Identification And New Drug Screening Of Anti-SARS-CoV-2 Protein-drugs Targeting The Six-Helix Bundle Structure Of Spike Protein Transmembrane Subunit

The novel coronavirus pneumonia(Corona Virus Disease 2019,COVID-19)broke out in Wuhan in the end of 2019.COVID-19 has been spread abroad and developed into kind of pandemic rapidly,although it was well contained in the initial stage.So far there have been more than 400 million confirmed cases worldwide,which makes devastating impact on the global health system and the world economy.With the emergence of multiple variant strains,the research and development of anti-SARS-CoV-2 drugs,neutralizing antibodies and vaccines have fallen into a bottleneck.The SARS-CoV-2 Spike protein is an important target for the development of antiviral drugs and Spike protein is responsible for virus participates in the recognition,binding,fusion and entry stages of the virus.HR1 and HR2 are two Heptad repeats in the S2 subunit of Spike protein,which can form a six-helix bundle(6-HB)and involv in the fusion and the entry of virus.Among the currently popular variant strains,the HR1 and HR2 in the S2 subunit are more conservative than the RBD in the S1 subunit,and the drugs targeting HR1 and HR2 in the S2 subunit are more broad-spectrum.Therefore,this project aims to develop a series of protein drugs with anti-SARS-CoV-2 activity targeting on HR1/2.Based on this,a rapid drug screening platform is established to screen potential antiviral drugs.Futhermore,broad-spectrum neutralizing antibodies were obtained based on 6-HB-derived proteins to deal with the problems of antibody resistance of currently popular variant strains.Based on the HR1 and HR2 sequences of SARS-CoV-2 Spike proteins,we designed and expressed 5Helix,HR121 and HR212 proteins,and tested the antiviral activity of the proteins through antiviral experiments and cell fusion inhibition experiments.Circular dichroism,ELISA and fluorescence polarization have proved that these three proteins exert antiviral activity by inhibiting the formation of 6-HB.A high-throughput drug screening method base on fluorescence polarization was established using the 5Helix,and Thonningianin A was selected out with high anti-SARS-CoV-2 activity,which inhibited cell fusion by blocking the formation of 6-HB.HR121 and HR212 were further used to immunize mouse,and the characteristics of antibodies in mouse serum were examined.It was found that there were antibodies that could recognize Spike protein and S2 protein in mouse serum,and it also showed activity in inhibiting cell fusion.Immortalized hybridoma cells were obtained,and 39 Hybridoma from HR121 and 23 Hybridoma from HR212 were screened and identified.Most of these 62 Hybridoma culture supernatants could inhibit SARS-CoV-2 Delta pseudovirus infection in varying degrees.Finally,we successfully obtained seven antibodies from Hybridoma from HR212,and except for the 3G1-1 mAb,the other six antibodies have neutralizing activity,which can recognize different mutant SARS-CoV-2 Spike proteins and SARS-CoV Spike protein,and they can bind tightly to the antigen.Among them,2E3-12 mAb showed strong broad-spectrum neutralizing activity and cell fusion inhibitory activity.Its antibody variable region was obtained through gene identification,and the 2E3-12-Fc antibody fused with human antibody Fc also had the same ability.Further identification of antibody binding sites suggested that 2E3-12 mAb could bind to HR2P and S2-Fc,and HR2P based on SARS-CoV-2 HR2 domain could compete with HR212 for the binding site on the 2E3-12 mAb,while the short peptide derived from HR2 domain had no competition.This indicated that the binding epitope of 2E3-12 mAb could be non-linelic.In addition,the combination of 2E3-12 Fab and trimeric Spike protein(Spike protein for six stabilizing proline substitutions,S6P)can change the conformation of trimeric S6P,making S6P tend to be unstable.In summary,this project has successfully developed a series of anti-SARS-CoV-2 protein,and used 5Helix protein to screen to obtain Thonningianin A,which also has anti-SARS-CoV-2 effect.And we used HR121 and HR212 proteins to obtain broad-spectrum neutralizing antibody 2E3-12 mAb,we provide a new therapeutic strategy for completely end the epidemic of COVID-19.