Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay
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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.
KeywordsHBV Polymerase Non-nucleoside Reverse transcription Replication
Aryl hydrocarbon receptor
Bovine serum albumin
Covalently closed circular DNA
HBV surface protein
Hepatitis B virus
Hepatitis C virus
Hepatitis D virus
Half-maximal inhibitory concentration
Large HBV surface protein
Moloney murine leukemia virus
Non-nucleos(t)ide analog reverse transcriptase inhibitor
Sodium taurocholate co-transporting polypeptide
Nucleos(t)ide analog reverse transcriptase inhibitor
Relaxed circular DNA
- RNase H
Surface plasmon resonance
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.
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.
- 1.World Health Organization. Global hepatitis report, 2017. https://apps.who.int/iris/bitstream/handle/10665/255016/9789241565455-eng.pdf?sequence=1. Accessed 17 Jun 2019.
- 18.Nakajima S, Watashi K, Fukano K, et al. High throughput screening of hepatitis B virus reverse transcriptase inhibitors. In: HBV international meeting, October 1–5. Melbourne, Australia [abstract P-310]; 2019.Google Scholar