The Screening And Antitumor Research Of Small Molecular Inhibitors Targeting PD-1/PD-L1 Interaction

Tumor immunotherapy has great potential to treat multiple types of cancer obtaining a durable,long-lasting response and low toxicity in cancer patients,which can substantially improve the overall survival of patients and represents a promising beginning for the cure of tumors.The discovery of a series of immune checkpoint inhibitors has equipped cancer therapy with novel and powerful"weapons".Checkpoint inhibitors can stimulate immune responses by removing inhibition signaling pathway between antigen-presenting cells and T cells,and subsequently restore T cell function to destruct cancer cells.Among the emerging checkpoint protein,programmed death-1（PD-1）/programmed death ligand 1（PD-L1）has already become one of the most promising targets in the field of immunotherapy.As immune checkpoint inhibitors,PD-1/PD-L1 monoclonal antibodies have been demonstrated their advantages in clinical applications,while due to the intrinsic limitations of antibodies,there are still great challenges in tumor response rate,highlighting the urgency and necessity for developing small molecular inhibitors currently.However,the development of small-molecule drugs blocking PD-1/PD-L1 interaction is inherent challenging because of the relatively large and flat interface of the protein-protein interaction（PPI）without well-defined pockets.It has become a conventional strategy for new drug development that applying virtual screening technology to screen potential conjugates for specific targets from a large number of known compounds,and then verifying their activities using biological experiments.Here,in an effort to discover potential PD-L1 small molecular inhibitors,we carried out a docking-based virtual screening strategy to rapidly identify the candidate compounds,with the combination of in vitro and in vivo experiment platforms,then comprehensively evaluated the PD-1/PD-L1 blocking activity of hit compounds,and explored the mechanism of action of them.The first part of the thesis is to identify small molecule compounds with novel structures that can effectively interact with h PD-L1 protein and inhibit the interaction of PD-1/PD-L1 from Specs compound database.Firstly,93 compounds having favorable interactions with h PD-L1 were selected from the Specs database using virtual screening method based on crystal structure of PD-L1 with ligand complex（PDB code:5J89）.Then based on the SPR and HTRF experiments,5 active compounds were identified that could effectively interrupt the PD-1/PD-L1interaction by binding to PD-L1 at the biochemical level,presenting the K_D and IC₅₀values at micromolar level.Subsequently,we discovered that two compounds APBC（Specs No.AG-690/11449006）and CBPA（Specs No.AN-465/42833793）with novel scaffold structures,could effectively interrupt the PD-1/PD-L1 interaction on the cell membrane surface based on flow cytometry assay.Meanwhile,based on the luciferase and T cell function assays,we further confirmed that both APBC and CBPA were capable of restoring PD-L1-mediated inhibition of T-cell function via interfering the PD-1/PD-L1 immune checkpoint pathway,elevating cytokine IFN-γand TNF-αsecretions of the primary T-lymphocytes in a dose-dependently manner.Altogether,these data suggested that the compounds APBC and CBPA represented two new promising hit compounds,thus the in vivo efficacy of APBC and CBPA warrant further exploration.For the second part of the study,in order to examine the therapeutic efficacy of the APBC and CBPA under complicated physiological conditions,and further study the mechanism of action of the hit compounds,different mouse tumor models were constructed.Firstly,utilizing well-established,murine tumor models,we demonstrated that both APBC and CBPA at a dose of 10 mg/kg significantly inhibited the growth of subcutaneous tumors in terms of both tumor weight and volume,displaying superior anti-tumor efficacy in different tissue types.Subsequently,based upon the RNA-seq data,we found that APBC and CBPA could induce enhanced antitumor immune responses-related signaling pathways and decreased tumor metabolic pathways such as aerobic glycolysis.After that,based on flow cytometry and immunohistochemistry assays,we observed that splenocytes harvested from mice treated with APBC showed remarkable increases in the CD4⁺and CD8⁺T cell sub-populations,and in the inflammatory cytokine production such as IFN-γ,TNF-?,and Perforin,relative to those taken from mice treated with vehicle control.In addition,it was found that CBPA did not affect the activation of CD4⁺and CD8⁺T cells in the spleen of mice,indicating that APBC could promote an on-going systemic immunity,while CBPA could not.Of note,analyses on the APBC/CBPA-treated mice further revealed significantly elevated levels of infiltrating CD4⁺and CD8⁺T cells,and inflammatory cytokines Gzm B and perforin secretion in tumor microenvironment.These inflammatory cytokines might orchestrate together to recruit more T cells into the tumor microenvironment to form the positive feedback loop against tumor growth.Altogether,these results supported the idea that APBC and CBPA could reinvigorate tumor infiltrating lymphocytes（TILs）by PD-1/PD-L1 PPI blockade,eventually suppressed tumor growth.In the third part of the study,we preliminarily evaluated the bio-safety and metabolic stability of compounds APBC and CBPA,and investigated binding modes between h PD-L1 and these two hit compounds by molecular dynamics simulation（MD）.Firstly,relatively good bio-safety was confirmed both for APBC and CBPA based on the cell viability assays and blood biochemical tests,as APBC/CBPA at therapeutic dose or high-dose administration（50 mg/kg）caused no observable hepatic and renal toxicity in murine models.In comparison,APBC had better security than CBPA.The metabolic characteristics showed that both of these two hit compounds possessed relatively good human blood plasma stability,while APBC had a shorter half-life in liver microsomes in vitro with higher liver clearance rate(T_1/2=17.4 min),CBPA possessed moderate stability in liver microsomes(T_1/2=44.6 min).These suggested that at the stage of structural optimization,we should consider not only improving the original activity,but also blocking or modifying the metabolic sites to improve the hepatic metabolic stability of compounds.In addition,the risk assessment of drug interactions using cytochrome P450 enzyme inhibition assay showed that APBC had a strong inhibitory effect on human CYP1A2 enzyme,and CBPA had a strong inhibitory effect on CYP1A2,CYP2C9,CYP2C19,and CYP2D6 enzyme,suggesting that we should avoid the risk of drug interacting with other drugs whose main metabolic pathways are above enzymes.Finally,the MD simulation results indicated that APBC/CBPA may have function through a PD-L1 dimer-locking mechanism,occluding the PD-1 interaction surface of PD-L1.Van der Waals interaction and electrostatic interaction were the main driving forces of the interaction between hit compounds and h PD-L1 protein dimer.Moreover,APBC formed two key hydrogen bonds with the key residue A121 and D122 from A chain,CBPA formed one hydrogen bond with Q66 from B chain.Thus,the number and strength of hydrogen bonding interactions may affect the binding ability of the ligand,which indicated that appropriately increasing the number of similar hydrogen bond acceptors and donors of the compounds may bring about a qualitative improvement in the activity ability of hit compounds in the further optimization.Exploring the advantages and problems of our hit compounds will provide useful clues and new guidance strategy for further structural optimization.In conclusion,the discovery of APBC and CBPA enriches the diversity of small molecule inhibitors of PD-1/PD-L1,which could serve as privileged scaffolds for further design of potent inhibitors targeting the PD-1/PD-L1 pathway,thus laying a solid foundation for immuno-oncology new drug development.