| Viral infectious diseases pose a serious threat to human health.With the rapid development of the world economy and the acceleration of globalization,new and emerging viral infectious diseases have been found and spread.The safe and effective vaccines preventing those viral infection had not been developed yet.Thus,small molecule drugs which have great potential for the treatment of viral infections are needed.In the present study,we screened natural or synthetic compounds to find antiviral drugs with high specificity and low toxicity.It would solve the blindness of drug development under the condition that the pathogen infection mechanism of infectious diseases is unknown.In present study,the traditional Chinese medicine chemical extract reference library and approved drug library were screened with a safe and sensitive high-throughput cell screening platform using Hantaan pseudoviruses?Adenovirus type 5 and Respiratory Syncytial Virus.The positive compounds were verified with in vivo animal model.This work would provide new clues for the development of antiviral drugs and new strategic material reserve for emergency disposal.Objective1.To screen the traditional Chinese medicine chemical extract reference library andapproved drug library for compounds with antiviral activity.2.The antiviral effects of the compounds were verified by in vivo and in vitro experiments,which would provide clues for the development of antiviral drugs.Methods1.High-throughput cell screening method was used to screen the compounds with antiviral activity from 556 traditional Chinese chemical extract and 1813 approved drug.2.The drugs screened in vitro were verified by using in vivo imaging technology of small animals,Real-time fluorescence quantitative PCR,Western Blot and Time of addition to further evaluate the antiviral effect of drugs.Results1.Three anti-Ads compounds were screened out at the cells level using live virus,named 11-40?ZY-28 and ZY-103,respectively.The EC50 of these compounds are 1.20?M?4.04?M and 4.5?M,respectively.Among them,ZY-28 also has anti-Ad3 activity,of which the EC50 value is 2.4?M,Animal tests showed that ZY-28 could greatly inhibit the liver tissue necrosis caused by adenovirus infection and had a protective effect on the tissues of infected mice.2.Four anti-HTNV compounds were screened out at the cells level using pseudovirus,named 2-21?18-10?18-13 and ZY-128,respectively.The EC50 of these compounds are 0.1?M?0.2?M?0.2?M and 0.9?M,respectively.In vivo validation experiments showed that only ZY-128 had antiviral effect.The inhibition rate of the virus can reach 94%at the dosage of 150mg/kg/d.The live virus verification experiment suggested that the compound had a certain inhibitory effect on the virus at 20?M.ZY-128 inhibited virus entry and membrane fusion with cells.3.Six anti-RSV compounds were screened out at the cells using live virus,named 2-21?20-37?8-11?7-55?11-40 and 21-67,respectively.The EC50 of these compounds are 0.15?M?0.17?M?0.23?M?1.38?M?1.78?M and 1.38?M,respectively.In Western blot verification experiments,2-21?20-37?8-11?7-55 and 21-67 all inhibited F protein expression at different levels.The action stages of the 3 drugs,2-21?8-11 and 21-67,are the membrane fusion period and post-entry inhibition,the action stages of drug 7-55 are virus entry and membrane fusion.Conclusions1.Three anti-Ad5 compounds,four anti-HTNV compounds and six anti-RSV compounds were discovered by screening 556 traditional Chinese chemical extract and 1813 approved drug at the cellular level.2.One compound with good anti-Ad5 activity,one compound with anti-HTNV activity and five compounds with inhibiting the expression of RSV F protein were verified by animal experiments,Western blot,Real-time quantitative PCR and Time of addition. |
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