Mechanism And Screening Of The Anti-virus Natural Small Molecule Drugs Targeting PEDV Replicase

Porcine epidemic diarrhea（PED）is a highly contagious,intestinal infectious disease caused by porcine epidemic diarrhea virus（PEDV）,which causing lethal diarrhea in suckling piglets,and leading to severe economic losses worldwide.The passively acquired maternal immunity can provide protection against PEDV infection in sucking piglets.But the available commercial vaccines fail to protect against highly virulent PEDV variants infection.It is of great theoretical significance and application value to study on the anti-viral drugs and clarify its mechanism of inhibition of PEDV replication.1.Growth characteristic of PEDV MSCH isolate in porcine intestinal epithelial cells and its full-length sequencing analysisPEDV infects intestinal epithelial cells of pig small intestine.IPEC-J2 cells were infected with PEDV MSCH isolate and CPE was observed.The virus titer reached up to10^3.8/ml TCID₅₀ at 18 hours after infection,and then gradually decreased.The full genome of this isolate（F25）was sequenced and analyzed by PCR and Sanger sequencing.The results showed that the genome sequence has the typical structural characteristics ofα-coronaviruses（Gen Bank ID:MT683617）with full-length of 28,066 bp,including a 32 bp poly-A tail.The isolate belongs to the Asian non-S-INDEL subgroup of the G2 subtype.It derived from the recombination of CH/GDGZ02/1401 and ZJCZ4 at the end of ORF1b and the S protein.This study provided the foundation for the virus research.2.Tomatidine inhibits PEDV replication by targeting 3CL proteaseTo find anti-PEDV natural drugs,a library of 911 natural products were subjected to two rounds of screening.Four of the compounds produced negligible cytotoxicity and inhibited PEDV infection by over 80%as determined by IFA.Tomatidine had the highest SI and was chosen for further study.The IC₅₀ of tomatidine against PEDV in cells was 3.447μM,the CC₅₀ was about 45.68μM,and SI was 13.33.Tomatidine had similar inhibitory effects on the different PEDV variant isolates（YZ,MS,and SH）,as well as on the classic CV777 isolate.Moreover,tomatidine inhibited PEDV proliferation in IPEC-J2 cells.The PEDV life cycle is composed of four stages:attachment,entry,replication,and release.Our results indicated that tomatidine significantly inhibits the viral infection at the replication stage.The interaction between tomatidine and active pocket of PEDV 3CL protease was tentatively predicted by molecular docking（binding energy=-9.14 kcal/mol）.A 10 ns molecular dynamic simulation was carried out for the assessment of receptor and ligand stability.Distances between tomatidine and the active pocket of 3CLpro were highly conserved and tight during simulation calculations,and tomatidine did form hydrogen bonds with the active pocket of 3CLpro,which may contribute to the stability of the tomatidine-3CLpro complex.PEDV 3CLpro was expressed in soluble form using an E.coli expression system.The purified recombinant 3CLpro was acquired by using Ni-sepharose purification method.And the interaction between 3CLpro and tomatidine was determined by a fluorescence quenching assay.The results showed that the fluorescence emission intensity of3CLpro decreases distinctly in a dose-dependent manner with increasing concentration of tomatidine and the Kq value was calculated to be 2.7×10¹¹ L·mol^-1·s^-1.It indicated that the static-quenching occurs and the interaction between 3CLpro and tomatidine was strong.The results of ITC showed that tomatidine directly interacted with PEDV 3CLpro with a Kd of2.78μM and N of 0.723 sites.The recombinated plasmids expressing 3CLpro and GFP_nsp5/6containing the nsp5/nsp6 junction（YGVNLQ^SG）were constructed.After co-transfection of the plasmids,vero cells were treated with increasing concentrations of tomatidine.Western blot showed that the cleaved fragments of GFP_nsp5/6 decreased in a dose-dependent manner,indicating that tomatidine inhibited the activity of PEDV 3CL protease.FRET assay was used to confirm the effect of tomatidine on 3CLpro activity.The dose-dependent increase of fluorescence intensity showed that the 3CLpro obtained possesses catalytic activity.In order to explore the inhibition of viral 3CLpro activity of tomatidine in PEDV-infected cells,anti-3CLpro serum antibody was prepared by vaccination of mice with the purified recombinant3CLpro.Western blot indicated that tomatidine significantly inhibited the activity of PEDV3CLpro.In addition,tomatidine also had antiviral activity against transmissible gastroenteritis virus（TGEV）,porcine reproductive and respiratory syndrome virus（PRRSV）,encephalo myocarditis virus（EMCV）and seneca virus A（SVA）in vitro.This study demonstrated that tomatidine inhibits PEDV replication by targeting 3CLpro,providing a theoretical basis for the research of anti-PEDV drugs.3.Natural compound ZINC12899676 reduces PEDV replication by inhibiting the viral NTPase activityTo discover more antiviral compounds against PEDV,the potential PEDV replicative enzymes inhibitors were identified by virtual screening of 187,119 compounds contained in ZINC natural products database.It was observed that the viral NTPase has the most hits among the targets after two-round virtually screening by using Autodock vina and Ledock software.Considering binding energy and drug-like characteristic of compounds,seven small compounds were selected to ascertain the effect of compounds to PEDV NTPase.The prokaryotic expression plasmid containing PEDV nsp13 gene was constructed and the recombinated nsp13 was purified,and the ATPase activity of recombinant nsp13 proteins was detected by using a Kinase-Glo Plus Luminescent Kinase Assay kit.Among seven compounds,ZINC12899676 was identified to significant inhibit the viral NTPase activity.Ligplot software visualized the two H-bonds and some residues involving hydrophobic interaction between ligand and receptor.H-bond between compound and the key active site S290 were also visualized by Pymol.It is observed that the head and backside of ligand ESP surfaces were fully compatible with the protein ESP surfaces in relevant position,which explained the ZINC12899676 and PEDV NTPase interaction using a quantum mechanical.To assess the receptor and ligand stability,a 20ns molecular dynamic simulation was carried out.Protein-compound complex reached stable throughout 8 ns simulation with relative RMSD in acceptable range,with few fluctuations observed.Distances between the compound and the active pocket of NTPase were highly conserved in range of 0.153-0.154nm and tight during simulation calculations.The stable existence of H-bonds in the complex began with the 12ns during the simulation.The average of binding free energy of protein-ligand complex obtained using MM-PBSA was-154.702 k J/mol,indicated a strong interaction.Residues locating in active pocket contributed dominating binding free energy.The structure of viral NTPase can be induced to enter the local energy minimal state by ZINC12899676.Furthermore,it was obviously thatβ-sheet located in R568 had been transformed intoα-helix and Q405 shifting out from the active pocket leaded to the expansion of pocket.The conformation change caused by ZINC12899676 might finally lead to the dysfunction of viral NTPase.ZINC12899676 inhibited PEDV replication in IPEC-J2 cells.The CC₅₀ of ZINC12899676 on cells was 64.63μM,the IC₅₀ of ZINC12899676 against PEDV in cells was 4.489μM and SI was 14.4.An artificial intelligence model was built to predict drug property and 768 compounds with high binding energy to the viral NTPase（<-9.5 kcal/mol）were quickly screened from the 13,651,290 compounds through AI and VS combination.This study first demonstrated that ZINC12899676 inhibits PEDV replication by targeting the viral NTPase.And the NTPase can be used as a novel target for developing anti-PEDV drugs.