Study On The Structure Optimization And Anti-hepatitis B Virus Activity Of Novel Human La Protein Inhibitor HBSC11

Background: Previously,we found HBSC11 can combine with La protein conferring anti-HBV effects.The purpose of this study was to optimize,synthesize and evaluate anti-HBV activity of compounds as well as to get favorable physiologic properties of potential molecular non-nucleoside agents in anti-HBV therapy.Methods: 1.10 derivatives(3a-3f,5a-5d)were obtained by 2 sections-scaffold and kept the active site which predicted by computer.The new compounds were then docked to the human La protein by molecular doking.2.Cytotoxicity and anti-HBV activities were assayed with cell counting kit-8(CCK-8),enzyme linked immunosorbent assay(ELISA)and real-time PCR.3.An HBV mouse model established by hydrodynamic injection was used to assess anti-HBV activity of promising candidates.HBc Ag in liver tissue were assayed by immunohistochemical staining.The level of ALT was detected by bioluminescence.Pathological changes in liver carried by Hematoxylin and eosin(HE)staining.4.RT-PCR,Western blot,ELISA and immunohistochemical(IHC)were used to detect the changes of m RNA level,protein expression level and cellular localization of La protein.5.Leading compound HBSC11 and its active derivatives 3a,5a and 5b were predicted for their compliance with the Lipinski’s rule of five using free online software.Molecular dynamics simulation studies on candidate compound 5a and Leading compound HBSC11 to verify the combine with La protein.Results: 1.10 derivatives(3a-3f,5a-5d)were obtained by 2 sections-scaffold successfully and were docked to the human La protein using the Glide software.2.we identified 3 active derivatives that showed improved anti-HBV activity and lower cytotoxicity,and they all conformed well to the Lipinski’s rule of five.Especially,candidate compound 5a showed similar activity as entecavir by reducing the level of HBV antigen approximately to 50%.And in a mouse model 5a has 98.9% inhibition rate of HBV DNA,57.4% of HBs Ag,46.4% of HBe Ag,compared 90.8%,3.8%,9.8% respectively in control group.3.The results of molecular dynamics simulation studies show the scroe of leading compound HBSC11 is-4.1443 and candidate compound 5a is-4.1123,HBSC11 is combine better than 5a with La protein.4.The HBV model was successfully constructed.The level of La protein in this model is related to HBV expression.The m RNA level of La protein in the liver of the mice was generally higher than that in the control group.However,Western bolt results showed that there was no significant difference in intracellular protein levels,but the level of La protein in serum of mice was generally higher than control group,especially on the 7th day(P < 0.0001).Immunofluorescence assay showed a decreasing trend of La protein in the hepatocytes after administration,especially in the nucleus.Conclusions: 10 derivatives(3a-3f,5a-5d)of HBSC11 were obtained successfully.All results indicated that candidate compound 5a was a promising anti-HBV compound,which has higher anti-HBV activity and lower cytotoxicity.Our study not only provides novel experimental evidence of anti-HBV therapy but also offers the molecular characterization of La protein involved in the pathogenesis of HBV.