| Cancer immunotherapy has attracted widespread attention because of its efficacy in targeting and eliminating cancer cells by stimulating host immune system.It includes chimeric antigen receptor T cells（CAR-T）,immune checkpoint inhibitors（ICIs）and cancer vaccines.Among them,programmed cell death protein-1（PD-1）antibodies as the representative of ICIs therapy have developed most rapidly and become one of the important approaches for tumor treatment.PD-1 is an important immunosuppressive molecule,expressed on a variety of immune cells,including T cells,B cells,tumor-associated macrophages（TAMs）and natural killer cells（NK cells）.In healthy immune cells,PD-1 can bind to its ligand PD-L1,which can inhibit T cell proliferation and cytokine secretion,and negatively regulate lymphocyte activation.In the tumor microenvironment（TME）,PD-L1 protein is overexpressed on the surface of tumor cells and inhibits T-cell mediated immune response by binding PD-1,contributing to tumor cells’immune escape.Currently,the approved monoclonal antibodies（m Abs）have shown clinical effects in a variety of tumors,including melanoma,lung cancer,Hodgkin’s lymphoma,urothelial carcinoma,bladder cancer,colorectal cancer and renal cell carcinoma.In recent years,small molecule PD-1/PD-L1 inhibitors have been reported,but no small molecule inhibitors have been approved.We conducted the structure-activity relationship studies of PD-L1 inhibitors and designed,synthesized and evaluated the novel PD-L1 inhibitors.The specific work is as follows:Comparative studies of activities of small molecule PD-L1 inhibitors with different structures:the biological activities of three small molecule inhibitors with different structures,compound 2（Incyte-001）,3（Incyte-011）and 4（BMS-1001）,were compared.In the HTRF experiment,compound 4 had the best inhibitory activity for PD-1/PD-L1 binding(IC50=0.9 nM),followed by compound 3(IC50=5.293 nM)and compound 2(IC50=11 nM).In the 293T-OS-8-h PD-L1&T cells co-incubation assay,only compound 3 induced the production of IFN-γin T cells,which could enhance the immune response.Compound 4 exhibited lower cytotoxicity than the other two compounds in HPDE6C7 cells（human normal pancreatic ductal epithelial cells）and HL7702 cells（human normal hepatocytes）.Molecular docking and simulation studies showed that all three compounds can bind to the interfaces pocket of two PD-L1 dimers.In further pharmacokinetics experiment,compound 2（intravenous injection of 2 mg/kg）showed great blood-brain barrier（BBB）permeability and high drug exposure in brain tissue,while the other two compounds had no significant BBB permeability.This part of the work shows that small molecule PD-L1 inhibitors have different biological characteristics due to their different skeleton structures,and these characteristics will facilitate targeted structural optimization.The discovery and structure-activity relationship of novel small-molecule PD-L1 inhibitors:We have focused on the structural skeletons of amide PD-L1 small-molecule inhibitors reported by the Incyte and other companies,and explored the influence of linker groups and pharmacodynamic groups.Firstly,two series of 29compounds were designed and synthesized by bioelectronic isosteric and conformational restriction of amide groups in the molecular skeleton of Incyte company.HTRF results showed that the substitution of amides made the activity of the obtained compounds lost or significantly decreased.The direct removal of the amide group and direct connection of the triaryl ring have maintained the activity:the IC50 values of compounds B8 and B9 were 19.6 and 16.22 nM,respectively.Furthermore,compounds A10、A11 and B8-B10 were dimerized.Considering the poor physicochemical properties of the four benzene ring molecules,17 molecules were designed and synthesized by introducing different hydrophilic groups into the tail chain.As expected,three compounds C15(IC50=4.23 nM)、C16(IC50=3.902 nM)and C17(IC50=4.008nM)were obtained.In the 293T-OS-8-h PD-L1&T cells co-incubation assay,the three compounds can increase the production of IFN-γin T cells,which has an immune enhancement effect.At the same time,all three compounds showed low toxicity on PBMC,HPDE6C7 and HL7702 cells,and did not inhibit the h ERG channel.Finally,from the perspective of computer-aided drug design,the interaction between compound C16 and PD-L1 protein was analyzed in detail.It was found that the interaction between benzene ring skeleton and Tyr56 of PD-L1 protein formedπ-πstacking to stabilize the skeleton;the interaction between serine in the tail and PD-L1 protein formed hydrogen bond to further strengthen the binding.In general,compounds C15-C17 have low toxicity as well as high activity in vitro and can enhance immune effects,which have the potential to be further developed as small molecule inhibitors of PD-L1.In conclusion,we have systematically evaluated the relationship between the skeleton and activity of small molecule PD-L1 inhibitors and designed and synthesized a new type of high-efficiency leading compounds C15-C17 targeting PD-L1,which provides promising leading compounds for immune checkpoint blocking therapy. |
