Study On The Dissociation Mechanism Of Acid-induced Capsid And The Discovery Of Novel 3D Polymerase Inhibitors In FMDV

Foot-and-mouth disease is a highly contagious viral disease among the cloven hoofed animals.The outbreak of foot-and-mouth disease can cause huge economic losses.When it happens,vaccination is the main control strategy.However,the acid sensitivity of foot-and-mouth disease virus capsid has a very adverse effect on the preparation and transportation of traditional inactivated vaccines and recombinant vaccines.It requires expensive cold chain and frequent inoculation to achieve effective protection of vaccine integrity.Therefore,it has great theoretical value to study the molecular mechanism of capsid protein cracking under acidic conditions.There is no universal vaccine for foot-and-mouth disease and emergency vaccination requires at least 7 days to trigger an effective immune response.Therefore the vaccination is more likely the prevention strategy for the uninfected animals and slaughtering is almost the only way for the infected animals in fact.In order to prevent infected animals from being killed,it is very important to develop the new FMDV inhibitor.3D polymerase is an RNA-dependent RNA polymerase of FMDV,which is the key enzyme that copies the genes in the process of virus replication.Therefore,it is an important target for the development of anti-FMDV drugs.Most drugs targeting 3D polymerase are nucleotide analogues,which are broad-spectrum mutagenic antiviral drugs with very strong toxicity.Therefore,the discovery of lead compounds that selectively target 3D polymerases is of great significance for the treatment of foot-and-mouth disease.In the first part of this paper,a brief overview of the outbreak of foot-and-mouth disease and its prevention and control strategy were firstly summarized.Then,the structural basis of FMDV capsid protein and its 3D polmerase are introduced.And the research progress of the acid stability study on FMDV capsid protein and the discovery of inhibitors targeting FMDV 3D polmerase are also reviewed.In the end,the main research methods,including molecular dynamics simulation and virtual screening,as well as the development of their applications are described.The second part focuses on the molecular mechanism of FMDV capsid's acid sensitivity.Through 200 ns conventional molecular dynamics simulation of two protomers under the condition of pH=6 and pH=7.4,we found that protein VP2 and VP3 interface under the acidic condition has a large conformational change.The hot residues,which have an important influence on the capsid protein stability,are identified by residue free energy decomposition and virtual alanine scanning mutagenesis.The obtained result indicates that residue H3141,H3144,and H2021 were closely associated with pH conditions change and turned to be the hotspot residues on the interface.Then the molecular mechanism of the release of capsid protein VP2 and VP3 in acidic conditions is further analyzed by performing residue interaction networks analysis and the specific interaction analysis.The result suggested that protonated H3141 resulted in electrostatic repulsion between H3141 and K2088,which led to the dissociation of the ?-helix region in the adjacent VP2 and VP3 center.The protonation of H3191 resulted in the dissociation of VP2 and VP3 edges.Meanwhile,it can be predicted from the results that the protonation of H3144 would result in the dissociation of the ?-sheet region,which were VP2 and VP3 core region.The study on the molecular mechanism of FMDV capsid's acid sensitivity at the molecular level has important theoretical guiding value to design the acid vaccine of foot-and-mouth disease.The third part is based on the discovery of the second part,which mainly focuses on the molecular mechanism of dissociation of virus capsid induced by protonation of H3144 on protein VP3 under acidic condition.The molecular mechanism of FMDV capsid protein acidic sensitivity is further explored by molecular dynamics simulation method.It's found that the protonated H3144 on the protein VP3 under the acidic condition promoted the further dissociation of the core ?-sheet region in VP2 and VP3 by exploiting molecular dynamics simulation of 500 ns.The results of binding mode and residue interaction network analysis showed that the electrostatic repulsion between the protonated H3144 and the positively charged K2063 on the adjacent pentamer increases the distance of ?-sheet that contains H3144 and ?-sheet that contains K2063.As a result,the increased distance between H3144 and K2063 caused dissociation of protein VP2 and VP3 further.In the fourth part,the virtual screening of the non-competitive inhibitors targeting FMDV 3D polymerase is studied.Firstly,molecular dynamics simulation of 50 ns was used to obtain relatively rational crystal structure conformation of protein-ligand complex.The molecular docking based virtual screening was performed on five compound databases in order to discover novel non-competitive inhibitors of 3D polymerase.Based on the evaluation of MM-GBSA,cluster analysis and the binding model analysis,65 candidate compounds with structural diversity were selected.Then 34 candidate compounds was selected for next virus inhibition experiments,and the results showed that 6 candidate compounds have good activity against 3D polymerase of FMDV.In this paper,the molecular mechanism of capsid protein dissociation under acidic condition is theoretically instructive for design of acid fast vaccine on foot and mouth disease.The non-competitive inhibitors of FMDV 3D polymerase have been found through molecular docking based virtual screening,which has important theoretical value for the design and development of drugs targeting FMDV 3D polymerase.In summary,the results of this study provided an important guidance for the development of better foot-and-mouth disease vaccines and drugs.