Screening And Evaluation Of Potential Inhibitors Against Rabies Virus And Vaccinia Virus

Rabies is a fatal disease caused by rabies virus.About 60,000 people worldwide die of rabies every year,of which 40%are children under the age of 15,mainly in Asia and Africa^[1].The incidence and mortality of rabies in China ranks second in the world,second only to India^[2].Rabies is one of the diseases with the highest mortality,with a fatality rate of nearly 100%.Few cases of successful treatment have been reported^[3].Finding an effective anti-rabies drug and treatment to reduce the incidence and mortality of rabies,so as to eliminate people's"rabies phobia"has become one of the important challenges faced by drug researchers all over the world.Smallpox,one of the most devastating human diseases,was eradicated in 1979^[4].Presently,the remaining smallpox viruses are preserved in only two WHO-designated laboratories:the Centers for Disease Control and Prevention(CDC)in Atlanta,USA and the State Research Center of Virology and Biotechnology VECTOR in Koltsovo,Russia^[5].However,other smallpox virus discoveries have been reported outside of these laboratories,e.g.,USA in 2014^[6].Notably,as the largest double-stranded DNA virus in genome,smallpox virus is so stable in the environment that once it is used as a biological weapon,the consequences will be unimaginable.In addition,the interruption of vaccination also provides strong conditions for the spread of the virus.Therefore,it is necessary to screen and evaluate the drugs against smallpox virus.It usually takes many years to develop new drugs,and there are a variety of production requirements,and the reuse of existing drugs is an attractive alternative^[7].These approved drugs have advantages because their pharmacological and toxic characteristics are known,safety data in humans have been established,and the feasibility of production and formulation has been demonstrated^[8,9].In this study,we established a high-throughput screening method based on rabies virus pseudovirus and replicated vaccinia virus Tiantan strain to screen approved drugs on the market and to identify effective antiviral candidates in vitro and in vivo.In the first part of this study,767 compounds from the Institute of Standard Materials and Standardization of China Institute of Food and Drug Control were screened with high throughput to find compounds against rabies virus.After three rounds of in vitro screening,11 hits were obtained.Among the 11 compounds,clofazimine ranked first.At the same time,the inhibitory activity of clofazimine was confirmed by RFFIT test with rabies virus live virus CVS strain.In vitro,the EC₅₀ of CVS strain was 2.28?M,CC₅₀>2200?M,and the treatment index SI>967(patent number:N112279815A).At the same time,through time-of-addition analysis,Biacore molecular interaction analysis docking and other experiments,it was determined that clofazimine was mainly used as a membrane fusion inhibitor to play an antiviral role.In addition,the infection model of rabies virus CVS strain in BALB/c mice was established by intramuscular injection,imitating the way that humans were bitten by dogs.The results of prevention after exposure showed that clofazimine could prolong the time of onset and death of mice,and improve the survival rate by 10%in vivo.Clofazimine,as a first-line drug in the clinical treatment of tumorous leprosy,its pharmacokinetics and safety have been widely confirmed,but it has the side effect of skin pigmentation.In order to improve the solubility of clofazimine and reduce its accumulation in adipose tissue,a series of clofazimine salts were synthesized based on the parent nucleus of clofazimine.Through the evaluation of in vitro and in vivo activity,it was found that clofazimine salicylate was a more ideal candidate for anti-rabies virus.In order to further clarify the structure-activity relationship of clofazimine against rabies virus,a series of new clofazimine analogues were synthesized by means of chemical modification,and their activities need to be further verified in vivo.In the second part of this study,the antiviral activity of 767 drugs were screened by vaccinia virus Tiantan strain.Mycophenolate mofetil and tranilast,which have potential efficacy in treating smallpox virus,were found and the antiviral efficacy of mycophenolate mofetil and tranilast was tested in animals.The results showed that after 5 times of administration,the inhibition rate of mycophenolate mofetil on vaccinia virus replication in mice was 99%(10 mg/kg),and that of tranilast was 59%(45 mg/kg).Time-of-addition analysis showed that both drugs played a major role in the process of virus replication.In a word,based on the screening and verification of drugs against rabies virus and vaccinia virus based on"drug repositioning",this study found a rabies virus inhibitor-clofazimine.And two vaccinia virus inhibitors,mycophenolate mofetil and tranilast,the activities of these drugs have also been verified at the animal level.The overall framework study on the antirabies activity,action mechanism and chemical basis of phenoxazine derivatives based on clofazimine mother nucleus was completed.In order to guide the reorientation of the new medicinal function of clofazimine against rabies virus and provide important material basis and scientific basis for the research and development of new rabies virus drugs.