Hydroxylated Tropolones Inhibit Hepatitis B Virus Replication By Blocking The Viral Ribonuclease H Activity

BackgroundHepatitis B virus(HBV) infection is a major cause of viral hepatitis worldwide. Every year, HBV infection accounts more than 1 million people deaths from cirrhosis, liver failure, and hepatocellular carcinoma. HBV is a hepatotropic DNA virus that replicates by reverse transcription. HBV has eight genotypes(A–H) that differ by ﹥8% at the sequence level. Two kinds of drugs including interferon α and nucleos(t)ide analog drugs have been approved for treating HBV infection. However, interferon suffer from a number of limitations including the poor long-term responses, very low cure rates and the high frequency of adverse side effects. The loss of the viral surface antigen(HBs Ag) from the serum in only 3–6% of patients, resurgence of viral replication almost always following termination of therapy, long term drug administration and emergence of HBV variants resistant to antiviral therapy make nucleos(t)ide analog drugs far from an ideal anti-HBV drug. It is therefore important to continue research to identify new anti-HBV targets and discover novel compounds with unique mode of action for controlling viral replication.HBV encodes two enzymes essential for its reverse transcription: a DNA polymerase and a ribonuclease H(RNase H). HBV DN A replication can be inhibited by blocking either the DNA polymerase or the RNase H activity. The nucleos(t)ide analogs targeting DNA polymerase inhibit HBV DNA synthesis and suppress viral replication often to below the standard clinical detection limit. The failure to clear the virus is due to the residual and low- level viral replication. The additive suppression of HBV DNA replication from combining DNA polymerase and RNase H inhibitors should substantially increase the possibility to clear the ccc DNA. So the purification of HBV RNase H with activity is the first step to screen the HBV RNase H inhibitor in vitro.The screening of HBV RNase H inhibitor from so many natural and synthesized compounds is the second step to develop anti-HBV RNase H medicine. It is reported that many troponoids can inhibit the human immunodeficiency virus(HIV) RNase H. Both the HBV and HIV are the virus that replicate by reverse transcription. It is demonstrated that many nucleos(t)ide analogs can inhibit both HIV and HBV polymerase. Both the HBV and HIV RNase H enzymes belong to the nucleotidyl transferase superfamily. So the hypothesis is that many troponoids may also inhibit the HBV RNase H.Aims1. To develop a biochemical screening system for inhibitors of the HBV RNase H by producing active recombinant HBV RNase H.2. To screen the troponoids compounds inhibiting the HBV RNase H and HBV DNA replication in preparation for anti-HBV RNase H drug development.3. To assess the activity of multiple genotypes HBV RNase H and compare the inhibitory activities of troponoids compounds to different genotype HBV RNase H.Methods1. Construction of plasmids for HBV RNase H and human RNase H1 expression. Codon-optimized c DN A sequences for genotype D and C HBV RNase H(HRHLP-gt.D &HRHLP-gt.C) were cloned by gene synthesis into p Trc His2 B with a C-terminal hexahistidine tag. Codon-optimized c DNA sequences for genotype C HBV RNase H(MBP-gt.C) were cloned by gene synthesis into p MAL-c5 x His with a N-terminal MBP tag and C-terminal hexahistidine tag. The human RNAse H1 gene was cloned with an N-terminal hexahistidine-tag into p Rset B by gene synthesis.2. RNase H expression and enrichment. HBV RNase H and human RN Ase H1 were expressed in LO BSTR E. coli BL21 cells. RNase H proteins were enriched by nickel–agarose affinity chromatography.3. RNase H identify. The RNase H proteins purified were identified through SDS-PAGE gel electrophoresis and Western Blot.4. Activity assay of HBV RNase H and human RNase H1. RNase H activity was measured using an oligonucleotide-directed RNA cleavage assay. Protein extract was mixed with an internally 32P-labeled 264 nt RNA derived from the Duck Hepatitis B Virus genome(DRF+ RNA) plus complement oligonucleotide. C leavage products from the DRF+ RN A were resolved by denaturing polyacrylamide gel electrophoresis, detected by autoradiography.5. Inhibition assay of troponoids against HBV RNase H and human RNase H1. Fifty-one troponoid compounds were screened for their ability to suppress HBV RNase H activity using the oligonucleotide-directed RNA cleavage assay. Compounds with ability to suppress RNase H activity decreased the products from the cleavage of DRF+ RNA. C leavage products were resolved by denaturing polyacrylamide gel electrophoresis, detected by autoradiography, and quantified using Image J.6. HBV replication assay. Inhibition of