Sulfamoylbenzamide derivatives inhibit the assembly of hepatitis B virus nucleocapsids

Abstract

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.

Fig 1

Experimental demonstration of the utility of the AML12HBV10 cell-based assay for the discovery of antiviral drugs against HBV. (A) Structures of three known anti-HBV compounds. (B) AML12HBV10 cells were seeded into a 96-well plate and cultured in the absence of Tet for 24 h. Cells were then left untreated (control, columns 11 and 12) or treated with 1 μg/ml Tet (column 10), serial 2-fold dilutions of lamivudine (100 μM to 0.78 μM; columns 1 to 3), Bay 41-4109 (5 μM to 0.039 μM; columns 4 to 6), and AT-61 (25 μM to 0.39 μM; columns 7 to 9) for 2 days. Cell lysates were blotted onto a nylon membrane and hybridized with a ³²P-labeled full-length riboprobe specific to the minus strand of HBV. The HBV DNA hybridization signals were revealed by a phosphorimager and quantified with QuantityOne software (Bio-Rad). (C) The HBV DNA amounts in the drug-treated wells were expressed as percentages of the values of the mock-treated controls. The means and standard deviations (n = 3) were plotted.

Fig 2

Structures of representative hits from the high-throughput screening. General formulas of the two classes of sulfamoylbenzamides and representative hit compounds of each class are presented.

Fig 3

The antiviral activity of two representative sulfamoylbenzamide compounds against HBV in human hepatoma cells. (A) HepDES19 cells were left untreated (0) or treated with the indicated concentrations (μM) of DVR-01, DVR-23, lamivudine, or Bay 41-4109 for 4 days. Cytoplasmic core-associated HBV DNA replication intermediates were extracted and determined by Southern blot hybridizations. (B) The ratios of single-stranded (SS) to relaxed circular (RC) HBV DNA were determined.

Fig 4

Antiviral mechanism of sulfamoylbenzamide derivatives against HBV. AML12HBV10 cells were left untreated (control) or treated with the indicated concentrations of the compounds of DVR-01, DVR-56, and DVR-23 for 2 days. Bay 41-4109 (5 μM) or AT-61 (25 μM) served as a positive control. (A) Intracellular viral RNA was determined by Northern blot hybridization. 28S and 18S rRNA served as loading controls. (B) The total amounts of nucleocapsids were determined by a particle gel assay. (C) Encapsidated pgRNA was extracted and measured by Northern blotting. (D) Nucleocapsid-associated HBV DNA was quantified by alkaline treatment of nucleocapsids on the membrane following the particle gel assay and hybridized with a ³²P-labeled HBV-specific riboprobe. (E) HBV DNA replication intermediates were extracted and determined by Southern blot hybridizations. Relative HBV RNA, capsids, or DNA level in each sample is expressed as the percentage of the average level of RNA, capsids, or DNA in the two mock-treated controls and is presented underneath each of the blots. RC, DSL, and SS indicate relaxed circular, double-stranded linear, and single-stranded HBV DNA, respectively.

Fig 5

Antiviral spectrum of sulfamoylbenzamide derivatives against hepadnaviruses. HepG2 cells were transfected with plasmids pCMV-HBV, pCMV-WHV, and pCMV-DHBV. Six hours after transfection, the cells were mock treated or treated with 10 μM lamivudine, 2.5 μM Bay 41-4109, 25 μM AT-61, 10 μM DVR-01, or 5 μM DVR-23 for 3 days. Cytoplasmic core-associated viral DNA replication intermediates were extracted and determined by Southern blot hybridizations with a ³²P-labeled full-length riboprobe specific to the minus strand of HBV, WHV, and DHBV. Relative viral DNA level in each sample is expressed as the percentage of the average level of viral DNA in the two mock-treated controls (NT) and is presented underneath each of the blots. RC, DSL, and SS indicate relaxed circular, double-stranded linear, and single-stranded viral DNA, respectively.

Fig 6

Identification of the molecular target of sulfamoylbenzamide derivatives by genetic complementation assays between HBV and WHV. HepG2 cells were cotransfected with plasmids pCMVHBV/C⁻ and pcDNA3/HBVcore (A), pCMVHBV/C⁻ and pcDNA3/WHVcore (B), or pCMVHBV/C⁻ and pCMVWHV/P⁻ (C and D). Six hours after transfection, the cells were mock treated or treated with 10 μM lamivudine, 2.5 μM Bay 41-4109, 25 μM AT-61, or 10 μM DVR-01 for 3 days. Cytoplasmic core-associated viral DNA replication intermediates were extracted and determined by Southern blot hybridizations with a ³²P-labeled full-length riboprobe specific to the minus strand of HBV (A, B, and C) and WHV (D). RC and SS indicate relaxed circular and single-stranded HBV DNA, respectively. The sources of the capsid protein, polymerase, pgRNA, and probe for hybridization for each of the experiments are presented below each blot.