Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase

Although several nucleo(s)tide analogs are available for treatment of HBV infection, long‐term treatment with these drugs can lead to the emergence of drug‐resistant viruses. Recent HIV‐1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non‐nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI‐resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)‐reverse transcriptase (RT) (TP‐RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP‐RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA‐binding assay system. Then, we used TP‐RT in cell‐free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6‐hydroxy‐DL‐DOPA and N‐oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell‐based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP‐RT domain to treat HBV infection.