| Tuberculosis(TB)is an infectious disease that has plagued mankind for thousands of years and its causative bacterium is Mycobacterium tuberculosis(MTB).Due to the long treatment period of TB,MTB has developed different level of drug resistance to both first-line and second-line anti-TB drugs.The multidrug-resistant tuberculosis(MDR-TB)and extensively drug-resistant tuberculosis(XDR-TB)are the biggest obstacles in TB treatment,which also continues to threaten public health and safety.Therefore,exploring the interaction mechanism between MTB-related targets and their drugs or inhibitors can not only obtain drug resistance mechanisms at the molecular level,but also provide theoretical basis for the design of new anti-TB drugs.In recent years,with the rapid development of computer technology,Computer Aided Drug Design(CADD)has gradually matured.Molecular dynamic(MD)simulation can not only explore the drug resistance mechanism caused by target mutation at the molecular level,but also explore the binding and dissociation process of drugs.These studies can provide important theoretical foundations for drug engineering and molecular design,which cannot be obtained by traditional experiments.In this thesis,we explored the interaction of MTB-related targets and their inhibitors through different MD simulation methods and binding free energy calculation,dynamic network analysis,hydrogen bond analysis and other trajectory analysis methods.This thesis mainly includes the following four aspects of work:First of all,we explored the molecular mechanism of rifampicin resistance caused by S456 L,D441V and H451 Y mutations in MTB RNA polymerase by Gaussian accelerated MD simulation.The results showed that the binding affinity of rifampicin was reduced in the three mutants.In addition,the disappearance of the hydrogen bond between rifampicin and R454 residue also led to a significant reduction in the energy contribution of the R454.Binding mode analysis revealed that the conformation of the R454 residue was significantly changed in the mutants.Further dynamic network analysis showed that the R454 residue could form interactions with residues at the three mutant sites in the wild-type system,but these interactions were weakened or even disappeared in the mutant systems.Therefore,the flexibility of the R454 residue was increased,eventually leading to its conformational changed and breaking the hydrogen bond between it and rifampicin.Based on the analysis results,we speculated that enhancing the interaction between the R454 residue and rifampicin may overcome the resistance caused by the mutations.Then,we combined thermodynamic integration(TI)and conventional MD simulations to explore the cross-resistance mechanism of isoniazid(INH)and ethionamide(ETH)caused by the S94A mutation of InhA.TI simulation as well as MM/GBSA calculation suggested that the S94A mutation in the InhA target reduced the binding affinity of INH-NAD and ETH-NAD.In addition,the S94A mutation directly led to the disappearance of the hydrogen bond between it and INH-NAD and ETH-NAD,reducing the energy contribution of this residue.In the INH-NAD system,the S94A mutation also resulted in the disruption of the hydrogen bonds between Thr196,Leu197 and INH-NAD,resulting in a significant reduction in their energy contributions.Dynamic network analysis revealed that the S94A mutation weakened its interaction strength with surrounding residues and increased pocket flexibility.In addition,in the mutants,the adenine group of INH-NAD and the pyrophosphate region of ETH-NAD moved towards the outside of the binding pocket,resulting in their reduced binding capacity.Later,we investigated the binding mode of InhA to its direct inhibitors TCL,PT70,PT91,PT119,PT501 and PT506 by means of conventional MD simulations.Residue energy decomposition showed that hydrophobic residues such as Phe149,Met155,Tyr158,and Met161 had higher energy contributions to the binding of InhA direct inhibitors.In addition,Tyr158 can not only form pi-pi stacking interaction with the benzene ring of TCL and PT501,but also form hydrogen bonding interaction with the hydroxyl group on PT70 and PT91 molecules.Therefore,the interaction between Tyr158 and the molecule is one of the key points in inhibitor design.Compared with PT70 with the highest binding affinity,the α-helix of the substrate binding loop(SBL)regions in the TCL,PT91 and PT501 systems were converted to the loops,thereby increasing the flexibility of the binding pocket and reducing the molecular binding affinity.In addition,the SBL and H7 in the PT119 and PT506 underwent a large positional shift,resulting in the exposure of the ligands to the solvent,which also reduced their binding stability.Finally,the dissociation pathways and residence times of direct inhibitors of InhA were predicted by Tau random acceleration MD(τRAMD)simulations.The results showed three possible dissociation pathways for the six inhibitors.The two main dissociation channels were path1 which dissociated along the H7 direction and path2 which dissociated along the H6 direction.Among them,TCL could dissociate from two dissociation channels,while PT70,PT91 and PT501 mainly dissociated along path1,and PT119 and PT506 mainly dissociated along path2.Furthermore,the order of the residence times of the inhibitors predicted by τRAMD simulation method was consistent with the experimental order.Then,we used Steered MD to explore the key intermediate states in the dissociation process of these six inhibitors.The results showed that some hydrogen bonds and van der Waals interactions between these inhibitors and InhA were the main obstacles in the dissociation process.Collectively,the above four research works have investigated the resistance mechanisms of rifampicin and isoniazid at the molecular level,as well as the thermodynamic and kinetic information of the binding of InhA targets to their direct inhibitors,which is helpful for understanding drug resistance mechanism and provide imporant theoretical basis for the design of new anti-TB drugs. |
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