Virtual Screening And Activity Evaluation Of Small Molecule Inhibitors Against PD-L1

Programmed cell death protein 1（PD-1）,an immune checkpoint,could combine with its ligand（programmed cell death protein ligand 1,PD-L1）in the intricate tumor microenvironment,which always inhibits the anti-tumor immunity of T cell.Therefore,it is effective to block the interaction between PD-1 and PD-L1 to avoid the tuomor immunosuppression and activate immune responses against tumor.The high level of PD-L1 expression could be found on the surface of many kinds of tumour cells,therefore,it can be regarded as an ideal anti-tumor target and don’t influence the immune activities of T cells.At present,although PD-L1 monoclonal antibody inhibitors have good anti-tumor effect,they are high-cost and have poor stability.These antibody inhibitors,in many cases,could cause the serious immune-related adverse reactions due to its high binding specificity.Therefore,there is an emerging need for developing small molecular weight compounds to target and inhibit the activities of PD-L1.In this study,we employed virtual screening techniques,including the pharmacophore model and molecular docking,to screen small molecular weight PD-L1 inhibitor from traditional Chinese medicinal ingredient and existing drugs,and subsequently evaluated the binding affinity of candidate compounds with PD-L1,as well as the anti-tumor effects in vitro and in vivo.Finally,the immune activities of candidate compound and another drug,anidulafungin,were assessed.Herein this dissertation demonstrated the virtual screening of PD-L1 small molecular weight inhibitors and related experiments.PartⅠ.IntroductionThe aim of this chapter is to summarize the current studies on many aspects,including the clinical traits of PD-1/PD-L1 blockade,the development of drug virtual screening technology and the current methodology for the discovery of the biomolecular interactions.Firstly,this review introduced the function and mechanism of PD-1/PD-L1 pathway,as well as global research progress of PD-1/PD-L1checkpoint inhibitors.Next,the development of compounds virtual screening methods,including pharmacophore model and molecular docking as well as common software applied for compound virtual screening,were outlined.Lastly,this chapter summed up the approaches for the determination of biomolecular interactions,and showed the related application.Taken together,this chapter introduced relative scientific research involved in this study,which would open the way for our study.PartⅡ.Screening of small molecular weight inhibitors of PD-L1based on the pharmacophore modelIn this chapter,small molecular weight inhibitors of PD-L1 were preliminarily screened by pharmacophore model.Firstly,32 small molecular weight inhibitors with various structures were used as the training set to build a pharmacophore model,which was successively evaluated by the training set and test set containing other 30inhibitor.Then the pharmacophore model was further verified by the analysis of the structure features of reported PD-L1 small molecular weight inhibitors.Subsequently,the optimal pharmacophore model IL-2 containing five pharmacophoric features（RHHda）was determined to screen the novel PD-L1 inhibitors from 3906 small molecular weight compounds（including 1876 components of traditional Chinese medicine and 2030 existing drugs）.Finally,460 compounds which matched the pharmacophoric features were hit,and considered as candidates for further screen.PartⅢ.Screening of PD-L1 small molecular weight inhibitors based on molecular dockingIn this chapter,the potential PD-L1 inhibitors were further screened from the 460candidate compounds obtained by pharmacophore model.Firstly,the interaction center of PD-L1 for the binding of small molecular weight inhibitors,including 16residues（Phe19、Thr20、Asp26、Ile54,Tyr56、Glu58、Glu60、Asp61、Gln66、Arg113、Met115、Ala121,Asp122,Tyr123、Lys124 and Arg125）,was determined based on the 3D crystallization structure of humanized PD-L1 molecule and small molecular weight inhibitors complex,humanized PD-1 and PD-L1 complex,humanized PD-L1 and monoclonal antibody inhibitor complex.And related 21 pairs of interaction mode between them were regarded as a positive reference to screen novel compounds.Then all atoms of PD-L1 molecule were selected as the docking site to investigate the binding affinity of 460 candidate compounds;and 247compounds were screened according to the standards