Theoretical Study On The Design Of Antiviral Inhibitors Based On The Infection Mechanism Of SARS-CoV-2

In 2019,the pandemic of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection has posed an unprecedented crisis to global health.The severe medical and health situation urgently demands the development of effective therapies(drugs,vaccines)to curb the rapid spread of the virus.Scientists worldwide have tirelessly devoted efforts to this cause.As of today,there has been a significant enhancement in the understanding of the biology of the novel coronavirus,demonstrating that drug development targeting key proteins in the lifecycle of the novel coronavirus is a viable therapeutic strategy.SARS-CoV-2 belongs to the family Coronaviridae,subfamily Orthocoronavirinae,and genus Betacoronavirus.It possesses the largest RNA genome among known viruses.The genome encodes 29 proteins,including structural,nonstructural,and accessory proteins.These proteins are involved in host cell entry,genome replication and transcription,and viral assembly and release.SARS-CoV-2 proteins can also exert their physiological roles individually as components of the viral replication process or interact with many host cell receptors.Over time,SARS-CoV-2 has continuously evolved into multiple variants through genome mutations,which have challenged established antiviral strategies.In this paper,molecular dynamics(MD)simulations were first performed to systematically elucidate the molecular mechanisms of the interaction between the spike(S)protein of SARS-CoV-2 and certain variants with its receptor,angiotensinconverting enzyme 2(ACE2),from structural and energetic perspectives.Subsequently,several miniprotein inhibitors targeting the S protein of SARS-CoV-2 and its Omicron variant were designed,and the antiviral mechanism of the non-covalent inhibitor WU-04 was explored.These results can provide reliable theoretical guidance for the rational design of novel antiviral drugs and vaccines at the atomic level.The main contents of this paper are summarized as follows:1.Investigation on the interaction mechanism of different SARS-CoV-2variants spike with ACE2: insights from molecular dynamics simulationsThe widespread prevalence of the COVID-19 pandemic has led to continuous genomic mutations and evolution of the SARS-CoV-2,resulting in the emergence of many new variants.In comparison to the original strain,these variants exhibit increased transmissibility and infectivity.Experimental studies have demonstrated that these mutant strains achieve enhanced infectivity by strengthening their binding affinity to host cell receptor proteins.In this work,we implemented all-atom MD simulations to study the binding and interaction of the receptor binding domain(RBD)of the SARSCoV-2 S protein singly(D614G),doubly(D614G+L452R and D614G+N501Y),triply(D614G+N501Y+E484K),and quadruply(D614G+N501Y+E484K+K417T)mutated variants with the ACE2 receptor protein in the host cell.The effects of the mutations and the differences between the individual complex systems were elucidated from multiple perspectives through a variety of analytical approaches,including the dynamic correlations,interaction patterns,collective movement of residues,free energy landscape,and charge distribution on the electrostatic potential surface between the ACE2 and all RBD variants.Meanwhile,the binding affinities of these RBD mutants with ACE2 were assessed by calculating the binding free energy between the proteins using MM/PBSA method.The results indicated that the D614G+N501Y+E484K mutant has the lowest binding free energy(highest affinity)compared to D614G+N501Y+E484K+K417T,D614G+L452R,D614G+N501Y,and D614 G mutants.The residue-based energy decomposition also showed that the energy contribution of residues at the mutation site to the total binding energy was highly variable.The interaction mechanisms between the different RBD variants and ACE2 elucidated in this study will provide important guidance for the development of drugs targeting the new SARS-CoV-2 variants.2.Computational design of miniprotein inhibitors targeting SARS-CoV-2spike proteinThe first step of SARS-CoV-2 virus invasion into the human body involves the interaction between the receptor binding domain(RBD)of the S protein and the peptidase domain(PD)of ACE2,followed by entry into host cells.Therefore,blocking the binding of RBD and ACE2 is a promising strategy to inhibit the invasion and infection of the virus in the host cell.In the study,we designed several miniprotein inhibitors against SARS-CoV-2 by single-,double-,and triple-point mutant based on the initial inhibitor LCB3.Molecular dynamics(MD)simulations and trajectory analysis were performed for an in-depth exploration of the structural stability,dominant motions of protein residues,dynamic