Discovery Of Novel Natural Agonists Targeting Constitutive Androstane Receptor CAR Based On Virtual Screening And Biological Evaluation

Cholestasis is one of the most common clinical symptoms of liver disease.At present,there is a lack of drugs for the treatment of cholestasis.Constitutive androstane receptor（CAR）is an important regulator of hepatic bilirubin synthesis and transport,and plays an important role in the pathogenesis of cholestasis and jaundice.Therefore,it has become a potential drug target for the treatment of cholestasis.In this study,taking CAR as the target and compounds of traditional Chinese medicine as the research object,based on computer-aided drug discovery combined with experimental technology,the layer-by-layer screening and evaluation strategy of"in silico→in vitro→in vivo"was adopted to find novel natural compounds which are safe and effective and have"anti-jaundice"effect in the treatment of cholestasis.Methods（1）Virtual screening technique based on molecular docking method to find potential natural compounds with potential active activity to hCARThe crystal structure of hCAR is derived from PDB database（PDB code:1XVP）,and the compound molecules are derived from traditional Chinese medicine database（TCM Database）and natural product database（DNP Natural product）.The Schr?dinger software are used to pretreat the molecular structure of proteins and compounds,respectively.The position of CITCO in the crystal structure is used to define the docking lattice position,and calculate the lattice file.The known agonist CICTO is docked to the active site of hCAR,to verify the reliability of the docking method.Then layer-by-layer progressive screening strategy is used,that is,according to the accuracy of HTVS→SP→XP,and the molecules with the top 10%docking score are selected in each step;finally,the ADME/Tox properties of the selected compounds are predicted,and those compounds that bind well to hCAR are determined as candidate compounds for the next experiments.（2）To investigate the toxicity of candidate compounds and their activity of hCAR at the cellular levelThe cytotoxicity of the candidate compounds at the cell level in vitro were investigated by 24 h and 72 h MTT experiments.hCAR1 overexpression plasmids,CYP2B6 luciferase reporter gene plasmids and Renilla control plasmids were constructed and transfected into Hep G2 cells to obtain Hep G2-CYP2B6-hCAR1 cells.After transfection,10,20,40,80 and 100μmol/L compounds,5μmol/L PK11195,1μmol/L positive drug CITCO and control（DMSO）were added to the transfected cells.Finally,the luciferase activity was detected and the activation activity of the purchased compounds on hCAR was investigated.（3）To explore the effect of target compounds on ANIT-induced cholestasis model miceMale C57BL/6J mice were randomly divided into control group,model group,positive drug OCA group,positive drug TCPOBOP group,#004（L）（1 mg/kg）,#004（M）（5mg/kg）,#004（H）（10 mg/kg）,#006（L）（1 mg/kg）,#006（M）（5mg/kg）,#006（H）（10mg/kg）,#014（L）（1 mg/kg）,#014（M）（5mg/kg）,#014（H）（10 mg/kg）,with 8 mice in each group.The experimental period was one week.On the fifth day,the mice in the experimental group were given ANIT（75mg/kg）soluble in olive oil.Two hours after the last administration,the eyeball was removed,the blood was taken and the liver tissue was collected.ALT,AST,TBA,TBIL,DBIL,TC,TG and total bile acid were detected.The transcriptional levels of Car,Cyp2b6,Ugt1a1,Ntcp and Mrp2 m RNA in mouse liver were measured by Q-PCR technique.The pathological changes of liver tissue were detected by HE staining.（4）Study on the effect of candidate compounds on bilirubin level in vitroUsing the hyperbilirubinemia cell model previously established in our group,after the intervention of 10μmol/L,20μmol/L,40μmol/L,80μmol/L and 100μmol/L,the anti-yellowing effect of the target compound was evaluated by detecting TBIL and DBIL.Finally,Q-PCR method was used to detect the changes of CAR,CYP2B6 and bilirubin metabolizing enzyme m RNA in hyperbilirubin Hep G2 cells.Results（1）Screening of natural compounds with agonistic activity on hCAR based on molecular docking technology.The verification results of the docking method show that the RMSD value of the conformation obtained by docking and the conformation in the crystal structure of the complex is 0.224?,indicating that the Glide docking method can well reproduce the binding mode between agonist molecules and hCAR.The molecules from the prepared compound library were docked into the active pocket of hCAR,and the accurate docking of HTVS→SP→XP was used in turn.Because of the high docking score of compounds#006 and#014,we chose these two compounds as representatives.The hydrophobic bases formed by#006-hCAR complex,#014-hCAR complex and CITCO-hCAR complex are the same,indicating that compounds#006,#014 and CITCO are bound in the same hydrophobic pocket position of hCAR.