synovial histopathological changes,more importantly,CBED blocked activation of NLRP3 inflammasome and TLR4 signaling pathways in synovium tissues.Notbly,CBED exhibited no effects on all these indicators in normal animals,predicting its safety.Conclusions:As a innate immune receptor inhibitors with urate-lowering activity,CBED might play a causal role in both anti-hyperuricemia and anti-inflammation targeting XOD,NLRP3 and TLR4,and subsequently achieve the goal of improving gout.