Screening Of TNF-α/JAK1 Dual Inhibitors Based On Molecular Simulation

Rheumatoid arthritis（RA）is an autoimmune disease that primarily causes joint damage,with a higher rate of disability and bringing a heavy burden to society and patients.Tumor necrosis factorα（TNF-α）and Janus Kinase 1（JAK1）are two clinical targets in treating rheumatoid arthritis.In this design,the molecular simulation method was used to screen TNF-α/JAK1 dual inhibitors from the compound database.The main subjects and results of the research are:1.TNF-αinhibitors screening pharmacophore construction:Three crystal complexes of TNF-αand its allosteric inhibitor were selected to analyze critical amino acids and their interactions with inhibitors via molecular dynamics simulation.Molecular dynamics clustering analysis selects the representative structures that protein-ligand stable binding during dynamics simulation to construct pharmacophore based on the structural characteristics of receptor-ligand complexes.Pharmacophore characteristics were then screened based on the results of the interactions analysis.Molecular dynamics analysis showed that the critical amino acids of TNF-αwere C:Tyr59,C:Tyr119 and C:Tyr151.The main interactions were hydrogen bonding,hydrophobic andπ-πstacking interactions.The representative structures obtained by cluster analysis were constructed pharmacophore then combined with critical interactions to screen the characteristics of the pharmacophore.Finally,three TNF-αscreening pharmacophore models were obtained.2.JAK1 inhibitors molecular docking screening:JAK1 activity inhibitor and bait set molecules were used to dock with five JAK1 protein crystal structures through two docking programs SP and XP in Schr（?）dinger.Docking scores were subjected to ROC curve analysis to analyze the selectivity and sensitivity of docking program and protein crystal models as screening tools.Finally,the SP molecular docking program was selected to conduct molecular docking screening between the compounds in the database and the crystal structure of JAK1（PDB:6BBU）,and a total of 9509 compounds were obtained.3.Dual-inhibitor screening and ADMET analysis:The compounds obtained from the JAK1 molecular docking screening were matched with 3 TNF-αpharmacophore respectively,and then 2232 compounds were screened according to the Phase screen score value.After the candidate compounds were molecularly docked with TNF-α,the compounds whose docking score with JAK1 protein was lower than-9.00 kcal/mol and the docking score with TNF-αprotein were lower than-11.00 kcal/mol were used for ADMET evaluation.Finally,a total of8 candidate compounds was obtained through the Lipinski rule and ADMET screening.4.Induced fit docking and metadynamics simulation studies:Induced fit docking was used to accurately predict the binding conformation of 8 candidate compounds to TNF-α/JAK1 protein.Before the docking experiment,molecular dynamics simulation was used as the main method to analyze the interaction between the inhibitor and TNF-α/JAK1 protein.The results showed that the critical amino acids and the interaction between TNF-αand inhibitor were consistent with the dynamic analysis results of the pharmacophore experiment.The JAK1 inhibitor had strong hydrogen interactions with the critical amino acids Glu957 and Leu959,and formed a strong hydrophobic interaction with Leu1010.Combined with the critical amino acids and interaction,the conformations produced by the induced fit and docking were screened,and the optimal conformations of 8 candidate compounds were obtained than metadynamics were simulated.After analyzing the stability of the binding conformation of 8 candidate compounds to TNF-α/JAK1 by metadynamic simulation,5candidate compounds were retained.5.Molecular dynamics simulation and binding free energy analysis:The complex of TNF-α/JAK1 and candidate compound was determined the stability of the critical interactions between the compound and the protein by molecular dynamics simulation,and the binding free energy calculation was used to determine whether the compound has potential active.Molecular dynamics simulation showed that silibinin,entecavir and compound 5 interacted with several critical amino acids of TNF-α/JAK1,and these interactions keep superior stability and durability during the dynamic simulation.At the same time,the three compounds have superior binding free energy with two target proteins.6.Analysis of anti-inflammatory activity:The anti-inflammatory activity of silibinin and entecavir was studied by constructing the RAW 264.7 inflammation model induced by LPS（Lipopolysaccharide）.Experiments show that silibinin and entecavir can significantly reduce NO expression in the inflammation model compared with the LPS group,that indicates two candidate compounds have anti-inflammatory activity.And the IC₅₀values of silibinin and entecavir are 30.35±9.8 and 49.45±60.10μM,respectively.