Study On The Activity And Mechanism Of Novel Nucleoside Compound GY006 Against Coxsackie Virus B3

Purpose:Coxsackievirus B3(Coxsackievirus B3)is a single-stranded positive-stranded picornavirus belonging to the genus Enterovirus,Picornaviridae.CVB3 infect the body through the digestive tract and respiratory tract can cause viral myocarditis(VMC),pancreatitis and other diseases.It mainly affects children and adolescents and about 20% of VMC patients and 10% of pancreatitis patients are related to CVB3 infection.However,there is still a lack of effective treatments.Therefore,it is extremely important to find safe and effective therapeutic drugs for the clinic.Methyl 4-alanine-1-(2'-deoxy-2'-?-fluoro-4'-?-azido-?-D-furanosyl)cytosine(GY006)is a novel nucleoside compounds,in vitro experimental results show that it has anti-CVB3 activity.Therefore,this project explores the anti-CVB3 activity of GY006 in vivo and in vitro and its potential mechanism of action by establishing CVB3 infection cell models and in vivo animal models.Method:1.Study on the anti-CVB3 activity of GY006 in vitroThe MTT method was used to detect the proliferation inhibition rate of GY006 on He La cells,and the half-toxic concentration(CC50)was calculated.The end-point dilution method was used to determine the half tissue culture infective dose(TCID50)of CVB3.After CVB3 infected He La cells were treated with GY006,the median inhibitory concentration(IC50)was calculated by detecting cell survival.Quantitative Real-time PCR(q RT-PCR)was used to detect CVB3 gene expression,and the inhibitory effect of GY006 on CVB3 replication was investigated.2.Study on the anti-CVB3 activity of GY006 in vivoBalb/c mice infected with CVB3 were randomly divided into 5 groups: model group(normal saline),GY006 group(20,10,and 5 mg/kg),and ribavirin control group(100 mg/kg).At the same time,a blank control group(normal saline)without virus infection was set up.The mice in each group were given intragastric administration continuously for one week.Observe the changes in the mental state and body weight of the mice.Use a small animal ultrasound imaging system to detect left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)to evaluate cardiac function.The serum levels of myocarditis markers lactate dehydrogenase(LDH),creatine kinase(CK)and cardiac troponin 1(c Tnl)were measured.H&E staining and histopathological examination were performed on the heart,pancreas,spleen and other tissues to evaluate the protective effect of GY006 treatment on tissue damage.Measure the expression of CVB3 gene in tissues by q RT-PCR method to evaluate the inhibitory effect of GY006 treatment on CVB3 replication.3.Research on anti-CVB3 mechanism of GY006SYBYL-X software was used to evaluate the drug-protein interaction between GY006 Triphosphate(GY006 Triphosphate,GY006-TP)and CVB3 RNA-dependent RNA polymerase(Rd Rp).The effect of GY006 on the activity of CVB3 Rd Rp.Detect changes in the expression of inflammation-related genes NOD-like receptor family 3(NOD-like receptors,NLRP3),cysteinyl aspartate specific proteinase 1(Caspase-1),interleukin 1?(IL-1?)and interleukin 6(IL-6).Western Blot method and immunohistochemistry were used to detect NLRP3,previous cysteinyl aspartate specific proteinase1(pro-Caspase-1),IL-1?Tumor necrosis factor-?(tumor necrosis factor-?,TNF-?)protein expression changes in cells and myocardial tissues.CVB3 infected mouse macrophages RAW264.7.The q RT-PCR method was used to detect the innate immunity-related genes Interferon-?(Interferon-?,IFN-?)and its effector gene Interferon Stimulating Gene 15(ISG15)and Myxovirus 1(Mx1)after the intervention of GY006.Results:1.GY006 anti-CVB3 active in vitroThe CC50 of GY006 for He La cells is greater than 1000 ?M,the IC50 for CVB3 is 25.34±1.711 ?M,and the TI is greater than 39.46.After treatment with 12.5,25,and 50 ?M GY006,the CVB3 gene expression was 0.58,0.28,and 0.08 times that of the virus group,respectively.After 100 ?M ribavirin treatment,the CVB3 gene expression was 0.57 times that of the virus group,indicating that GY006 can significantly inhibit CVB3 virus replication in vitro.2.GY006 anti-CVB3 active in vivoHigh levels of CVB3 gene expression were detected in the myocardium and pancreas tissues of CVB3 infected mice,the body weight was significantly decreased,LVEF and LVFS were significantly decreased,the concentrations of myocarditis markers LDH,CK and c Tnl were significantly increased,and the myocardium,pancreas and spleen tissues were significantly present Inflammatory infiltration,and with serious tissue damage.After treatment with GY006,the expression of CVB3 gene in the myocardium and pancreas tissues of the mice was significantly decreased,the body weight was not significantly reduced,the LVEF and LVFS were significantly increased,the concentrations of LDH,CK and c Tnl were significantly reduced,and the lesions of the myocardium,pancreas and spleen tissue were significantly reduced.And the improvement of the above indicators are better than the ribavirin treatment group.Shows that GY006 can significantly inhibit CVB3 replication,restore heart function in mice,reduce myocardial inflammation and damage,and the effect is better than ribavirin.3.The mechanism of action of GY006 against CVB3(1)The effect of GY006-TP on CVB3 Rd RpThe molecular model docking results showed that GY006-TP interacted with the protein residues in the active site of CVB3 Rd Rp(ARG188,GLY293,SER295,ASP111 and ALA109),indicating that GY006 can inhibit viral replication by inhibiting the activity of CVB3 Rd Rp(2)The effect of GY006 on inflammation-related genes and protein expressionAfter CVB3 infected He La cells were treated with GY006,the expression of NLRP3,Caspase-1,IL-1?,and IL-6 genes were significantly reduced,indicating that GY006 can effectively down-regulate the expression of inflammation-related genes.Consistent with the results of genetic testing,the protein expression of NLRP3,pro-caspase-1,TNF-? and IL-1? decreased significantly after GY006 treatment.In mice,the expression of NLRP3,pro-caspase-1,TNF-? and IL-1? protein in myocardial tissues was also significantly reduced after GY006 treatment.It shows that GY006 can significantly reduce the expression of inflammation-related proteins in vivo and in vitro,inhibit the inflammation reaction,and reduce the inflammatory damage of various organs such as myocardium.(3)The effect of GY006 on the expression of macrophage innate immunity related genes.After CVB3 infected RAW264.7 cells,the expression of IFN-? gene increased,and the expression of IFN-? effector genes ISG15 and Mx1 also increased,indicating that the virus stimulated the innate immune response of macrophages.After adding 50 ?M GY006 intervention,the expression of IFN-? and its effector genes ISG15 and Mx1 further increased.It shows that GY006 enhances the innate immune response of macrophages to CVB3 and strengthens the antiviral response.Conclusion:1.The novel nucleoside compound GY006 has significant anti-CVB3 activity in vitro.2.The novel nucleoside compound GY006 has significant anti-CVB3 activity in vivo,and significantly reduce inflammation,improve histopathological damage3.GY006 inhibits virus replication by binding to CVB3 Rd Rp,and reduces inflammatory damage in tissues and organs by inhibiting the expression of NLRP3 pathway genes and proteins.In addition,GY006 can also enhance the innate immune response of macrophages to CVB3 and strengthen the antiviral response.Highlights:This study reported for the first time the anti-CVB3 activity of a novel nucleoside compound GY006 in vivo and in vitro.For the first time,the antiviral molecular mechanism of GY006 was clarified from three aspects: inhibition of virus replication,improvement of inflammatory response and enhancement of macrophage innate immune response.