HBV replication was measured in Hep DES19 cells. Cells were seeded into 6-well plates and incubated. The test compound was applied to duplicate wells 48 hours later and medium containing the compound was refreshed daily for the following two days. Cells were harvested and HBV DNA was purified from capsids. Taq Man PCR was performed to quantify the plus-polarity strand and minus-polarity strand of the HBV DNA.7. MTT cytotoxicity assays. Hep DES19 cells were seeded in 96-well plates and incubated. Compounds were diluted in medium to the indicated concentrations and added to cells 24 hours after plating, with each concentration tested in triplicate. Medium containing the compound was refreshed daily for the next two days. Thiazolyl blue tetrazolium bromide(MTT) was added to the well, the cultures were incubated for 60 minutes, metabolites were solubilized in acidic isopropanol, and absorbance was read at 570 n M.8. Assess activity of the RNase H inhibitors against mutiple HBV genotypes. Eighteen expression plasmid for genotype/sub-genotype B, C and D HBV RNase H were cloned. HBV RNase H with different genotype were purified by nickel–agarose affinity chromatography. The oligonucleotide-directed RNA cleavage assay was used to assess the activity of mutiple HBV RNase H and compare the inhibitory activities of compound #46 to different genotype HBV RNase H.Results1. Only small amounts of soluble recombinant HBV RNase H( HRHLP-gt.D and HRHLP-gt.C) were expressed and enriched, but detectable by Western blot analysis. Large amounts of soluble recombinant HBV RNase H(MBP gt.C) were expressed and purified. Purified MBP gt.C could be detected by both Coomassie staining and Western blot analysis.2. Data demonstrate that all the three HBV RNase H( HRHLP-gt.D、HRHLP-gt.C and MBP gt.C) and human RNAse H1 have specific RNase H activity.3. The activity of purified HBV RNase H was stable in a certain temperature and time.4. Fifty-one troponoids were screened for anti- HBV RNase H activity. Eight compounds had activity at only 60μM against the genotype D and/or C enzymes. Three additional compounds inhibited the genotype D and/or C enzymes at 20μM, and the two most active compounds(#46 and #106) inhibited the RNase H from both genotypes at 10 μM.5. IC50 values were determined for the six most active compounds against both genotype D and C enzymes. Four compounds(#46, 106, 107, and 110) had IC50 values ≤40μM against both the genotype D and/or C enzymes.6. Thirty- five of the 51 compounds were counter-screened against recombinant human RNase H1 in an initial effort to evaluate specificity of inhibition. Four compounds were active against the human enzyme at 10 μM, 14 had activity at 20μM or 60μM and 17 had no activity at 60μM.7. The data confirmed the different patterns of inhibition between the HBV and human enzymes, with compound #46 being 10- to 20-fold more active against the HBV enzyme, #106 being almost two- fold more active against the HBV enzyme, #110 and #112 having similar IC50 values, and #113 being about four- fold more active against the human RNase H1.8. EC50 values were determined for the six compounds which preferentially suppressed the plus-polarity DNA strand(compounds #46, 56, 106, 110, 112, and 113). All EC50 values were <10 μM, with the best value being 0.34 μM for compound #110. The minus-polarity DN A strand in the same samples was either not suppressed by treatment with the co mpounds or was suppressed at much higher compound concentrations for all compounds. This led to the therapy indexes(TI, CC 50/EC50) up to a high of 94 for compound #110.9. Cytotoxicity of the six compounds that selectively suppressed the plus-polarity DNA strand was assessed in Hep DES19 cells using an MTT assay. Moderate cytotoxicity was observed for all compounds, with CC50 values ranging from 25 to 79μM.10. Statistically difference were observed among the activity of genotype/sub- genotype B, C and D HBV RNase H(P<0.05)。There were no significantly difference among the activity of compound #46 against genotype B, C and D HBV RNase H(P>0.05)。Conclusions1. Recombinant HBV genotype B, C and D RNase H proteins with specific activity can be expressed in E.coli and enriched by nickel–agarose affinity chromatography.2. A low throughput biochemical screening system for inhibitors of the HBV RNase H is developed.3. HBV RNase H is a new anti-HBV drug target.4. Hydroxylated tropolones inhibit HBV replication by blocking the viral RNase H activity, which indicate that troponoids, and specifically ?- hydroxytropolones are promising lead candidates for development as anti- HBV drugs.5. Although the different activity among mutiple genotypes HBV RNase H, the genetic variation does not affect its sensitivity to RNase H inhibitors.