Discovery Of Novel Inhibitors Targeting The PD-1/PD-L1 Interaction As Potential Anticancer Agents

Immunotherapy has been widely used for the treatment of cancer and is currently revolutionizing the treatment of various types of cancers.Antibody-mediated immune checkpoint blockade has demonstrated clinical benefits in different types of tumors.However,therapeutic antibodies suffer from several disadvantages such as immunogenicity,long circulation half-life,poor tissue and tumor penetration,lack of oral bioavailability,and costly production.Moreover,currently available antitumor monoclonal antibodies target only extracellular receptors/proteins,which account for only a small fraction of cancer biomarkers.Compare to monoclonal antibodies,small molecule programmed cell death-1/programmed cell death-ligand 1(PD-1/PD-L1)modulators for cancer immunotherapy in general have reasonable pharmacokinetic properties including better tissue and tumor penetration,higher oral bioavailability,acceptable half-lives etc.In addition,small molecules can be administered orally,which provides the possibility of improved clinical dosing within the therapy regimen.Therefore,small molecule PD-1/PD-L1 modulators as cancer immunotherapies have the potential to overcome the above-mentioned disadvantages of therapeutic antibodies,and may also be combined with antibodies to achieve synergistic anticancer effects.Based on molecular docking or the cocrystal structures of inhibitors with PD-L1,we successfully identified a series of novel PD-L1 inhibitors with good bioactivities and pharmacokinetic properties through structure-based drug design and scaffold hopping strategy.Part 1:Starting from a hit compound Bavachinin,we designed and synthesized a series of chalcones,flavonoids,naphthalene,and resorcinol dibenzyl ethers as novel PD-1/PD-L1 inhibitors by using the docking-based drug design,as well as an extensive SAR study.Among them,we found the resorcinol dibenzyl ether compounds potently inhibited the PD-1/PD-L1 interaction with IC50 in the low nanomolar range,for example,compound NP19 inhibited the PD-1/PD-L1 interaction with IC50 values of 12.5 nM in HTRF binding assays.Furthermore,NP19 significantly promoted T cell response.In a well-established T cell-tumor co-culture assay,treatment with NP19 induced IFN-y release in a dose-dependent manner.Moreover,NP19 displayed significant in vivo antitumor efficacy in two different mouse models of cancer(a B16-F10 melanoma tumor model and an H22 hepatoma tumor model).In addition,H&E staining and flow cytometry(FCM)data suggested that NP19 activated the immune microenvironment in tumor,which may contribute to its antitumor effects.Overall,this work shows NP19 is a promising lead compound for further development as a new generation of small molecule inhibitors targeting the PD-1/PD-L1 pathway.To further improve the drug-like and pharmacokinetic property of NP19,we designed a series of resorcinol diphenyl ether-based PD-L1 inhibitors,by incorporating hydrophilic moiety into side chain,and evaluated for their inhibitory activity against the PD-1/PD-L1 pathway.Among them,compound P18 inhibited the human PD-1/PD-L1 interaction with IC50 values of 9.1 nM in HTRF binding assays.In addition,P18 dose-dependently promoted HepG2 death in a co-culture model of HepG2/hPD-L1 and Jurkat T cells.Furthermore,P18 displayed high water solubility(>10 mg/mL).Molecular modeling study indicated that P18 binds well to the PD-L1 binding site.Our results suggest that the novel resorcinol diphenyl ether-based PD-L1 inhibitors deserve further investigation as potential anticancer agents.Part 2:Starting from the analysis of the co-crystal structure of the dimeric PD-L1 protein in complex with BMS-8,we designed,synthesized and evaluated a novel series of resorcinol diphenyl ether-based PROTAC molecules as potential dual inhibitors and degraders of PD-L1 to overcome potential drug resistance of PD-L1 inhibitors.Most of the compounds exhibited excellent inhibitory activities against PD-1/PD-L1 interaction with IC50 values ranging from 25 nM to 200 nM.Among them,compound P22 is one of the best with an IC50 value of 39.2 nM.In addition to inhibiting PD-1/PD-L1 interaction,P22 also significantly restored the immunity repressed in a co-culture model of Hep3B/OS-8/hPD-L1 and CD3 T cells,as assessed by the IFN-? secretion levels.P22 strongly promoted IFN-? secretion in a dose-dependent manner,with the effects being more remarkable than that of Keytruda,a marketed monoclonal antibody(mAb)drug targeting PD-1/PD-L1.Moreover,flow cytometry and western-blot data suggested that P22 could moderately reduce the protein levels of PD-L1 in a lysosome-dependent manner,which may contribute to its immune effects.Collectively,these results suggest that it is possible to build PD-L1-targeting PROTAC-like molecules based on PD-1/PD-L1 inhibitors,and that P22 may serve as a starting point for exploring the degradation of PD-L1 by PROTAC-like strategy.Part 3:Novel CA-4 analogs based on PD-1/PD-L1 inhibitor and CA-4 were synthesized as dual inhibitors of tubulin and PD-1/PD-L1 to enhance the effective response rate of PD-L1 inhibitors.Among them,compound TP5 showed strongest inhibitory effects against five cancer cell lines with an IC50 value of 0.8 ?M in HepG2 cells and negligible cytotoxicity to normal cells(IC50>40 ?M for HEK-293 and NCM460).In addition,mechanism of action studies suggested that TP5 exerted its effects by inhibiting tubulin polymerization,suppressing HepG2 cell migration and colony formation,causing cell arrest at G2/M phase,and inducing apoptosis.Furthermore,TP5 exhibited moderate anti-PD-1/PD-L1 activity with IC50 values of 48.76 ?M.In vivo efficacy studies indicated that TP5 could significantly suppress tumor growth in an immune checkpoint humanized mouse model with a Tumor Growth Suppression(TGI)of 57.9%at 100 mg/kg without causing significant toxicity.Moreover,TP5 did not cause in vivo cardiotoxicity and myelosuppression in BALB/c mice.These results suggest that the novel CA-4 analogs may serve as a starting point for developing more potent dual inhibitors of tubulin polymerization and PD-1/PD-L1.In summary,we successfully identified a series of novel PD-L1 inhibitors with good bioactivities and pharmacokinetic properties through structure-based drug design and systematic SAR study.The representative compounds,NP19,P22,P18,and TP5,may serve as promising lead compounds for further development as a new generation of small molecule inhibitors targeting the PD-1/PD-L1 pathway.