Synthesis Of N-(benzoyl)-L-cysteine Derivatives And Their Negative Regulation Of TLR2 Anti-in Flammatory Activity

Research BackgroundToll-like receptors(TLRs)are an important type of pattern recognition receptors,that could recognize a variety of pathogenic microorganisms and play an important role in the body’s anti-infection immunity.As an important member of the Toll-like receptor family,TLR2 is widely involved in the immune process of bacterial and viral infections.The moderate inflammatory response mediated by TLR2 is beneficial,but excessive activation can lead to many inflammatory diseases,such as sepsis,systemic lupus erythematosus,atherosclerosis and so on.Therefore,inhibition of TLR2 may become an important target for the treatment of related inflammatory diseases,but so far no TLR2 inhibitor drugs have been successfully marketed,and the study of small molecule inhibitors of TLR2 with novel structures is of far-reaching significance.Research PurposesNatural products play a pivotal role in drug discovery,and 50%of clinically small molecule drugs are directly or indirectly derived from natural products.Drawing on this research thinking,we focused our attention on two natural small molecules with anti-inflammatory activity:Gallic acid and S-allyl-L-cysteine(SAC).Using the principle of splicing,combining the two into a skeleton with a new structure through chemical bonding at a special site,exploring and discovering novel TLR2 small molecule inhibitors to enhance its anti-inflammatory activity.The purpose of this study is to connect two small molecule components through a chemical bond without affecting the activity of a single natural product small molecule,and to modify their structure in order to discover novel TLR2 small molecule inhibitors with anti-inflammatory activity.Research Content(1)Designed to chemically link Gallic acid and S-allyl-L-cysteine through an amide bond to form a new structure of compound SGA,and use the backbone of the linker(SGA)as the parent to synthesize a series of N-(benzoyl)-L-cysteine derivatives.The structures of these compounds were confirmed by 1H NMR,13C NMR and MS.(2)Use mouse macrophages(Raw264.7)to activate the inflammatory signal nitric oxide(NO)with the TLR2 specific positive activator Pam3CSK4,and judge by investigating the nitric oxide signal which is related to the inhibitory activity and toxicity of the compounds.At the same time,HEK-Blue hTLR2/3/4(transcribed human TLR2/3/4 and alkaline phosphatase reporter gene)cell lines were used for target verification of the compounds.(3)The relationship between the compound and TLR1/2,TLR2/6 and TLR2/MyD88 protein expression was studied by western blot and immunoprecipitation methods.And the use of human myeloid leukemia monocytes cell(THP-1)and human peripheral blood mononuclear cell(PBMC)to study the effects of compounds on the downstream signal factor TNF-α of TLR2.Finally,the molecular docking model is used to study the binding sites between the preferred compound and TLR1/2.Analysis Conclusion(1)We found the preferred compound SMU-8c through structure design,synthesis and activity analysis,which has a certain inhibitory effect on the inflammatory signal NO activated by the TLR2-specific positive activator Pam3CSK4 in mouse peritoneal macrophages(Raw264.7)(IC50=22.54±2.60 μM).In the cytotoxicity analysis,SMU-8c did not show obvious cytotoxicity even at concentrations as high as 100 μM,suggesting that the inhibitory activity of the compound has nothing to do with toxicity.The study also found that SMU-8c can inhibit the alkaline phosphatase(SEAP)signal in HEK-Blue hTLR2 cells,while it has no inhibitory effect in HEK-Blue hTLR3/4.(2)In Raw264.7 cells and THP-1 cells,when the concentration of SMU-8c was 25 μM,the release of TNF-α induced by Pam3CSK4 was inhibited by 50%.In PBMC cells,the inhibition rate at a concentration of 12.5 μM has exceeded 95%.In addition,we also observed the dose-inhibiting effect of SMU-8c on TLR2 protein and the negative regulation of TLR1/TLR6/MyD88 protein associated with TLR2 protein at the protein level.Molecular simulation docking shows that SMU-8c can be closely attached to the binding site of TLR1/2 and Pam3CSK4,competitively hindering the binding of Pam3CSK4 and TLR2,so as to achieve the purpose of anti-inflammatory.