Study On The Interaction Mechanism Between The Surface Proteins Of H1N1 Influenza A Virus And Inhibitor Or Antibody

Influenza A virus is one of the pathogens that pose serious threat to human health and has been widely concerned in the field of disease control.The hemagglutinin?HA?and neuraminidase?NA?are major surface glycoproteins of influenza virus and play essential roles in the virus life cycle.Due to the special function of them,they have become especially attractive targets for inhibitors for therapeutics.The H1N1 influenza A viruses have caused a number of worldwide pandemics,and the research of novel inhibitory molecular focus on the subtype virus is a hot topic in this field.With the continuous development of experimental screening and synthesis technology and computer-aided tools,more and more potential small molecule inhibitors and broad-spectrum neutralizing antibodies,which are targeted by HA and NA,are found.This provides great convenience for the development of anti-flu drugs.However,to make full use of these new inhibitory molecules to develop new drugs,it is necessary to have a deep understanding of its microscopic mechanism.But the pace of the current theoretical research is far less than the speed of new inhibitory molecules found and known number of inhibition of molecular mechanism is still very limited.Therefore,it is particularly necessary to further study the mechanism of inhibitors based on experimental screening results.This article will apply the molecular docking,molecular dynamics simulation,steered molecular simulation and free energy calculation to study the microscopic mechanism of small molecules and antibody which targeted on the H1N1 influenza A virus surface protein HA and NA.The specific research contents of this paper include the following three parts:Part1:Exploration of binding and inhibition mechanism of a small molecule inhibitor of influenza virus H1N1 hemagglutinin.In previous studies,Arnab et al.obtained a novel compounds,named MBX2329,using a high-throughput screening assay.The molecule could inhibit the conformation changes of HA and further destroy the fusion of virus and cell.The discovery of MBX2329 has important significance for the development of novel H1N1 influenza A inhibitors.However,it's still not clear about the inhibition mechanism from the theoretical aspect.To explore the inhibition mechanism of this new inhibitor,in this study,molecular docking,molecular dynamics simulation and the binding free energy calculation approach were applied under the neutral and acidic conditions.The results indicated that MBX2329 can stably bind with HA under neutral conditions via van der Waals interactions and has the ability to enter the endosome following HA binding during endocytosis.While the interaction of the molecule with HA at low-pH conditions at a new binding site employs a different mechanism to carry out its inhibitory effect.After a series of confirmation,we believe that the binding of MBX2329 can prevent HA1 from disassociating from HA2,further maintain the rigidity of the HA2 loop and protect the salt bridge between Glu101 and Lys50,stabilize the distance between the long helix and short helix.The combination can effectively prevent the depolymerization of HA trimer under acidic conditions.The investigated residues in the new binding site are highly conserved,implying that the new binding site has the potential to be an new effective drug target.The results in this study will provide theoretical clues for the mechanism of new influenza virus HA inhibitors.Part2:Exploration of inhibition mechanism of a novel inhibitor of H1N1 influenza A virus neuraminidasesIt has been reported recently that one of the novel small molecule named 7a,could inhibit both A/Puerto Rico/8/34?H1N1?NA(NA_PR)and A/California/04/09?H1N1?NA(NA_CA).However,potential structure differences in the active site could be easily detected between the NA_PR and NA_CA.Crystallization experiments reported that most classical group 1 NAs,including N1 NA,own the 150-cavity near their active center.However,a recent crystallographic result on the NA_CA determined that the 150-cavity might be loss in NA_CA.Therefore,it might be puzzling that how 7a could play its inhibition role on both N1 NAs with possible different structure in the active pocket.For exploring the inhibition mechanism of this new NA inhibitor,in this study,molecular dynamics simulation and steered molecular dynamics simulation approach were applied.The study shows that 7a can combine with the two kinds of NA via hydrogen bond and cationic conjugate interaction.It is indicated that 7a could be adopt a relatively extended pose in the active center of NA_PR.While in NA_CA-7a complex,the molecule preferred to be recognized and located on the side of active center near the 150-loop.According to the theoretical results,it is speculated that the process of7a ring structure adapting to the active site determines its binding position to some extent,and the boric acid structure can further stabilize the joint.Due to the existence of the 150-cavity,NA_PR has a relatively wide active center in which the ring of 7a could be more easily combined with the positive and electrical amino acids at the active site.Comparatively,due to the lack of 150-cavity,the activity center of NA_CA is relatively narrow,and the ring of 7a tends to be first combined with the positive charged residues exposed to the vicinity of 150-loop and 430-loop,following the boric-acid group makes H-bond interaction with the external residues for further stabilizing the binding.Due to the interactions between 7a and the NA_CAA 150-loop,the closed 150-loop of NA_CA is constructed with a150-cavity-like structure.The results reveal the possible broad spectrum inhibiton mechanism of 7a,and provide theoretical clues for the novel inhibition mechanism and developing future anti-flu inhibitors.Part3:Exploration of dynamics mechanism of a broad-neutralizing antibody targeted on the hemagglutinin of influenza virus H1N1.The interaction mechanism between targeting molecules and influenza virus surface proteins has been widely concerned.The development of therapeutic monoclonal antibodies and small molecular inhibitors is urgently needed.Recently,it has been confirmed by Wang et al.that a human monoclonal antibody called 3E1could inhibit the structure changes of HA2 from H1 and H5 under low p H condition.The crystal structures between two H1N1 HAs and 3E1 Fab have been obtained.However,the dynamic mechanism of 3E1 antibody is still not further studied.This part will apply the homology modeling and molecular dynamics simulation to study the dynamic inhihitory mechanism of 3E1.In addition,relevant biochemical data show that the I45F mutation of HA2 would abolished the binding with 3E1,however,the specific mechanism has not been further explored.Thus,in this part,the effect of I45F mutation on the stability of HA_I45F-3E1 complex has been investigated as well.The theoretical results show that the combination of 3E1 can reduce the disturbance at the upper end of HA2 long helix;at the same time,3E1 can protect the salt bridge between Glu101 and Lys50,maintain the stability of HA2 long and short helixes,and then inhibit the turning back at the bottom of long helix near the 105-107residues.In addition,through the dynamics behavior study between HA_I45F and 3E1,it was found that I45F mutant could destroy the hydrophobic interactions between the short helix of HA2 and HCDR3 of 3E1,and Trp32 of light chain of 3E1.This destruction will weaken the binding potential of 3E1 and HA,and further make the heavy chain of 3E1 move down.At same time,the effective contact between HA and the light-chain of 3E1 has been also broken.The above results indicated the behavior of 3E1 and can be used as the basis for improving the broad-spectrum design of the antibody,and provide theoretical clues for the modification and design of therapeutic antibodies based on 3E1.The results will be of great significance for the prevention and treatment of influenza virus.The explanation of interactions between inhibitor molecules and HA/NA could determine the design idea of specific anti-influenza drugs;guide the modification direction of broad spectrum and antibody;even could provide the theoretical clues to the design of candidate immunogenicity.