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

概要

〔目　的(Purpose)〕
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 NRTl-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.

〔方法ならびに成績(Methods/Results)〕
We expressed and purified the HBV TP-RT with high purity using an E. coli expression system and established an in vitro epsilon 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-DLDOPA and N-oleoyldopamine, which inhibited the binding of epsilon RNA with the HBV polymerase. Furthermore, these drugs reduced IIBV 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.

〔総　括(Conclusion)〕
We successfully purified HBV polymerase TP-spacer-RT domain expressed in E. coli with high purity and established a system to find new compounds or chemicals to inhibit binding of TP-spacer-RT with a packaging signal of epsilon RNA. We found two compounds; DL-DOPA and OLDA to inhibit pgRNA packaging into the capsids followed by HBV genome replication with the assay system. Although these chemicals would not be applicated for clinical use directly, our system should be very useful to explore new anti-HBV agents in terms of packaging inhibitors.