ABSTRACT
This is a biphasic project to develop and implement a novel high throughput screening (HTS) system for
identification of inhibitors of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) (R21 phase),
followed by preclinical drug development studies (R33 phase). HBV cccDNA is essential to the virus life cycle,
its elimination is considered critical to a cure but has not been achieved by the FDA-approved drugs that
exclusively target the viral polymerase. Due to the limitations of current HBV experimental systems, including
the impracticality of detecting cccDNA itself, cccDNA has not been rigorously targeted in HTS of small molecule
libraries. In the R21 phase of this proposal, a novel cell line expressing a split luciferase tag HiBiT-labeled HBV
precore (pC) in a cccDNA-dependent manner will be used to establish an HTS assay for screening of cccDNA
inhibitors. This cell line inducibly produces viral pregenomic RNA from a stably integrated HBV genome
(transgene) with the HiBiT sequence inserted in the pC region, leading to viral DNA replication and cccDNA
formation; subsequently, HiBiT-tagged-pC mRNA is only transcribed from the cccDNA template, then the
translated HiBiT-pC protein is detected by split luciferase reconstitution assay. In such system, compounds that
lower the HiBiT-pC would be considered candidate inhibitors of cccDNA formation, expression or longevity. We
will first miniaturize the cell-based assay to the 384-well format, the performance characteristics and the
robustness of the assay under HTS conditions will be determined. Next, an HTS screen will be conducted against
a library containing 20,000 “cherry-picked” compounds, the first round hits will be filtered through dose-ranging
activity and cytotoxicity analyses, and through counter-screening in a cell line constitutively expressing transgene
(not cccDNA)-dependent HiBiT-pC to remove the off-target hits. Finally, the HTS-derived hits will be validated in
multiple cccDNA-producing cell models by directly measuring the levels of cccDNA and its transcripts. The
confirmed hits and their analogs will be repurchased and retested for their activity against cccDNA to obtain the
final hits. In the R33 phase, hit-to-lead optimization will be conducted through structure activity relationship (SAR)
study, and lead compounds will be prioritized according to activity, cytotoxicity, chemical tractability, in vitro
pharmacology, mechanism of action (MoA), and synergistic effect and off-treatment effect in combination with
tenofovir (TDF). Up to ten compounds will be advanced for in vivo pharmacology assessment, and the best
candidate(s), ideally one cccDNA destabilizer and one cccDNA silencer, will progress to in vivo antiviral efficacy
test in HBV-infected humanized mouse model. Successful completion of our goals will deliver at least one lead
compound to advanced preclinical studies toward development of novel therapy to cure chronic hepatitis B.