Studies On The Biochemical Properties Of Human Programmed Death-Ligant 1(PD-L1) And The Discovery Of Small Molecular Active Compounds Targeting PD-L1

Objective:PD-L1,a human programmed death-Ligand 1（hPD-L1）,interacts with its receptor PD-1 to form a co-stimulatory signal pathway that transmits negative regulatory signals to regulate the activity of T lymphocyte.Studies have shown that this signal pathway is closely related to the occurrence of a variety of tumors.The aim of this thesis is to obtain a large number of homogeneous;stable hPD-L1 proteins and to determine their biochemical properties;to study their interaction with hPD-1proteins and to screen the small molecular active compounds targeting hPD-L1.Methods:The recombinant strain hPD-L1^19-238was successfully constructed by molecular cloning and expressed in large quantities.SDS-PAGE and Western blot were used to detect the expression.Ni-NTA Gel affinity chromatography and Gel filtration chromatography were used to purify the target protein.and CD was used to detect the secondary structure of the target protein(hPD-L1^19-238).The binding of hPD-1 Protein to hPD-L1 Protein was determined by Gel filtration chromatography,and the melting temperature（Tm）of hPD-L1 Protein,hPD-1 Protein was determined by Protein thermal shift Assay（TSA）.Determination of EC₅₀of hPD-L1 and hPD-1 by non-competitive ELISA.Using virtual screening and computer drug design,a virtual screening model was established to screen 10 million small molecules by DeepBindVec method,and finally 15 candidate compounds were obtained.The specific affinity of hPD-L1 with 15 candidate compounds was tested by TSA,and three potentially active compounds（Compound-13,Compound-14 and Compound-15）were obtained.Results:1.A cell line BL21-pET-21b-hPD-L1^19-238was successfully constructed,which was able to express hPD-L1 protein in large quantity.IPTG of 0.5 m M was induced at37°C for 6 hours.At the elution concentration of 250 m M imidazole,the hPD-L1protein was purified by Ni-NTA Gel,purified by molecular sieve and its stability was investigated.The results of CD showed that the main secondary structure of hPD-L1^19-238wasβ-sheet,the content of which was 50.4%.2.The results of Gel filtration chromatography showed that the recombinant hPD-L1 and hPD-1 could combine well.The results of TSA show that the melting temperature of hPD-L1^19-238was 52.46°C,the melting temperature of hPD-1 was53.05°C and the melting temperature of hPD-1-hPD-L1^19-238was 55.66°C,the results showed that hPD-L1^19-238could bind specifically to hPD-1.The EC₅₀of hPD-1-Biotin was 0.2065 and 0.2973,when the concentration of hPD-L1 was 1.0μg/mL and 0.5μg/mL,respectively.3.A virtual screening model was established based on hPD-L1-hPD-1 Complex Structure（PDB code:4ZQK）,and 10 million small molecules were screened by DeepBindVec method（patent acceptance number:201910117693.5）,more than 1000small molecules with a binding probability of 0.99 were selected for further screening,and Vina docking was performed for these more than 1000 candidate small molecules.In the first screening of 1000 small molecules,combined with Moe flexible docking and Vina molecular docking.In the screening of 1,000 small molecules,combining the results of MOE flexible docking and Vina small molecule docking experiments and visual experience（such as the number of hydrogen bonds）,three compounds with potential binding ability were found:Compound-13 interacts with Ile54,Met115,and Ala121 and Ser117 in hPD-L1 through hydrogen bonds and Hydrophobe,compound14 interacts with Tyr56,Met115 and Ala121 and Ser117 in hPD-L1 through hydrogen bonds,hydrophobicity,and-conjugation;Compound-15 interacts with Ala52,Ile54,Val68,His69 and Gly119 of hPD-L1 by hydrophobic interaction,-conjugation and hydrogen bonding.4.The in vitro binding ability of Compound-13,Compound-14 and Compound-15 to hPD-L1 was tested by TSA.When the concentrations of Compound-13,Compound-14 and Compound-15 were 135μM,the concentrations of Compound-13,Compound-14 and Compound-15 were-2.77°C,-3.61°C and-3.86°C,respectively.The effect of these three compounds on the binding pathway of hPD-1-hPD-1 was investigated.The results showed that the ability of blocking hPD-1binding was Compound-15,Compound-13 and Compound-14,respectively.Conclusion:In this thesis,the hPD-L1^19-238protein was successfully expressed and purified,and its conformation was homogeneous and stable.The results of circular dichroism showed that the secondary structure of hPD-L1^19-238wasβ-sheet,which accounted for 50.4%.And the results of molecular sieves,TSA and ELISA showed that hPD-L1 could bind specifically to hPD-1.The virtual docking model was built with hPD-L1 Protein Structure（PDB code:3BIS）,and the results of Vina docking of three potential active compounds were demonstrated.The results of TSA showed that these three compounds could bind to hPD-L1 protein specifically.