Study On The Action Sites And Mechanisms Of Selective Antagonizing TNFR1 Of Hydrostatin-SN10 As A Candidate Drug For The Treatment Of Inflammatory Bowel Disease

BackgroundTumor necrosis factor type I receptor（TNFR1）mediates TNF-αThe main receptors of pro-inflammatory biological functions play an important role in the occurrence and development of inflammatory diseases,and are popular targets for the treatment of such autoimmune diseases.However,there is currently no selective TNFR1 antagonist drug available on the market.Our research group previously obtained a class 1 candidate drug-the selective TNFR1 antagonist peptide Hydrostatin-SN10.Currently,we have completed preclinical research on its indication for inflammatory bowel disease（IBD）and are about to enter clinical phase I.However,the interaction sites and antagonistic mechanisms of SN10 with the target TNFR1 are still unclear.ObjectiveThe key amino acid sites of SN10 interacting with TNFR1 were analyzed based on the structural model of SN10-TNFR1 complex.The binding sites on TNFR1 were analyzed using site directed mutagenesis,protein expression and purification,and molecular interaction techniques.The key sites on SN10 were analyzed through the animal model of IBD disease to clarify the interaction and antagonistic mechanism between SN10 and target TNFR1,To provide experimental basis for guiding the structural optimization and modification of SN10 and developing new TNFR1 selective antagonists.Methods1.The prokaryotic expression vector of TNFR1 mutant extracellular domain protein was constructed by site directed mutagenesis.2.Expression of TNFR1 and TNFR1 mutant recombinant protein in E.coli BL21,and purification of the target protein by denaturation,renaturation,Ni²⁺affinity chromatography and molecular sieve chromatography to obtain the recombinant target protein.3.Detection of TNFR1-R106A,TNFR1-N139A and ligand TNF-αusing microcalorimetry（MST）.The binding ability of TNFR1,TNFR1-R106A,TNFR1-N139A and SN10,the activity of TNFR1-R106A,TNFR1-N139A recombinant protein and the binding site on TNFR1 were analyzed.4.Using a DSS induced mouse model of ulcerative colitis disease,investigate the in vivo anti-inflammatory activity of SN10 mutant peptides and analyze key binding sites on SN10.Results1.Prokaryotic expression vectors p MCSG7-TNFR1-R106A and p MCSG7-TNFR1-N139A were constructed for the extracellular domain protein of the TNFR1 mutant.The recombinant protein was expressed in Escherichia coli BL21.Through denaturation,renaturation,nickel column affinity chromatography and gel filtration chromatography,the TNFR1 mutant recombinant protein（purity>96%）was purified.The yield of TNFR1-R106A was 6.1 mg/L,and the yield of TNFR1-N139A was 8.83 mg/L.2.The TNFR1 mutant recombinant protein has good activity,and the recombinant protein TNFR1-R106A and TNF-α,The affinity K_Dvalue is 450 n M,and the recombinant protein TNFR1-N139A is associated with TNF-α,The affinity K_Dvalue is 740 n M.3.Use MST to detect the activity of TNFR1 mutant protein and the affinity between TNFR1,NFR1 mutant protein and SN10.The results indicate that the K_Dvalue of SN10-TNFR1 is 4.42μM.The K_Dvalue of SN10-TNFR1-R106A is 7.74μM.There was no significant change compared to SN10-TNFR1;And the K_Dvalue of SN10-TNFR1-N139A is≥374μM.The binding ability of ASN139 was significantly lower than that of SN10-TNFR1,indicating that ASN139 may be a binding site on TNFR1.4.Using a DSS induced mouse model of ulcerative colitis disease,the results showed that the mutant peptide SN10-M1 had no significant anti-inflammatory activity and no significant anti-inflammatory therapeutic effect compared to SN10,indicating that GLU8may be a key binding site on SN10.ConclusionThe experimental results of this study indicate that the potential binding sites for selective antagonism of TNFR1 targets by the candidate drug Hydrostatin-SN10 for the treatment of inflammatory bowel disease are ASN139 on TNFR1 and GLU8 on SN10.