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. 2014 Aug:108:48-55.
doi: 10.1016/j.antiviral.2014.05.007. Epub 2014 May 20.

Hepatitis B virus replication is blocked by a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) inhibitor of the viral ribonuclease H activity

Catherine W Cai  1 , Elena Lomonosova  2 , Eileen A Moran  3 , Xiaohong Cheng  4 , Kunjan B Patel  5 , Fabrice Bailly  6 , Philippe Cotelle  7 , Marvin J Meyers  8 , John E Tavis  9
Affiliations

Hepatitis B virus replication is blocked by a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) inhibitor of the viral ribonuclease H activity

Catherine W Cai et al. Antiviral Res. 2014 Aug.

Abstract

Nucleos(t)ide analog drugs profoundly suppress Hepatitis B virus (HBV) replication but rarely cure the infection, so therapy is usually life-long. The nucleos(t)ide analogs inhibit the viral DNA polymerase and often push HBV to the brink of extinction, so it may be possible to eradicate HBV by suppressing HBV replication further. The HBV ribonuclease H (RNaseH) is a logical new drug target because it is the second of only two viral enzymes essential for viral replication. We recently developed a low throughput screening pipeline for inhibitors of the HBV RNaseH and viral replication. Here, we screened a series of twenty-three nitrogen-based polyoxygenated heterocycles including sixteen 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives for anti-HBV RNaseH activity. Nine compounds inhibited the HBV RNaseH, but activity was marginal for eight of them. Compound #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione, HID] was the best hit with an IC50 of 28.1μM and an EC50 of 4.2μM. It preferentially suppressed accumulation of the viral plus-polarity DNA strand in replication inhibition assays, indicating that replication was blocked due to suppression of HBV RNaseH activity. It had a CC50 of 75μM, yielding a therapeutic index of ∼18. The EC50 value was 7-fold lower than the IC50, possibly due to cellular retention or metabolism of the compound, or higher affinity for the full-length enzyme than the recombinant form used for screening. These data indicate that the 2-hydroxyisoquinoline-1,3(2H,4H)-diones will have different structure-activity relationships for the HBV and HIV RNaseHs. Therefore, HID compounds may provide a foundation for development of more effective RNaseH inhibitors of HBV replication.

Keywords: 2-Hydroxyisoquinoline-13(2H,4H)-diones; Hepatitis B virus; Ribonuclease H; Viral replication.

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Figures

Fig. 1
Fig. 1. Structures of the compounds used or discussed in this study
Fig. 2
Fig. 2. Representative primary biochemical screening assays for the HBV RNaseH
A. The oligonucleotide-directed RNaseH assay. Internally radiolabeled RNA is bound to a complementary DNA oligonucleotide, and the RNaseH cleaves the RNA within the DNA:RNA heteroduplexes. RNA, black line; DNA, grey line; S, RNA substrate; P1 and P2, RNaseH cleavage products. B. Representative primary screening assay employing HBV genotype D RNaseH. DMSO, vehicle control. C. E. coli RNaseH activity was not affected by compound #1 in the oligonucleotide-directed cleavage assay under identical conditions.
Fig. 3
Fig. 3. Dose-response curves with compound #1 in the oligonucleotide-directed RNA cleavage assay against the HBV RNaseH
A. Genotype D RNaseH. B. Genotype C RNaseH. The curves are from representative assays. The numerical values are the average ± one standard deviation from three or four independent assays.
Fig. 4
Fig. 4. Effect of compound #1 on HBV replication
A. Basis for the strand-preferential PCR reactions. The minus-polarity preferential primers/probe are upstream of the start site for the plus-polarity DNA, and the plus-polarity preferential primers/probe cross the gap in the minus-polarity DNA. Grey, minus-polarity DNA strand; black, plus-polarity strand; oval, the covalently-attached viral polymerase protein; arrows, 3’ ends of the DNAs. B. Relative sensitivity of the plus- and minus-polarity preferential primers against a double-stranded HBV DNA template. A linear HBV DNA was serially diluted and used as a template for quantitative PCR employing the strand-preferential primers. Filled circles, plus-polarity preferential primers; open circles, minus-polarity preferential primers. Error bars are ± one standard deviation from three independent assays. C. HBV induction kinetics in HepDES19 cells. Tetracycline was withdrawn from the medium, cytoplasmic HBV capsid particles were purified 1 to 6 days later, and plus-polarity HBV DNA derived from the capsid particles was measured by quantitative PCR. D. Inhibition of HBV plus-polarity synthesis by compound #1. Viral nucleic acids were purified from cytoplasmic capsid particles from cells replicating HBV in the presence of varying concentrations of compound #1. Quantitative PCR preferential for plus- and minus-polarity strands of HBV DNA was performed to measure the effect of compound # 1 on accumulation of plus- (black) and minus- (grey) strand HBV DNA. The results were normalized to the DMSO vehicle control. Data are the mean ± one standard error of the mean from three independent experiments each performed in duplicate.
Fig. 5
Fig. 5. MTT toxicity assay for compound #1
An MTT assay was conducted in the presence of varying concentrations of compound #1 under culture conditions analogous to the replication assay in Fig. 4D. The curve is from a representative assay. The CC50 value is the average ± one standard deviation from three independent assays.
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