Docking-based Virtual Screening And Bioassay Of Brucella Abortus Threonyl-tRNA Synthetase Inhibitors With Novel Skeleton

In this research,3D structure of Brucella abortus Threonyl-tRNA Synthetase(ThrRS)(BaThrRS)was built using homology modeling and molecular dynamics simulation.Then BaThrRS was used as target for docking-based virtual screening,and potential compounds of BaThrRS novel inhibitors were obtained.Finally,activaties of all the potential compounds were tested using experiment.This research included three parts as follows:1.Obtain of BaThrRS 3D structure3D structure of the target was required for docking-based virtual screening,and the 3D structure of BaThrRS has not been analysed.In this study,amino acid sequence of BaThrRS was obtained from Protein database in NCBI web server.Then the protein whose amino acid sequence has high similarity with that of BaThrRS was obtained using blast tool in NCBI web server.And Modeller 9.16 was used to build initial 3D structure of BaThrRS.Moreover,model quality was assessed using MolProbity 4.3,and molecular dynamics simulation for 20 ns was used to refine the model.All the results showed that Escherichia coli ThrRS(EThrRS)and BaThrRS share 51%sequence identity,sufficient to construct a reliable model.The structure of EThrRS was used as template.Before optimization,95.1%(623/655)of all residues were in favored regions,98.9%(648/655)were in allowed regions,and 1.07%were in disallowed regions.The ERRAT score was 76.425.Verify3D revealed that 90.27%of the residues had average 3D-1D scores.After refinement of the model,92.2%(604/655)of all residues were in favored regions,99.2%(650/655)were in allowed regions,and 0.76%were in disallowed regions.The ERRAT score was 85.440.Verify3D revealed that 93.76%of the residues had average 3D-1D scores.After optimization,the overall quality factors were increased and the error values were decreased by satisfying special constraints.The final model could be used for virtual screening.2.Virtual screening of potential BaThrRS novel inhibitorsActive center(binding site)was required for docking-based virtual screening.In this study,ATP was docked to BaThrRS to identify the amino acid residues around ATP,which make up the active center.Then borrelidin was docked to BaThrRS to confirm the binding site of the selective inhibitor of ThrRS and binding energy.All the results showed that the active center of BaThrRS could be bonded by inhibitors and could be used for docking-based virtual screening.The binding free energy of borrelidin was-9.7 kcal/mol which was greater than that of ATP(-9.8 kcal/mol).It means that the compounds with binding free energy less than-9.7 kcal/mol should be selected in virtual screening.Finally,99 compounds with binding free energy less than-11 kcal/mol were selected using virtual screening.3.Identification of BaThrRS novel inhibitorsPotential compounds selected by previous virtual screening were tested using plate experiments and MIC analysis.All the results showed that 4 compounds in 42 potential compounds could inhibit Brucella abortus.It indicated that the effective rate of inhibitors identification using the method in this research was about 10%.And MICs of E1 and F4 were 1 mg/mL and 0.5 mg/mL,respectively.The research should facilitate future experimental effects to find novel drugs for use against bovine brucellosis.