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

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 28.1 µM and an EC50 of 4.2 µM. 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 75 µM, 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.

Synergistic Interactions between Hepatitis B Virus RNase H Antagonists and Other Inhibitors

“…HepDES19 cells are HepG2 hepatoblastoma cells with a tetracycline-repressible expression cassette for a replication-competent HBV genotype D genome (28) that express high levels of HBV. We determined anti-HBV activity of the compounds by treating cells replicating HBV following release of tetracycline suppression with the test compounds and quantifying the HBV minus-and plus-polarity DNA strands in core particles by quantitative PCR (qPCR) (19,20). Two HBV RNase H inhibitors that we previously identified from different chemical scaffolds were selected for this study: #1 (19) and #46 (20) (Fig.…”

“…Encapsidated HBV DNA was then extracted using the Qiagen QIAamp cador Pathogen minikit (Qiagen). Quantitative PCR (qPCR) was performed with a strand-preferential assay with TaqMan reagents as described previously (19). The amounts of plus-and minus-polarity DNA strands were calculated as percentages relative to the quantity of DNA in DMSOtreated cells.…”

“…Therefore, the RNase H is an attractive target for new anti-HBV drugs that may be used in combination with the NAs to increase effectiveness of treatment (15,16). Recently, we identified several promising compounds that inhibit both HBV RNase H activity and viral DNA synthesis (17)(18)(19)(20). In addition to RNase H inhibitors, several other new agents to inhibit viral replication that target different critical steps in the HBV replication cycle are being developed.…”

Tratamiento actual y nuevas terapias contra la infección crónica por el virus de la Hepatitis B

“…La proteína transmembrana estimuladora de genes de IFN (STING) es una proteína adaptadora de múltiples receptores citoplasmáticos y también actúa como un PRR que reconoce segundos mensajeros bacterianos (43). La estimulación de STING ha mostrado un aumento significativo del IFN-I con supresión de la replicación viral del VHB en los hepatocitos de ratones infectados (32), lo que abre un nuevo campo de estudio terapéutico.…”

“…La inhibición de esta enzima está ampliamente conocida como parte del tratamiento contra el VIH, pero poco se ha estudiado sobre su uso como tratamiento para el VHB, con algunos estudios in vitro que sugieren su efectividad (43,73). El objetivo de los medicamentos que comparten este mecanismo de acción es suprimir la replicación viral para detener el reaprovisionamiento de ADNccc en el hígado, lo que permite eliminar las células infectadas por el sistema inmune o por otro medicamento concomitante (66,74).…”