Discovery Of Foot-and-Mouth Disease Virus 3C Protease Inhibitors And Molecular Mechanism Of The Effect Of Mutation On Activity

Foot-and-mouth disease(FMD)is a kind of disease that can be widely circulated in pigs,cattle,sheep and many wild cloven-hoofed animals.The outbreak of foot and mouth disease will cause great economic losses.At present,the only response to foot-and-mouth disease is to slaughter and bury infected and suspected animals,and mass vaccinations to block the recurrence of foot-and-mouth disease.Foot-and-mouth disease is caused by the foot-and-mouth disease virus(FMDV).The complete capsid protein of FMDV requires the cleavage of the FMDV non-structural protein 3C protease(3Cpro).Based on the key role of 3C protease in the life cycle of foot-and-mouth disease virus,it is considered to be an important target for the discovery of a very potential anti-foot-and-mouth disease virus drug.Up to now,there are still no marketed drugs for FMDV 3C protease,and it is necessary to find inhibitors with novel framework and strong inhibitory ability.In addition,production of the new foot-and-mouth disease vaccine requires 3Cpro to cleft the structural protein P1 into capsid proteins to produce non-infectious virus-like particles,but the damage to host cells of wild-type FMDV 3Cpro limits its large-scale production and development.Studies have shown that mutations at the I22,V124 and L127 sites of 3C protease can effectively reduce the damage to the host and increase the production of capsid proteins,but the relevant molecular mechanisms are still unclear.Understanding these mechanisms could lead to more effective development of novel FMDV vaccines with 3C protease.Therefore,combined with these problems,this paper mainly carried out the following work.In the first chapter of this paper,we summarized the current situation of FMD,epidemic prevention strategies and the structure of foot-and-mouth disease virus,focusing on the structure,biological functions,and research progress of related inhibitors and vaccines about 3C protease.Then,the virtual screening,molecular dynamics simulation and some trajectory analysis methods are introduced.In the second chapter,FMDV 3C protease inhibitors were found by virtual screening method combined with biological activity evaluation.Virtual screening was performed using the Glide software of semi-flexible docking in Schr?dinger.After three precision step-by step screening,17 compounds were selected from Chemdiv,Specs and other databases for virus activity inhibition experiments.Five compounds with antiviral activities were detected by real-time PCR.Two small molecules with novel structures,C1 and C5,were selected and 39 compounds with similar structures were obtained by similarity search on the Sci Finder platform.In the third chapter we used conventional molecular dynamics simulation methods to study the interaction mechanisms between compounds C1 and C5 with FMDV 3C protease obtained by virtual screening.Equilibrium analysis,hydrogen bond analysis and other methods summarized the interaction mode of compounds C1 and C5 with 3C protease.The results show that these inhibitors bind to the hydrophobic cavity formed by the catalytic triplet Cys-Asp-His and ?-barrel,which occupy the substrate binding pocket,and can form ?-? stacking interaction with His46 and hydrogen bond interaction with Ile30,Gly161 and other residues.These results can provide important theoretical guidance for the further design of effective FMDV 3C protease inhibitors.In the fourth chapter of this article,we used accelerated molecular dynamics(AMD)to study the molecular mechanisms of mutations such as I22,V124,L127 and others that affect the activity of FMDV 3C protease,and explore the conformational changes of 3Cpro caused by the mutations.Through molecular dynamics simulation,we found that the wild-type 3C protease had greater conformational flexibility and the mutation enabled 3Cpro to recognize and cleavage substrates through more stable hydrogen bonds.At the same time,we found that the lack of hydrogen bonds between C163 and Gln211 in the I22P/L127 P double mutation system and the reduced contact probability with ?-ribbon made 3Cpro almost unable to cleave the substrate P1.In summary,five FMDV 3C protease inhibitors with high activity were found based on the virtual screening method of molecular docking combined with the biological activity evaluation method.The interaction mechanisms between small molecule inhibitors C1 and C5 with FMDV 3Cpro were further analyzed by molecular dynamics simulation method.Secondly,we revealed the molecular mechanisms of the effects of 3Cpro mutation on its own activity based on accelerated molecular dynamics simulation.The obtained results can not only provide candidate compounds and important theoretical guidance for future drug discovery of FMDV 3Cpro inhibitors,but also provide a possible direction for the production of novel vaccines based on 3C protease.