Discovery,Optimization And Bioactivity Of TLR2-Specific Small Molecule Modulators

Toll-like receptors(TLRs)are a kind of pattern recognition receptors,which can identify pathogen-related molecular patterns and play an important role in the body’s anti-infection,anti-cancer and tumor immunity.More and more evidence shows that there is a close relationship between TLR and the occurrence and development of autoimmune diseases.On the one hand,inhibition of TLR can inhibit the release of inflammatory factors and can treat rheumatoid arthritis,systemic lupus erythematosus,et al.On the other hand,proper activation of TLR receptors can promote immune protection in some cases.For example,TLR4 activator MPLA can be used as a vaccine adjuvant in vaccines for cervical cancer.TLR7/8 activator R848 can be used in stromal cells.Cancer treatment.Therefore,the forward and reverse regulation of TLR has very important clinical therapeutic significance.However,there are only two successful precedents for small-molecule drugs for TLR.There is a lack of research on small molecule modulators of these membrane proteins.The purpose of this study was to investigate the synthesis of TLR2 positive and negative regulators and its application in tumor immunomodulation.All compounds were confirmed by 1H-NMR and 13C-NMR as well as HR-ESI-MS.1.A total of 48 new compounds that activate TLR1/2 were synthesized,of which SMU-Z1 performed the best.The design of TLR1/2 agonists was based on CU-T12-9 reported earlier in this research group,retaining its parent nuclear structure and synthesizing a total of 48 series of agonists.The first series of thiazole compounds were 20 compounds.Compounds 1-20 were synthesized by two-step reaction using different substituents of thiobenzamide and α-carbonyl bromides with different substituents as raw materials.The second series was modified with 1,3-diphenyl-1H-imidazole as the mother nucleus to obtain imidazole compounds 21-48 in 28 total.All compounds were tested for the activity of alkaline phosphatase release signal in HEK-Blue hTLR2 cells using CU-T12-9 as a control.The series of biologically active final test results showed that Compound 23(SMU-Z1)had the best activity with an EC50 of 4.88±0.79 nM,a 10-fold increase over CU-T12-9.The results of deep structure-activity relationship studies indicate that the imidazole ring,nitro or sulfone groups,and hydroxyl groups are the key factors for maintaining the activation effect.2.Pharmacological activity studies have shown that SMU-Z1 targets TLR1/2 to activate downstream signaling pathways.Western blotting experiments also showed that SMU-Z1 can directly act on TLR2 protein.Antibody-specific experiments demonstrated that the compound SMU-Z1 is a TLR1/2 agonist rather than TLR2/6.ELISA,mRNA and NO tests showed that SMU-Z1 can upregulate the expression of TNF-α,IL-1β and promote the secretion of NO,IL-6 and 8.It was confirmed that SMU-Z1 promotes the release of downstream inflammatory cytokines through the NF-κB signaling pathway after acting on TLR1/2.Next,biotin was introduced to label the compound SMU-Z1 to investigate the biophysical activity of binding of the small molecule to the TLR1/2 protein.Of all the biotin-labeled compounds,Compound 45 had the best activity when it was attached to biotin based on SMU-Z1.Biophysical experiments showed that 45 could specifically bind to the proteins on the surface of TLR1/2 cells,further confirming the ability of compound SMU-Z1 to specifically bind to proteins.3.In addition to the TLR1/2 activator,we also studied TLR2 inhibitors.Based on the structure of C29 studied by the Vogel’s group,a total of 50 TLR2 inhibitors were synthesized based on computer-aided design.At the same time,the activity was measured.The results showed that compound K35(SMU-A0B13)can well inhibit the expression of TLR2,and its IC50=18.21±0.87 μM is the best activity.Structure-activity relationship studies have shown that the carbon-nitrogen double bond and the benzene ring and hydroxyl groups on both sides and the size of the substituents play a decisive role in the inhibitory activity.Western blot experiments and specific experiments demonstrated that SMU-AOB13 is a TLR2 inhibitor.ELISA and nitric oxide assays showed that SMU-A0B13 was found to act on TLR2 cells and was found to inhibit iNOS and TNF-α signaling up-regulated by Pam3CSK4.Through the study of this project,we have developed two TLR2 regulators.It is expected that these efforts will lay a foundation for the future research on immune vaccine adjuvants and immunotherapy of small molecule anticancer drugs.The research focus of subsequent agonists will focus on improving the water solubility and reducing the toxicity of SMU-Z1 and verifying the tumor immune function in vivo;we look forward to finding anti-inflammatory and immune-activating precursors that are not easily hydrolyzed and have better activity.