Biological Activity And Binding Mode Study Of Peptide Inhibitors Targeting PD-L1

Monoclonal antibodies targeting the PD-1/PD-L1 immune checkpoint signaling pathway have achieved great success in the treatment of clinical malignancies.This has promoted the development of peptide and non-peptide small molecule inhibitors which have stronger effects,fewer side effects and better druggability than monoclonal antibodies.Because peptide molecules combine the advantages of antibodies and non-peptide small molecules,the development of peptide inhibitors targeting the PD-1/PD-L1 signaling pathway has distinctive advantages in drug discovery.In this paper,we screened 10 peptide inhibitors targeting PD-L1 obtained by computer-aided drug design,and selected two peptides with better activity for subsequent activity evaluation.In addition,structural biology and molecular dynamics simulation were used to study the combination mode between peptides and PD-L1.The paper mainly carried out the following two aspects of work:In the first part,cell-free and cell-based assays were performed to evaluate the biological activity of peptide inhibitors in vitro.Surface plasmon resonance technology was used to measure the binding affinities of 10 peptides with PD-L1.The results showed that the peptides 1681 and 1682 had the highest affinity,and the equilibrium dissociation constant（K_D）were 5.42μM and 13.06μM,respectively.Homogeneous time-resolved fluorescence technique demonstrated that 1681 and 1682 can obviously inhibit the interaction between PD-1 and PD-L1,and the half-maximal inhibitory concentration(IC₅₀)were 25.98μM and 48.37μM,respectively.After cyclizing the peptides,the binding affinity and inhibitory activity were slightly increased.The K_D value were 3μM and 7.91μM,and the IC₅₀ values were 14.56μM and 32.07μM,respectively.Furthermore,we also evaluated the activity of these peptides at the cellular level.The results of T cell activation experiments and cell co-culture luciferase experiments indicated that 1681 can significantly restore T cell activity.These results proved that 1681 can effectively inhibit the PD-1/PD-L1 interaction in cells.In the second part,in order to further study the binding modes between peptides and PD-L1,we utilized the X-ray crystallography to analyze the structure of PD-L1 and peptide complex.After screening a total of 194 crystallization conditions of Index,Screen1 and Screen2 series by the sitting drop method,we used the hanging drop method to optimize the crystallization conditions and obtained crystals with better diffraction effects.After the diffraction and structure analysis of the crystals,we found that all crystal structures were PD-L1 dimers,and no peptide molecules was found.Finally,the molecular dynamics simulation was employed to predict the binding modes of 1681 and 1682 with PD-L1.It found that the interaction between the terminal residue at 1681 and Glu58 and Arg125 of PD-L1 was weakened after cyclization.When combined with cyclic 1682,the residue with the largest energy contribution on PD-L1changed from Arg113 to Arg125.And the residues with the best energy contribution on1681 and 1682 are Ile10 and Arg1,respectively.In this study,we obtained an immune checkpoint peptide inhibitor 1681 that can significantly inhibit the interaction between PD-1 and PD-L1 at the biochemical level and cell level,and the binding mode of its interaction with PD-L1 was analyzed by molecular dynamics simulation.The obtained peptide 1681 can be used as a lead compound for further optimization of activity and druggability.