Molecular Simulation Of EPPIN/SEMG1 And P62-zz/IDR1 And Discovery Of Their Small Molecule Inhibitors

Protein-protein interaction(PPI)is the main way to regulate biological signals,and selective intervention on them can change the function and fate of cells.In drug research,PPI is a new target which is different from enzyme and receptor.It is of great significance to study the structure and function of PPI and to design as well as synthesize novel small molecule lead compounds.In this paper,the interaction between epididymal protease inhibiting protein(EPPIN)and sperm coagulation protein Semenogelin 1(SEMG1)was studied by means of homologous modeling,docking and molecular dynamics simulation.The results of molecular simulation showed that the disulfide bonds in EPPIN protein played an important role in the stability of its structure.The EPPIN/SEMG1 complex model was evaluated according to the calculated binding free energy,and it was found that EPPIN could interact with SEMG1 by Tyr107,Gly112,Asn116,Gln118,Asn122 and other key amino acid residues in its C-terminal,while Arg32 in N-terminal also contributed to the binding interaction.In addition,we found that the docking pocket formed by Arg32,Asn114,Asn116,Phe117 and Asn122 was of great significance for designing the new EPPIN ligand.This detailed PPI study may contribute to a better understanding of the biological function of EPPIN and provide the basis and support for the development of male non-hormonal contraceptivesSecondly,the structure model of the first p62-zz structural domain peptide ligand,natural defense regulator 1(IDR-1)was construction.After docking with p62-zz structure,the molecular dynamics simulation study of p62-zz/IDR-1 protein complex was carried out and their binding free energy was calculated.The results showed that the IDR-1 short peptide could interact with the key amino acid residues near the arginine ligands of the p62-zz domain(Ile127,Cys128,Asp129 and Asp147,Asp149)by multiple amino acid residues(Leu1,Ala7,Arg11,and Lys13).This binding interaction study may make for the better comprehensions of the structure and biological function of p62-zz.It also provided the key target sites for the development of p62-zz protein inhibitors or p62-zz/ IDR-1 protein interaction small molecule inhibitors.Finally,based on the first small molecule inhibitor of p62-zz domain: XIE1004,eight benzene 5-amino2-ether-benzamide derivatives were designed and synthesized,which enriched the diversity of parent skeleton structure.As lead autophagy inhibitor selected from eight synthesized compounds,the compound 4d showed the most obvious effect of LC3 B conversion.Further,the autophagy inhibition of 4d was evaluated using transmission electron microscopy and confocal microscopy,showing the fusion of autophagosomes and lysosomes at the final stage of autophagic flux was suppressed.We also found it could significantly enhance the chemosensitivity of vincristine in vincristine-resistant esophageal cancer cell line Eca109/VCR in a true synergistic association manner.Moreover,the computational study showed that 4d might bind with p62-zz to inhibit autophagy.We also found 4d showed more selectivity to cancerous cells than normal cells on the regulation of autophagy compared to the reported p62-zz inhibitor.The above study showed that 4d was a new autophagy degradation inhibitor with potential for development,and could be used as a sensitizer in combination with conventional chemotherapy drugs to treat esophageal cancer,laying a foundation for subsequent drug development.