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Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay

  • Shogo Nakajima
  • Koichi Watashi
  • Kento Fukano
  • Senko Tsukuda
  • Kousho Wakae
  • Hideki Aizaki
  • Masamichi Muramatsu
  • Takaji WakitaEmail author
  • Tetsuya ToyodaEmail author
Original Article—Liver, Pancreas, and Biliary Tract
  • 72 Downloads

Abstract

Background

Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro.

Methods

We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase.

Results

We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 μM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs.

Conclusions

We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

Keywords

HBV Polymerase Non-nucleoside Reverse transcription Replication 

Abbreviations

ADV

Adefovir

AhR

Aryl hydrocarbon receptor

BSA

Bovine serum albumin

cccDNA

Covalently closed circular DNA

dsDNA

Double-stranded DNA

DMSO

Dimethyl sulfoxide

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

ETV

Entecavir

FAM

6-Carboxyfluorescein

HBs

HBV surface protein

FITC

Fluorescein isothiocyanate

HBV

Hepatitis B virus

HCV

Hepatitis C virus

HDV

Hepatitis D virus

HRP

Horseradish peroxidase

IC50

Half-maximal inhibitory concentration

IFN

Interferon

LHBs

Large HBV surface protein

LMV

Lamivudine

MMLV

Moloney murine leukemia virus

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

NNRTI

Non-nucleos(t)ide analog reverse transcriptase inhibitor

NTCP

Sodium taurocholate co-transporting polypeptide

NRTI

Nucleos(t)ide analog reverse transcriptase inhibitor

PVDF

Polyvinylidene difluoride

pgRNA

Pregenomic RNA

rcDNA

Relaxed circular DNA

RNase H

Ribonuclease H

RT

Reverse transcriptase

SPR

Surface plasmon resonance

TAF

Tenofovir alafenamide

TAMRA

Carboxytetramethylrhodamine

TP

Terminal protein

Notes

Acknowledgements

We thank Dr. Y. Tanaka (Nagoya City University) for the gift of the HBV genotype-B plasmid. This study was supported in part by the Japan Society for the Promotion of Science KAKENHI (JP17H04085, 17K09447); the JST CREST and MIRAI program; the Ministry of Health, Labor and Welfare (15fk0310005h0004); the Japan Agency for Medical Research and Development, AMED (JP19fk0310114j0003, JP19fk0310101j1003, JP19fk0310103j0203, JP19fk0310103j0303, JP19fk0310103h1203, JP19fm0208019j0003, JP19fk0210036j0002, JP19fk0210053j1001, JP16fk0310503h1505, JP17fk0310103h0701, and JP18fk0310103h1202); and the Takeda Science Foundation.

Author contributions

Conceptualization: KW, TW, and TT; methodology: SN, KW, and TT; experiments: SN, KW, and KF; analysis: SN, KW, KF, ST, KW, HA, MM, TW, and TT; writing: SN, KW, ST, and TT; supervision: KW and TT; funding acquisition: KW, MM, and TW, TT.

Supplementary material

535_2019_1643_MOESM1_ESM.docx (734 kb)
Supplementary file1 (DOCX 733 kb)

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Copyright information

© Japanese Society of Gastroenterology 2019

Authors and Affiliations

  1. 1.Department of Virology IINational Institute of Infectious DiseasesTokyoJapan
  2. 2.Department of Applied Biological ScienceTokyo University of ScienceNodaJapan
  3. 3.Choju Medical InstituteFukushimura HospitalToyohashiJapan
  4. 4.CREST, JSTSaitamaJapan
  5. 5.MIRAI, JSTSaitamaJapan
  6. 6.Liver Cancer Prevention Research Unit, Center for Integrative Medical SciencesRIKENWakoJapan

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