Molecular Mechanism Of GC376 Inhibiting Alpha Animals Coronavirus Replication

Coronaviruses are a class of microbial pathogens that cause severe harm to humans and animals,which can cause serious harm to the economic and agricultural development,public health and safety construction of countries around the world.Among them,porcine epidemic diarrhea virus?PEDV?and feline infectious peritonitis virus?FIPV?pose serious threats to animal health,both of which belong to alpha coronavirus.The mortality of swine epidemic diarrhea is extremely high.Especially,the lethal rate for piglets whose immune function is not perfect,which reachs up to 100%.At present,the prevention and control of the disease mainly depends on vaccines,but it cannot provide 100%protection clinically.And there is a lack of specific drugs which can effectively prevent and control this disease.And feline infectious peritonitis is an important disease that threatens the health of felines.It is highly contagious and has a lethal rate of 100%.3CL^pro inhibitor GC376 is widely used in clinical treatment.The drug has achieved good results in the early stage of treatment,but recurrence will occur in the middle and late stages of treatment,which would affect the treatment effect and reduce the cure rate.This study proved that the inhibitory effect of 3CL^pro inhibitor GC376 on PEDV in physiological and biochemical levels for the first time,in this way,providing new ideas for the treatment of swine epidemic diarrhea.By the method of molecular docking,the binding mode of PEDV 3CL^pro-GC376 was simulated,and the molecular mechanism of inhibition was elucidated.At the same time,pharmacy was performed on the basis of GC376 of low concentration,and the efficacy of the improved drug was evaluated,providing guidance for drug improvement.Finally,through the passage of FIPV under drug pressure,drug-resistant strains were obtained to clarify the molecular mechanism of coronavirus resistance to the inhibitor GC376.The specific work of this study is divided into the following four aspects:1. The inhibitory effect of inhibitor GC376 on PEDV.At the biochemical level,the IC₅₀ of GC376 to PEDV 3CL^pro was determined and the value was 966 n M.At the cellular level,the EC₅₀ of GC376 against PEDV YN144 strain was determined and the value was 14.66?M.Western blot was used to detect the change of virus content under each drug concentration gradient,which revealed that the N protein of PEDV could not be detected when the GC376 concentration reached 25?M.Meanwhile,the IFA was used to further verify the inhibitory effect of GC376 on PEDV virus.The results showed that GC376 of 1.56?M could reduce virus infection when the final concentration reached12.5?M,GC376 could completely inhibit virus replication.2. Simulation of the combination of PEDV 3CL^pro and GC376.The molecular docking method was used to simulate the binding mode between the substrate GC376 and the receptor protein PEDV 3CL^pro.There are extensive hydrophobic interactions between proteases and compounds.In the simulation results,GC376 forms hydrogen bond interactions with six amino acid residues of PEDV 3CL^pro:His41,Gly142,Ala143,Cys144,His162,Gln163.And it was found that GC376 could inhibit the cleavage activity of PEDV 3CL^pro by blocking the active site of PEDV 3CL^pro,at Cys144.In this way,3 CL^pro has a conserved catalytic core region,so the inhibitor GC376 could bind to the virus 3 CL^pro very quickly and inhibit the replication of different strains of PEDV.3. Evaluation of GC376 improved drug efficacy.By comparing GC376 and the peptide complexes of PEDV 3CL^pro,different fluorescent peptide substrates were designed.We could find that leucine is the most preferred amino acid residue at the P2site of the fluorescent polypeptide substrate during the FRET test.Later,this study tested the inhibitory effects of the improved drugs Target 1 and Target 2.It was believed that GC376 can be rationally designed and modified based on leucine at the S2 position to achieve better inhibitory effects.4. Whole-gene analysis of drug-resistant FIPV strains.In this study,the wild-type FIPV-791146 strain was subcultured under inhibitor pressure for 50 generations to obtain drug-resistant strains.Through the determination of EC₅₀,Western blot detection and indirect immunofluorescence technology,the inhibitory effects of inhibitor GC376 on wild-type and drug-resistant strains were compared.Analysis of the entire genome sequence of the drug-resistant strains revealed that the molecular mechanism of coronavirus resistance to the inhibitor GC376 was not caused by the targeted protease3CL^pro,but a synergistic effect of multiple viral protein.This provides guidance for the optimization and improvement of 3CL^pro inhibitors.In summary,this article validated the inhibitory effect of 3CL^pro inhibitor GC376 on PEDV,and explored the molecular mechanism of coronavirus resistance to GC376.It could provide not only a new scheme for the treatment of porcine epidemic diarrhea,but also the guidance for further optimization and development of 3CL^pro inhibitors.