Discovery of Novel Hepatitis B Virus Nucleocapsid Assembly Inhibitors

Abstract

Hepatitis B virus (HBV) core protein is a small protein with 183 amino acid residues and assembles the pregenomic (pg) RNA and viral DNA polymerase to form nucleocapsids. During the last decades, several groups have reported HBV core protein allosteric modulators (CpAMs) with distinct chemical structures. CpAMs bind to the hydrophobic HAP pocket located at the dimer-dimer interface and induce allosteric conformational changes in the core protein subunits. While Type I CpAMs, heteroaryldihydropyrimidine (HAP) derivatives, misdirect core protein dimers to assemble noncapsid polymers, Type II CpAMs, represented by sulfamoylbenzamides, phenylpropenamides, and several other chemotypes, induce the assembly of empty capsids with global structural alterations and faster mobility in native agarose gel electrophoresis. Through high throughput screening of an Asinex small molecule library containing 19 920 compounds, we identified 8 structurally distinct CpAMs. While 7 of those compounds are typical Type II CpAMs, a novel benzamide derivative, designated as BA-53038B, induced the formation of morphologically "normal" empty capsids with slow electrophoresis mobility. Drug resistant profile analyses indicated that BA-53038B most likely bound to the HAP pocket but obviously modulated HBV capsid assembly in a distinct manner. BA-53038B and other CpAMs reported herein provide novel structure scaffolds for the development of core protein-targeted antiviral agents for the treatment of chronic hepatitis B.

Keywords: antiviral; capsid assembly; core protein allosteric modulator; hepatitis B virus; nucleocapsids.

Fig. 1.

Structure of representative HBV core protein allosteric modulators (CpAMs).

Fig. 2.. The novel antiviral compounds induced the capsid/nucleocapsid mobility shift and reduced the amount of hypophosphorylated core protein.

(A and B) AML12HBV10 cells were cultured in the absence of tetracycline (tet) and treated with 5 μM of compounds 1 to 7, BA-53038B, DVR-23 or 2 μM of Bay 41–4109, 1 μM of ETV. Two days post treatment, cytoplasmic HBV capsids were resolved by a 1.8 % agarose gel electrophoresis followed by transferring on to a nitrocellulose membrane. core protein was detected by antibody Dako (A, upper panel). Capsid-associated HBV DNA was detected by using a ³²P-labeled riboprobe to detect minus strand HBV DNA. (A). Intracellular core protein was detected with antibody HBc-170A in a Western blot assay. (B). (C) AML12HBV10 cells were cultured in the absence of tet and treated with 5 μM of DVR-23 or BA-53038B for 2 days. Cell lysates from HepG2 cells transfected with plasmids expressing wild-type or mutant core proteins (pCI-HBc-WT, pCI-HBc3A or pCI-HBc7A) were run in parallel to serve as size markers for core protein with different level of phosphorylation. Intracellular core protein was detected with antibody HBc-170A in a Western blot assay. β-actin served as a loading control.

Fig. 3.. BA-53038B inhibited HBV replication in both AML12HBV10 and HepDES19 cells.

AML12HBV10 (A) and HepDES19 (B) cells were cultured in the absence of tet and treated with BA-53038B for 2 and 6 days, respectively. (A) Cytoplasmic HBV core DNA were quantified by dot blot hybridization. (B) Cytoplasmic HBV core DNA was detected by a real-time PCR assay. Data were plotted as a fraction of that in mock-treated cells (n=3). Cytotoxicity of the compound were determined by a MTT assay.

Fig. 4.. BA-53038B inhibited the assembly of pgRNA-containing nucleocapsids.

AML12HBV10 cells were cultured in the absence of tet and treated with the indicated concentrations of BA-53038B, 5 μM of DVR-23, 2 μM of Bay 41–4109 or 1 μM of ETV. Two days post treatment, total HBV RNA (A) and encapsidated pgRNA (B) were detected by Northern blot hybridization with a ³²P-labeled riboprobe to detect minus-strand HBV DNA. 28S and 18S rRNA served as loading controls. (C) Cytoplasmic HBV capsids were resolved by a 1.8 % agarose gel electrophoresis followed by detection of capsid with antibody against core protein and HBV DNA, as described in Fig.2A legend. (D) HBV DNA replication intermediates were detected by Southern blot hybridization. rcDNA, dslDNA and ssDNA are relaxed circular, double-stranded linear and single-stranded forms of HBV DNA, respectively.

Fig. 5.. BA-38058B induced the formation of distinct HBV capsids in HepG2 cells.

HepDES19 cells were culture in the absence of tetracycline and treated with Bay 41–4109 (2 μM), DVR-23 (5 μM), BA-38017 (5 μM), BA-53038B (5 μM), AT-61 (25 μM) or ETV (1 μM) for 6 days. (A) The total amounts of capsids and the capsid-associated HBV DNA were determined using a particle gel assay in a 1.8 % agarose gel electrophoresis as described in Fig. 2A legend. (B) HBV DNA replication intermediates were determined by Southern blot analysis. rcDNA, relaxed circular DNA. dslDNA, double-stranded linear DNA. ssDNA, single-stranded DNA.

Fig. 6.. Physical properties of capsids derived from BA-53038B treated cells.

(A) AML12HBV10 cells were cultured in the absence of tetracycline and treated with 5 μM of BA-53038B. Two days poster treatment, the intracellular capsids were sedimented on a 15% to 30% sucrose. Total of 24fractions were collected from the bottom. 1/30 volume of each fraction was spotted on Nylon membrane for detection of HBV core protein (HBc) with Dako antibody followed by detection of minus strand HBV DNA using HBV riboprobe. (B) HBV capsids from fractions 3–12 were purified by ultracentrifugation and subjected to Western blot to detect core protein with antibody HBc-170A and analyses of capsids and their associated HBV DNA in 1.8 % native agarose gel assay. (C) Electronic microscopic graphs of the capsids purified from AML12HBV10 cells in presence and absence of 5 μM of BA-53038B treatment. Scale bar is 100 nm.

Fig. 7.. Effects of BA-53038B derivatives on HBV replication in HepAD38 cells.

(A) Structure of BA-53038B and derivatives. (B) HepAD38 cells were cultured in the absence of tetracycline and treated with the indicated concentration of compound for 6 days. Cytoplasmic HBV core DNA was determined by a real-time PCR assay and plotted as a fraction of that in mock-treated cells (n=3).

Fig. 8.. Substitution of core protein valine (V) 124 with tryptophan (W) conferred resistant to BA-53038B induced capsid mobility shift and HBV DNA synthesis.

HepG2 cells were transfected with pHBV1.3 or pHBV1.3-derived plasmids expressing core protein with indicated substitution at V124. The cells were treated with 2 μM of Bay 41–4109, 5 μM of DVR-23 or BA-53038B starting at 6 h post transfection. Cytoplasmic capsids were analyzed 3 days post treatment by a particle gel assay. Capsid-associated minus-strand HBV DNA was detected with a ³²P-labeled riboprobe.