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Abstract
Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the “two phenolic hydroxyl groups at both ends” and the “caffeic acid-like structure” of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.
Highlights
Hepatitis B virus (HBV) infection is a major health issue worldwide, with approximately 248 million chronically infected individuals (CHB) [1]
nucleos(t)ide analogues (NAs) more strongly suppress HBV replication than IFN by inhibiting reverse transcription (RT) with less side effects, the discontinuation of NAs may result in the relapse of HBV
Plasmids p3×Flag CMV/pol (YE), an expression plasmid of 3×FLAG-Pol, was generated by Dr Sakaguchi and Dr Chayama (Hiroshima University, Japan). p3×FLAG-ISG20 was constructed as described below. p3×Flag CMV/pol (YE) was digested using HindIII and EcoRI to remove the cDNA of Pol, and subcloned ISG20 cDNA was inserted into the cleaved vector. p3×FLAG-ISG20 D94G was generated using the KOD -plus- Mutagenesis Kit (TOYOBO, SMK101)
Summary
Hepatitis B virus (HBV) infection is a major health issue worldwide, with approximately 248 million chronically infected individuals (CHB) [1]. 686,000 HBV-related deaths occur annually [2]. Interferon-α (IFN-α), pegylated IFN-α (PEG-IFN-α), and six nucleos(t)ide analogues (NAs), including lamivudine, entecavir, adefovir dipivoxil, tenofovir disoproxil fumarate, tenofovir alafenamide, and telbivudine, are currently approved for use in the clinical treatment of CHB patients [3]. Treatments with IFN have the potential to achieve HBsAg seroclearance by immunomodulation; not all patients respond to IFN. Life-long treatments with NAs are required, but may result in the emergence of resistant virus variants [4]. Since current therapeutics for CHB are insufficient, novel anti-HBV drugs are urgently required
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