Virtual Screening And Biological Activity Evaluation Of PD-1/PD-L1 Protein-protein Interaction (PPI) Regulators

BackgroundTumor immunotherapy was a treatment method that eliminated cancer cells by enhancing the function of immune cells.It was a breakthrough treatment after surgery,radiotherapy,and chemotherapy,including cancer vaccines,targeted antibodies,adoptive cell therapy（ACT）,Immune checkpoint inhibitor（ICB）,non-specific immunomodulator and other forms.Immune checkpoint molecules induced cytotoxic T cell incompetence by generating immune co-suppression signals and mediated tumor immune escape,and were currently important targets for tumor immunotherapy.Immune checkpoint inhibitors killed tumor cells by blocking co-suppression signals to reactivate T cells,and had been successfully used in the treatment of various malignant tumors such as melanoma.Among them,antibody drugs targeting the PD-1/PD-L1 signaling pathway have developed most rapidly,and multiple monoclonal antibody drugs such as Avelumab have been launched.However,there were still several inevitable disadvantages of mAbs including limited tissue and tumor penetration,long half-life time,poor oral bioavailability,and expensive production.Therefore,Small molecule inhibitors with inherent advantages such as greatly reduced production costs,good stability,and less immunogenic side effects are expected to become effective alternatives or supplements to antibody drugs.ObjectiveBased on the structure of PD-1 and PD-L1,this project carried out virtual screening research on peptides and small molecules,combined with molecular simulation technology to find theoretical candidate compounds and analyze the binding mode.The anti-tumor activity was evaluated by surface plasmon resonance（SPR）competitive analysis and biological activity measurement to discover small molecular lead compounds with potential applications.So as to lay the foundation for the application and development of small molecule immune checkpoint inhibitors.Methods and Results1.Virtual screening and biological activity evaluation of PPI regulators（polypeptides）（1）Determine the binding site of small molecule modulator based on hPD-1/PD-L1 structure.Using PD-1 as the molecular target site,22 PPI peptide modulators were virtually screened in the internal peptide library of the laboratory utilizing molecular docking and molecular dynamics（MD）simulation,and their blocking ability was predicted.After MD simulation,it was analyzed that Tyr68,Thr76,Glu136 and other key amino acids form additional hydrogen bonds with the polypeptide to promote stable binding between them.The MM/GBSA method was used to decompose the binding free energy,which showed that Gln75,Thr76,Glu136and other important residues played an important role in the interaction between the regulator and the protein.It was determined by the SPR experiment that 8 of the peptides had good affinity with PD-1,and further,4 peptides were screened by the SPR competitive inhibition test to block the PD-1/PD-L1 interaction.（2）The RRWWRR-NH₂ and RRQWFW-NH₂ with the best inhibition ability were evaluated by ELISA,LDH detection and MTT cell viability assay to have biological activity,and RRQWFW-NH₂ played a greater role.2.Virtual screening and biological activity evaluation of small molecule PPI regulators（1）For the ChemBridge database,the structural similarity search was used to construct and optimize a small molecule compound library（PD-1:813;PD-L1:414）.Using the selected peptide as a chemical probe,based on its binding mode,a total of 9small molecule PPI regulators were virtually screened in the compound library using molecular docking and MD simulation,and their blocking ability was predicted.Applying MM/GBSA method to calculated and decomposed the binding free energy,the key amino acids Glu58,Arg113,Tyr123（PD-L1）and other amino acids contributed to the total energy of the regulator,and the situation of PD-1 key amino acids was consistent with the peptide.The greater the contribution of binding free energy was electrostatic interaction and VWD potential energy,but the solvation process was not conducive to the role of small molecules;（2）SPR analysis showed that 5 small molecules have good affinity with the target protein,and further SPR competitive inhibition test demonstrated that these 5small molecules all had blocking effect,which was consistent with the MD prediction results.In addition,the biological activity test confirmed that the better potential ID7844239 and ID66236437 compounds had good biological activity,and the effect of ID66236437 was obvious than ID7844239.ConclusionsFor the peptide modulators RRQWFW-NH₂ and RRWWRR-NH₂ finally screened by molecular docking,MD simulation and SPR analysis.The biological activity test verified that they have a good inhibitory potential for PD-1/PD-L1interaction,indicating they could be used as a chemical probe to screen small molecule compounds with binding potential based on binding model information.Utilizing molecular docking,MD simulation and SPR experiments,five small molecule compounds with good inhibitory potential were found,and the anti-tumor activities of ID66236437 and ID7844239 compounds were further verified by in vitro activity evaluation.The results showed that they could be used as a promising small molecule lead compound for follow-up studies to overcome the limitations of therapeutic antibodies.In addition,the exploration of the interaction mode between the ideal regulator and the target protein could lay a foundation for the discovery and structural optimization of a new class of tumor immunotherapy small molecules.