（2）The toxicity and activation activity of candidate compounds to hCAR were investigated in vitro:the results of MTT assay showed that the cell survival rate decreased after Hep G2 cells were treated with compound#012 at the concentration of0～100μmol/L for 24 hours,and the calculated value of IC₅₀ was 82.1±2.27μmol/L.Compound#012 was excluded from luciferase reporter gene assay because of its cytotoxicity.The survival rate of Hep G2 cells did not change significantly after the other candidate compounds were treated for 24 hours.Luciferase activity results showed that among the remaining 17 candidate compounds,#006 was considered to be a strong agonist of hCAR,and its activity exceeded 40%of the CITCO-mediated activation activity,reaching a maximum activation value of 42.7%.Compounds#004and#014 were defined as moderate hCAR activators,accounting for 15%and 40%of CITCO-mediated hCAR activation,reaching the maximum activation values of 34.1%and 31.7%.Therefore,compounds#004、#006 and#014 were used as target compounds for follow-up in vivo experiments.（3）To explore the effect of target compounds on ANIT-induced cholestasis model mice:the results of body weight changes showed that from the 5th day to the 7th day,the body weight of mice in the model group decreased significantly,but there was no significant change after the intervention of TCPOBOP,OCA,compound#004（L,M,H）,compound#006（L,M,H）and compound#014（L,M）.The serological results showed that the activities of liver function-related enzymes and the levels of TBIL and TBA in the model group were significantly higher than those in the model group（P<0.001）,while the liver enzyme activity and the levels of TBIL and TBA in the positive drug group（OCA and TCPOBOP）were significantly lower than those in the model group（P<0.001）.Compared with the model group,the activities of liver function-related enzymes and the levels of TBIL and TBA in the three dose groups（L,M,H）and#006（H）of the target compounds#004 and#014 decreased significantly（P<0.001）.The histopathological results of the liver of mice showed that compared with the normal group,there were more inflammatory cells infiltration,uneven size and disordered arrangement of hepatocytes in the model group.After administration of positive drugs,the size of hepatocytes was uniform and the nucleus was clearly visible;after the intervention of#004（H）,#006（H）and#014（H）,the hepatocytes of mice were the same in size,close to the normal group in shape and structure,and arranged radially and neatly.The results of Q-PCR test showed that compared with the normal group,the transcription level of Oatp1b2 m RNA in the liver tissue of the model group was significantly increased,while the transcription levels of Mrp2,Ugt1a1,Ntcp and Sult2a1 m RNA were significantly decreased（P<0.01）.In the positive drug OCA and TCPOBOP groups,the gene transcription levels of Sult2a1,Ntcp,Ugt1a1 and Mrp2were significantly increased,while the gene Oatp1b2 transcription was significantly decreased（P<0.01）.Compound#004（L,M,H）significantly increased the transcriptional levels of Sult2a1,Ntcp,Ugt1a1 and Mrp2 genes in liver tissues,while decreased Oatp1b2 transcriptional levels.Compound#006（M,H）increased the transcriptional levels of Sult2a1,Ntcp,Ugt1a1 and Mrp2 genes in liver tissue,while decreased the transcriptional level of Oatp1b2 gene.The gene transcription levels of Sult2a1,Ntcp,Ugt1a1 and Mrp2 in liver tissue of compound#014（L,M,H）mice were significantly increased,while the gene Oatp1b2 transcription level was significantly decreased.（4）Study on the effect of candidate compounds on bilirubin level in vitro:The results of intracellular bilirubin assay showed that the levels of intracellular TBIL and DBIL in the model group were significantly higher than those in the model group.Compared with the model group,the intracellular TBIL and DBIL contents of target compounds#004,#006 and#014 were significantly decreased when the administration concentration was 20μmol/L.At the concentration of 20μmol/L,40μmol/L,80μmol/L and 100μmol/L,the level of TBIL was significantly decreased in a dose-dependent manner.The results of Q-PCR assay showed that positive drug CITCO could significantly induce the expression of UGT1A1,CYP3A4 and CYP2C9 genes.The expressions of UGT1A1 genes in Hep G2 cells treated with compound#004,#006 and#014 were significantly higher than those in model group（P<0.01）.Conclusions（1）Through the virtual screening technique based on molecular docking method,withCAR as the target,compounds with potential activation of hCAR were found from the library of traditional Chinese medicine monomers and natural compounds.（2）The activity and toxicity of the selected small molecules were investigated in Hep G2 cells.Finally,it was found that compounds#004,#006 and#014 had strong activity to activate hCAR.The experimental results showed that compounds#004,#006and#014 were potential agonists of hCAR.（3）The pharmacodynamics of candidate compounds#004 and#014 were evaluated by intrahepatic cholestasis mouse model.The results showed that the candidate compounds could significantly improve the serological indexes and pathology of mice.After the intervention of compounds#004,#006 and#014,the transcriptional level of bilirubin reabsorption transporter gene in the liver of mice decreased,but the transcription level of genes related to metabolism and efflux increased,indicating that these three compounds can alleviate cholestasis.（4）The hyperbilirubin cell model was used to further explore the mechanism of three compounds as potential agonists of hCAR to regulate bilirubin levels.The results showed that compound#004 and#014 were effective in reducing the level of bilirubin in the model,and there was no cytotoxicity,and the results of Q-PCR experiment showed that the candidate compounds might increase the expression of bilirubin metabolism genes,thus reducing the accumulation of bilirubin in cells.