correlations,and contributions of per-residue to the overall affinity involved in the interaction between inhibitors and RBD protein.The results showed that the inhibitors can effectively bind to the RBD region of the S protein,resulting in the formation of the stable complexes.These inhibitors displayed low binding free energy in the MM/PBSA calculations,substantiating their strong interaction with RBD.Among them,the miniprotein H6Y-M7L-L17 F was an optimal inhibitor with the highest binding affinity to provide highly stable inhibition of SARSCoV-2 RBD.Following H6Y-M7L-L17 F mutant,the inhibitors with strong binding activity are successively H6Y-L17 F,L17F,H6 Y,and F30 Y mutants.Our study demonstrated that miniprotein inhibitors maintain highly stable blocking(or binding)action on SARS-CoV-2 while preserving their secondary structure.The research proposed new miniprotein inhibitors with enhanced affinity for the S protein,providing novel insights and theoretical guidance for the rational design of new SARS-CoV-2 S protein inhibitors.3.Computational design of miniprotein inhibitors targeting SARS-CoV-2variant Omicron spike proteinOmicron is a novel variant of SARS-CoV-2 that is currently spreading globally as the dominant strain.The virus first enters the host cell through the RBD of the S protein by interacting with the ACE2.Thus,the RBD region remains an ideal target for drug design against the SARS-CoV-2 Omicron variant.In this study,we designed several miniprotein inhibitors in silico to combat the SARS-CoV-2 Omicron variant using single-and double-point mutation approaches,based on the structure of the initial inhibitor AHB2.Also,two parallel MD simulations were performed for each system to validate the reproducibility of the computational results,and the binding free energy was evaluated with the MM/PBSA method.The evaluated values showed that all inhibitors,including AHB2,M7 E,M7E+M43W,and M7E+M43Y,were energetically more beneficial to the binding with the RBD than ACE2.In particular,the mutant inhibitor M7E+M43Y exhibited the highest affinity with RBD and was selected as the optimal inhibitor among all tested inhibitors.In addition,the combination of multiple analysis methods,such as free energy landscape analysis(FEL),principal component analysis(PCA),dynamic cross-correlation matrix analysis(DCCM),and hydrogen bond,salt bridge,and hydrophobic interaction analysis,also demonstrated that the mutations significantly affect the dynamical behavior and binding pattern of the inhibitor binding to the RBD protein.The current work suggested that miniprotein inhibitors can form stable complex structures with the RBD protein and exert a blocking or inhibitory effect on the SARS-CoV-2 Omicron variant.Several novel Omicron RBD protein mutant inhibitors identified in this study provide important guidance and insights for the rational design of new drugs targeting the SARS-CoV-2 Omicron variant.4.Theoretical study on the molecular mechanism of non-covalent inhibitor WU-04 targeting the 3CLpro of multiple coronavirus mainstream strainsThe 3CLpro protein is an essential protease in the replication process of coronaviruses.Effectively inhibiting the replication of the virus by suppressing the3 CLpro protein can prevent the spread and infection of the virus.From this perspective,the 3CLpro protein is also an important antiviral drug target.In this study,we elucidated the molecular mechanism of a novel and highly effective non-covalent inhibitor,WU-04,targeting the 3CLpro protein of the original SARS-CoV-2 strain through MD simulations.The difference in dynamic behavior between the apo-3CLpro and the holo-3CLpro systems suggested that the presence of WU-04 can indeed influence the motion amplitude of the 3CLpro protein,thereby maintaining a stable complex state.The energy calculations and interaction analysis showed that the hot-spot residues Q189,M165,M49,E166,and H41 and the warm-spot residues H163 and C145,maintain strong intermolecular interactions with WU-04 by forming multiple hydrogen bonds and hydrophobic interactions,which stabilizes the binding of the inhibitor.This forms the foundation for the stability of the inhibitor with the 3CLpro protein.Meanwhile,we also investigated the inhibitory effects of WU-04 on six SARS-CoV-2 variants(Alpha,Beta,Gamma,Delta,Lambda,and Omicron)and two other mainstream coronaviruses(SARS-CoV and MERS-CoV)3CLpro proteins.Excitingly,the slight difference in binding energies indicated that WU-04 is still highly effective against the coronaviruses.This positions WU-04 as a promising pan-inhibitor of the 3CLpro protein targeting various SARS-CoV-2 variants and other mainstream coronaviruses.This study provides profound theoretical insights for the rational design of new non-covalent inhibitors targeting the 3CLpro protein.