| Koumine has significant development value of new drugs,but its target and action methods need to be studied.Transporter protein?18 kDa?[translocator protein?18 kDa?,TSPO]is expected to be an important drug target for the treatment of neuropsychiatric diseases and other diseases.Previous and contemporaneous research of this research group suggested that koumine may have a positive gender structure regulation effect on TSPO,but it remains to be demonstrated.In recent years,people increasingly attention from mark of interaction of biological molecules technology application,including the surface plasma resonance?surface plasmon resonance,SPR?technology as a new tag free,real-time online research of interaction of biological molecules of high sensitive sensor technology,drugs can be real-time dynamic analysis of the affinity of membrane proteins and interaction of dynamic change,has been applied in allosteric regulation research,and radioactive ligand binding assay can support each other,and make up for the latter to the limitations of isotope labeled endogenous ligand.In this paper,we first adopt molecular docking to analyze whether TSPO and koumine can be combined.On this basis,establish a SPR technology,detect the affinity of koumine combined with TSPO,explore koumine could enhance TSPO is a ligand and anthropogenic restructuring TSPO,the source of the rat restructuring the affinity of TSPO,from the perspective of chemical biology koumine further argument of TSPO allosteric regulation.The main research contents are as follows:1.Molecular docking was used to calculate the affinity of koumine,PK11195 and TSPO.Based on the molecular docking technique,the AutoDock program was used to predict TSPO protein and its ligand PK11195 affinity,and further analyzed the interaction between the two.First,using the AutoDock program to calculate TSPO and its ligand PK11195 affinity.According to the results of molecular docking technology,the ligand PK11195 and TSPO affinity 57.31?M,mainly through the van der chemical force and hydrophobic effect into the activity of the protein.The active center is PRO18,ALA19,THR20,THR21,GLY22,ALA23,LEU25,LYS26,PRO27,ARG43,TRP44,PHE46,PRO47,TRP50,THR51,THR54,PHE92 and other amino acids and PK11195interactions.Then,the affinity of the koumine and TSPO to the TSPO was determined by the molecular docking technique.According to the results of molecular docking technology,the koumine and TSPO affinity 25.34?M,mainly through the van der chemical force and hydrophobic effect into the activity of the protein.The active center is GLY22,ALA23,LEU24,LEU25,LYS26,PRO27,ARG43,TRP44,PHE46,PRO47,TRP50,PHE92 and other amino acids and koumine interactions.2.The affinity of human recombinant TSPO protein with the koumine.2.1 The affinity of Koumine and TSPO orthotopic ligands with human source recombinant TSPO.The surface plasmon resonance technology was used to determine the influence of the koumine element on the affinity of TSPO and its orthotopic ligand.The value of KD which the combination PK111195 with TSPO was 199.77±0.12?M.The value of KD which the combination Ro5-4864with TSPO was 176.93±2.70?M.The value of KD which the combination PPIX with TSPO was 156.93±5.50?M.The value of KD which the combination koumine with TSPO was 155.33±11.0?M.2.2 The effect of the method to determine the affinity of TSPO and its orthotopic ligand.When the running buffer containing 10-11,10-10,10-9,10-8,10-7,10-6,10-5,10-4,10-33 M koumine,the value is 20.0?M PK11195?17.7?M Ro5-4864?15.7?M PPIX flow through protein fixed channel and reference channel response signal.The koumine concentration of the buffer solution is 10-5M,the response value of the interaction between PK11195 and TSPO reaches the maximum value;The koumine concentration of the buffer solution is 10-77 M,the response value of the interaction between Ro5-4864and TSPO reaches the maximum value;The koumine concentration of the buffer solution is 10-66 M,the response value of the interaction between PPIX and TSPO reaches the maximum value.Multidynamic cycle detection was used to run buffer containing 10-55 M koumine measured the value of KD which the combination koumine with TSPO was 95.27±0.62?M.Koumine can significantly enhance the affinity between PK11195 and human TSPO,which is statistically significant?P<0.001?.Multidynamic cycle detection was used to run buffer containing 10-77 M koumine measured the value of KD which the combination Ro5-4864with TSPO was 77.73±0.87?M.Koumine can significantly enhance the affinity between Ro5-4864 and human TSPO,which is statistically significant?P<0.001?.Multidynamic cycle detection was used to run buffer containing10-66 M koumine measured the value of KD which the combination Ro5-4864with TSPO was 66.07±3.32?M.Koumine can significantly enhance the affinity between PPIX and human TSPO,which is statistically significant?P<0.001?.3.The affinity of rat source recombinant TSPO protein with the koumine.3.1 The affinity of Koumine and TSPO orthotopic ligands with rat source recombinant TSPO.The recombinant TSPO was obtained through the prokaryotic expression,via Ni2+affinity chromatographic column purified protein was preliminarily characterized by Western blot.The recombinant mTSPO was captured with His label on the CM5chip.The value of KD which the combination koumine with mTSPO was 97.37±1.76?M.The value of KD which the combination PK111195 with TSPO was 161.33±6.1?M.The value of KD which the combination Ro5-4864with TSPO was 169.75±6.07?M.The value of KD which the combination PPIX with TSPO was 95.85±4.36?M.3.2 The effect of the method to determine the affinity of TSPO and its orthotopic ligand.When the running buffer containing 10-11,10-10,10-9,10-8,10-7,10-6,10-5,10-4,10-3M koumine,the value is 16.1?M PK11195?17.0?M Ro5-4864?9.6?M PPIX flow through protein fixed channel and reference channel response signal.The koumine concentration of the buffer solution is 10-77 M,the response value of the interaction between PK11195 and TSPO reaches the maximum value;The koumine concentration of the buffer solution is 10-66 M,the response value of the interaction between Ro5-4864and TSPO reaches the maximum value;The koumine concentration of the buffer solution is 10-77 M,the response value of the interaction between PPIX and TSPO reaches the maximum value.Multidynamic cycle detection was used to run buffer containing 10-77 M koumine measured the value of KD which the combination koumine with TSPO was 95.32±1.80?M.Koumine can significantly enhance the affinity between PK11195 and human TSPO,which is statistically significant?P<0.001?.Multidynamic cycle detection was used to run buffer containing 10-77 M koumine measured the value of KD which the combination Ro5-4864 with TSPO was 67.68±11.09?M.Koumine can significantly enhance the affinity between Ro5-4864 and human TSPO,which is statistically significant?P<0.001?.Multidynamic cycle detection was used to run buffer containing10-66 M koumine measured the value of KD which the combination Ro5-4864with TSPO was 39.87±3.87?M.Koumine can significantly enhance the affinity between PPIX and human TSPO,which is statistically significant?P<0.001?.Combining relevant literature,we first used SPR technology to simulate the biofilm environment using chip surface reconstruction methods to further optimize TSPO protein activity.Single-cycle kinetics was used to study the affinity of PK11195and koumine to the rat TSPO recombinant protein.The KD value of PK11195 and mouse TSPO recombinant protein was measured to be 8.04±1.42 nM,and the KD value of the recombinant protein between klematerin and mouse TSPO was detected to be0.61±0.11 nM.Later work will determine the effect of konjac hormone on the affinity of TSPO anterograde ligand and TSPO.The results of this study suggest that the SPR technique can be used to detect the affinity of koumine and TSPO in combination with TSPO,and analyze the influence of koumine on the affinity of TSPO and TSPO protein.Koumine can significantly enhance the affinity of TSPO orthotopic PK11195,Ro5-4864 or PPIX and TSPO,suggesting that koumine has a positive gender structure adjustment for TSPO. |
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