Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly

Abstract

Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein.Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV.IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating, and covalently closed circular DNA (cccDNA) formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. The discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported here, and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B.KEYWORDS antiviral agents, capsid assembly, hepatitis B virus

Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation

“…7A and B). Also, consistent with previous reports (27,31) and the results presented in Fig. 1, while 17-DMAG treatment did not alter the migration of HBV capsids in the native agarose gel electrophoresis, ENAN-34017 treatment induced the formation of fastermigrating capsids (Fig.…”

“…Although we demonstrated previously that the CTD of HBV core protein did not play a role in CpAM-induced assembly of structurally altered empty capsids, its role in CpAM-induced suppression of pgRNA encapsidation remains to be determined (27).…”

“…To further investigate whether interference of core protein dimerdimer interaction at the HAP pocket during capsid assembly disrupts the dynamic phosphorylation and dephosphorylation of core proteins, we took advantage of wellcharacterized mutant core proteins with point mutations of critical amino acid residues on the wall of the HAP pocket that mimics CpAM effects on capsid assembly and pgRNA encapsidation (32,33). Specifically, as reported previously, while the wild-type core protein in pHBV1.3-transfected HepG2 cells supported the assembly of both slowand fast-migrating capsids, V124A and V124W mutant core proteins supported the assembly of predominantly slow-and fast-migrating capsids, respectively (27) (Fig. 8A).…”

Role of Hepatitis B virus capsid phosphorylation in nucleocapsid disassembly and covalently closed circular DNA formation

“…Some of these compounds can inhibit capsid assembly or pgRNA packaging. Furthermore, consistent with the pleiotropic role of HBc in multiple stages of viral replication beyond capsid assembly and pgRNA packaging, particularly in controlling NC uncoating, HBc-targeted compounds have recently been shown to affect CCC DNA formation, possibly via modulating NC disassembly/uncoating [74][75][76][77][78][79][80][81]. Our results here further suggest that HBc-targeted agents may affect CCC DNA formation beyond NC uncoating by influencing events inside the nucleus.…”

Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay

“…As a mechanism of action proposed by Zlotnick et al, CpAMs enhance but misdirect the kinetics of capsid assembly, preventing the packaging of viral pregenomic RNA and polymerase complex into the capsid (5). However, none of the reported CpAMs has shown enhanced capsid formation in particle gel assays (13,18,21,(45)(46)(47)(48)(49)(50)(51), and thus 20deoxyingenol represents a novel reference compound to demonstrate that a CpAM could promote capsid formation but inhibit HBV DNA replication.…”