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



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.


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.


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.


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


HBV Polymerase Non-nucleoside Reverse transcription Replication 





Aryl hydrocarbon receptor


Bovine serum albumin


Covalently closed circular DNA


Double-stranded DNA


Dimethyl sulfoxide




Ethylenediaminetetraacetic acid






HBV surface protein


Fluorescein isothiocyanate


Hepatitis B virus


Hepatitis C virus


Hepatitis D virus


Horseradish peroxidase


Half-maximal inhibitory concentration




Large HBV surface protein




Moloney murine leukemia virus


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


Non-nucleos(t)ide analog reverse transcriptase inhibitor


Sodium taurocholate co-transporting polypeptide


Nucleos(t)ide analog reverse transcriptase inhibitor


Polyvinylidene difluoride


Pregenomic RNA


Relaxed circular DNA

RNase H

Ribonuclease H


Reverse transcriptase


Surface plasmon resonance


Tenofovir alafenamide




Terminal protein



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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