High Throughput Screening Of Inhibitors And Construction Of Monoclonal Antibodies Against JEV NS3Protease

Japanese encephalitis (Japanese encephalitis, JE) is a central nervous disease caused by the Japanese encephalitis virus, which can infect people and many kinds of animals and cause irreversible neurological damage. In2006, Japanese encephalitis happened in yuncheng, shanxi province, resulting in60people infected, of which19people were killed. It brought a huge social impact. In the infection of animals, pigs get infected most easily. Pregnant sows can be manifested as abortion, fever, stillbirth and boar appears to have orchitis. Japanese encephalitis has therefore become a serious public safety issue. Currently, there have been no effective drugs and specific treatments for JEV. So, it is necessary for us to search for drugs and treatments against JEV.JEV NS3protease, which is one of the potential therapeutic drug targets, plays a very important role in replication and assembly of JEV. NS3and NS2B combined together to form a heterodimer which plays an important role in processing the polyprotein precursor. At present, two drugs-targeting HCV NS3protease are boceprevir(SCH-503034) and telaprevir(VX-950). However, there is no report about the inhibitors of JEVNS3protease.In this study, the soluble NS3protease was purified, and its activity was then detected by fluorescence resonance energy transfer (FRET). On this basis, the high throughout screening method was established. We began to screen the chemical library, in the end, we got ten small-molecule compounds which can inhibit the activity of JEV NS3protease. But, on the cell level, only2compounds (The numbers in compound library: AG-690/40639011, AC-907/25005011) can inhibit the proliferation of JEV. We validated the ten compounds on WNV and DENV NS2B/NS3protease, the result showed that two of ten compounds have inhibition on WNV and DENV NS2B/NS3protein. This study provides two kinds of lead compounds for the prevention and treatment of flavivirus.1. The purification and activity assay of the JEV NS2B/NS3protease and the determination of the condition for HTSJEV, WNV, DENV NS2B/NS3protease were obtained by Ni2+affinity chromatography. Identification of SDS-PAGE showed that the three proteins have a high concentration and purity. We detected NS2B/NS3protease activity by FRET method. High throughput screening for JEV NS2B/NS3pro has been determined with signal-to-background ratio of10.07, Z’-value of0.924.2. High throughput screening of NS2B/NS3protease inhibitorsWe used high throughput screening to find inhibitors from a chemical library, which contains4160low molecular weight compounds. By analyzing the result of primary screening, we removed the false-positive compounds and got10compounds, which inhibits the JEV NS2B/NS3protease activity. The inhibition rate of10small molecule inhibitors is between80%to100%. The result of secondary screening validated the inhibition of10compounds again. We further validated the10compounds on WNV and DENV NS2B/NS3protease, the result showed that,2compounds (The numbers in chemical library:AG-690/40639011, AC-907/25005011) can inhibit the WNV and DENV NS2B/NS3protease activity to some extent.3. Antiviral activity of the compounds on the cellular levelWe tested the antiviral activity of the10compounds in BHK cells, each compound was diluted to four concentrations, from100μM to12.5μM. The result indicated that, among the10compounds,6compounds have cytotoxicity,2compounds have no inhibition to JEV infection, and the remaining2compounds (The numbers in compound library:AG-690/40639011, AC-907/25005011) can inhibit the proliferation of JEV. We used indirect immunoinfluscent assay and western blot to detect the variation of JEV protein expression, further confirmed the antiviral effect of two chemical compounds AG-690/40639011and AC-907/25005011. The results showed that compared with the control groups, protein expression in the treated groups with chemical compounds added has decreased.With the increase of concentration of compounds, protein expression decreased. It suggests that the two compounds have good antiviral effect.4. The preparation of Monoclonal Antibodies against JEV NS2B/NS3proteaseWe immunized the BALB/c mice with JEV NS2B/NS3protease, after cell fusion and screening by indirect enzyme-linked immunosorbent assay, we got three hybridoma cell strains secreting McAbs, named as2E3,2C10,3C4, respectively. The results of ELISA and Western blot showed that these three McAbs were specific for JEV NS3protein.