that the binding energy should be less than-7.5 kcal/mol and the interaction residues of PD-L1 molecule were more than or equal to 3.Subsequently,the active center of PD-L1 molecule determined above was set as the docking site,and the single-stranded and double-stranded PD-L1protein were set as the receptor for further screen,respectively.Finally,according to the above standards,the spatial binding configuration of compounds and interaction mode between compounds and PD-L1,two compounds,gallotannic acid and anidulafungin,were identified as candidate PD-L1 small molecular weight inhibitors,which would be undertaken the next step investigations.PartⅣ.Evaluation of anti-tumor effect of the candidates in vitro and binding affinity between PD-L1 molecule and the candidate compoundIn this chapter,antitumor effect of each candidate compound and the binding affinity between compounds and PD-L1 were investigated using tumor cell assay in vitro and biolayer interferometry technique,respectively.Firstly,the human lung adenocarcinoma cell line（A549）and Lewis lung cancer cell line（LLC）were cultured and the antitumor effect of tannic acid and anidulafungin were evaluated by cell counting kit-8（CCK-8）assay.The results showed the high viability of two tumor cells under the exposure of gallotannic acid at the highest dose of 200μg/m L,indicating that the anti-tumor effect of gallotannic acid was not obvious.The possible reason may be that the tannic acid itself cannot exert the effective antitumor activity,however,our cellular tumor model might not exactly mimic the intrinsic tumor immune microenvironment and this compound failed to regulate the anti-tumor immune activities.On the contrary,another compound,anidulafungin,showed significant antitumor effect since its IC₅₀value on A549 and LLC were 170.6μg/m L and 160.9μg/m L,respectively.In addition,results of intermolecular interaction measurement by biolayer interferometry technique showed that the interaction affinity constant K_Dvalue of PD-L1 and anidulafungin was 7.69×10^-5M,indicating a good binding affinity.Moreover,they presented a dynamic process of fast association and fast dissociation(kon1/（Ms）=4.08×10²,kdis1/s=3.14×10^-2).To sum up,anidulafungin might have potential antitumor activity and inhibitory effect on the function of PD-L1.PartⅤ.Evaluation of anti-tumor immunity in vivo of the candidate compoundIn this chapter,in order to further evaluate the anti-tumor immunity of anidulafungin in vivo,the LLC-tumor bearing mice model was established,and the related immune responses were detected using the enzyme-linked immunosorbent assay（ELISA）and immunohistochemistry（IHC）.Firstly,the antitumor effect of anidulafungin in vivo was investigated in terms of tumor volume and body weight.The results showed that the tumor volume and body weight of mice,which were administrated at the middle（25mg/kg）and high（50mg/kg）dose of anidulafungin,with monoclonal antibody（Durvalumab,1.25mg/kg）and chemotherapy drug（5-FU,25mg/kg）,were significantly lower than those in the model group.Also,an increase in the mice weight without tumor of either middle or high dose group confirmed the significant antineoplastic effect of anidulafungin.Compared with the general medicine,anidulafungin had little effect on the body weight of mice.Moreover,the results of ELISA showed that the concentrations of IFN-γand IL-4 in serum of the anidulafungin group and positive control group were significantly higher than those of the model group and the chemical group（P<0.05）.And results of IHC showed that the expression levels of IFN-γand GZMB in the middle and high dose group and the positive control group were significantly higher than that in the model group and the chemical group（P<0.05）.Collectively,all results supported the fact that anidulafungin had the significant anti-tumor effect,and was able to activate the tumor immunosuppressive activities.In this study,the pharmacophore model and molecular docking technology were used to screen novel PD-L1 small molecular weight candidate inhibitors from the traditional Chinese medicine and existing compounds,and the binding with PD-L1 as well as antitumor activity and immune responses in vitro and in vivo were evaluated,identifying anidulafungin as a novel PD-L1 small molecular weight inhibitor.Results of this study would provide a new strategy for PD-1/PD-L1 tumor immunotherapy and the development of other small molecular weight inhibitors.