Acetophenone 4-nitrophenylhydrazone inhibits Hepatitis B virus replication by modulating capsid assembly
Introduction
Hepatitis B virus (HBV) is the causative agent of acute and chronic hepatitis in humans. According to the World Health Organization estimates in 2015, approximately 257 million individuals were chronically infected with HBV worldwide, resulting in 887,000 deaths due to subsequent hepatic cirrhosis and hepatocellular carcinoma, which is comparable to that of HIV and tuberculosis (World Health Organization, 2016, 2020). Current antiviral drugs approved by the US Food and Drug Administration include immunomodulators, such as interferon-α and pegylated interferon-α, and nucleos(t)ide analogs, which are useful for the suppression of disease progression. However, these drug classes have certain limitations. Immunomodulators have low response rates and frequent side effects. Nucleos(t)ide analogs must be administered long-term to prevent viral reactivation because of their inability to eliminate covalently closed circular DNA (cccDNA) in infected hepatocytes (Guo and Guo, 2015). Thus, there remains an unmet need for the development of novel anti-HBV drugs that allow treatment cessation.
A pragmatic treatment endpoint of chronic HBV infection is achieved by a functional cure defined as seroclearance of hepatitis B surface antigen (HBsAg) with sustained viral suppression, which is correlated with a dramatic reduction of the incidence of hepatocellular carcinoma (Yang et al., 2002; Durantel and Zoulim, 2016; Pei et al., 2017). To enhance the rate of a functional cure, a combination or sequential therapeutic approach using multiple classes of drugs with distinct modes of action is needed: profound suppression of viral genome replication to block cccDNA replenishment, inhibition of viral antigen secretion, and activation or restoration of host immune response against HBV (Seto and Yuen, 2016). Future drug development for HBV treatment is likely to depend on these strategies.
HBV genome replication is initiated by the transcription of pregenomic RNA (pgRNA) from cccDNA, followed by its translation into core proteins and viral polymerase. The polymerase-bound pgRNA is encapsidated into core particles and reverse transcribed into minus-strand DNA. The polymerase subsequently synthesizes the plus-strand DNA to generate a relaxed circular DNA (rcDNA). The mature nucleocapsids are then used to produce progeny virions or recycled to the nucleus to supply the cccDNA pool (Beck and Nassal, 2007; Seeger and Mason, 2015; Takizawa and Yamasaki, 2017). While current nucleos(t)ide inhibitors for anti-HBV treatment can be highly effective in preventing liver disease progression by competitively inhibiting DNA elongation, faint viral replication with undetectable levels by a commercially available PCR still occurs, which may give rise to cccDNA replenishment (Durantel and Zoulim, 2016).
Recently, we established a new system, named HBV103-AdV, which was successful in increasing the accumulation of viral DNA using an adenovirus vector (Suzuki et al., 2017). In addition to even and effective transduction of the viral genome into cells, the application of the CMV promoter and rabbit β-globin poly(A) signal in the construct supported high expression and stabilization of pgRNA, which resulted in 58-fold more transcripts as compared to using native promoter and poly(A) signal. Moreover, a replication-competent 1.03-copy HBV genome, generated by the deletion of most of the preS1 and preS2 regions, achieved a boost in DNA synthesis due to the lack of S protein production. Consequently, the system permits us to evaluate the inhibitory potency of the test compounds in a short experimental period of only 4 days, with a Z’ factor of 0.64, proving its suitability for hit identification in drug screening.
In this study, we screened an in-house compound library using the HBV103-AdV system to identify a distinct chemical structure from known inhibitors of HBV genome replication. We report that N-(4-Nitrophenyl)-1-phenylethanone hydrazone, abbreviated as ANPH, selectively inhibits HBV by modulating capsid assembly and inducing the formation of empty capsid devoid of pgRNA. Moreover, size exclusion chromatography (SEC) and transmission electron microscopy (TEM) studies revealed that ANPH accelerated capsid assembly in vitro with the same morphology and size as the normal capsid.
Section snippets
Compounds
ANPH was synthesized by refluxing a suspension of acetophenone (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and 4-nitrophenylhydrazine hydrochloride (Tokyo Chemical Industry Co., Ltd.) in ethanol for 1 h at 50°C. The precipitated product was filtered and washed with distilled water, and its structure and purity were verified by high-resolution mass spectrometry (LTQ-Orbitrap XL mass spectrometer; Thermo Fisher Scientific, Waltham, MA, USA) and NMR spectroscopy (Bruker AVANCE III 600
Screening for inhibitors of HBV genome replication
Improvement of current antiviral therapy by inhibiting the replication of the HBV genome might prevent the de novo replenishment of the cccDNA pool in the nucleus, which causes persistent infection (Durantel and Zoulim, 2016). In this study, to identify a novel class of selective inhibitors for HBV genome replication, we screened an in-house compound library using the HBV103-AdV system that has been previously established to facilitate the detection of the replicating viral genome using an
Discussion
Approved nucleos(t)ide analogs for the treatment of HBV can be highly effective in preventing liver disease progression, which provides a proof-of-concept for viral genome replication as a promising drug target. Although maximizing the suppression of cccDNA replenishment is crucial for achieving an increased rate of functional cure, the current nucleos(t)ide inhibitors do not significantly reduce cccDNA in patients due to residual viral replication (Doo and Ghany, 2010; Cheng et al., 2011;
CRediT authorship contribution statement
Manabu Yamasaki: Investigation, Writing – original draft, Funding acquisition. Norie Matsuda: Investigation. Kazuaki Matoba: Investigation, Resources. Saki Kondo: Methodology, Resources. Yumi Kanegae: Methodology, Resources, Funding acquisition. Izumu Saito: Methodology, Writing – review & editing. Akio Nomoto: Conceptualization, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We are grateful to Prof. Tetsuro Suzuki (Hamamatsu University School of Medicine) for generously supplying the anti-HBcAg antibody. We also thank Dr. Mariko Suzuki (The Institute of Medical Science, The University of Tokyo) for the preparation of AdVs, Dr. Kunio Ishiki for his technical support with the synthesis of ANPH, Dr. Kiyoko Iijima for HRESI-MS and NMR measurements, and Ms. Yui Yamamoto for her technical support with qPCR and Western blot analysis. We would like to thank Editage (//www.editage.com
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