Discovery Of Novel Aporphine Alkaloid As TLR2 Antagonist And Its Anti-acute Inflammation By Reversing Antigen-presenting Cell

Inflammation is the most common pathological process in human disease.Current research shows that many diseases are related to inflammation,such-as tumor,cardiovascular disease,autoimmune disease and so on.The clinical application of anti-inflammation drugs is mainly divided into non-steroidal anti-inflammatory drugs(NSAIDs)and steroidal anti-inflammatory drugs(SAIDs).Although SAIDs have strong anti-inflammatory and analgesic effect,their application is limited due to their severe side effects.NSAIDs are mainly cyclooxygenase(COX)inhibitors and the main side effects of these drugs are gastrointestinal reactions,central nervous system.disorders and cardiovascular adverse events.It can be seen that the major problem of anti-inflammatory drugs are the single anti-inflammatory target and significant side effect.Therefore,it is important to find new anti-inflammtion targets and develop an effective and safe anti-inflammatory drug.As an important pattern recognition receptor,Toll-like receptor2(TLR2)maintain the homeostasis of the internal environment through regulating innate and adaptive immune.The current studies revealed that the over-activation of TLR2 is associated with rheumatoid arthritis,systemic lupus erythematosus,Alzheimers disease and so on.Thus,TLR2 inhibitors are likely to be new therapy for inflammation treatment.However,the reported TLR2 inhibitors are limited,with moderate activity and unstable structure,which fails to satisfied the clinical application.Given that,we here devoted ourselves to develop novel structure of TLR2 inhibitors,interpret its anti-inflammtory mechanism and verify its anti-inflammatory effect through construsting inflammation model.The main results are-summarized as following:1.We found aporphine alkaloid Taspine inhibited TLR2 significantly through cellbased screening of 2100 natural products.We synthesized Taspine in 10 steps and carried out structure-activity relationship studies to obtaine derivative SMU-Y6.2.Compared with Taspine,SMU-Y6 exhibited better anti-TLR2 activit,IC50 value of 0.11±0.04 μM,with 20 times improvement in water solubility and less toxicity.Selectivity studies revealed that SMU-Y6 inhibited TLR2 rather than other TLRs and reduced the release of inflammatory cytokines,such as TNF-α,IL-6,IL-1βand so on.3.In vitro anti-inflammatory mechanism studies revealed that SMU-Y6 reduced the recruitment of TLR2 to MyD88,inhibited the phosphorylation of p-p65,p-38,IKKα/β,ERK1/2,and reduced the degradation of IKBa,which led to the blockage of NF-κB and MAPK signaling pathway as well as the less release of inflammatory cytokines.In addition,SMU-Y6 reversed the polarization of macrophage into M1 phenotype.4.In mice paw edame and colitis model,SMU-Y6 significantly reduced the infiltration of neutrophile in local tissue,polarization of M1 macrophage and production of inflammatory cytokines to carry out anti-inflammatory effect.Pharmacokinetic studies and in vivo safety evaluation suggested that SMU-Y6 had a long half-life,high bioavailability and low toxicity in vivo,and is expected to be a safe and long-acting anti-inflammatory drug.In short,we obtained an aporphine alkaloid SMU-Y6 through structure optimization to Taspine,with better anti-TLR2 activity and water solubility,less cytotoxicity as well as preference to TLR2 rather than TLRs.We revealed that it blocked TLR2/MyD88/NF-κB and MAPK signaling pathway to reduce the release of cytokines and polarization of macrophage into M1 phenotype as well as infiltration of neutrophil to perform anti-inflammtion.We provided a candidate for TLR2 small molecular inhibitors with novel structure,excellent activity and good druggability,as well as put forwared insights for the potential anti-inflammatory mechanism of TLR2 inhibitos.