Abstract
Chronic hepatitis B remains difficult to cure because the viral covalently closed circular DNA (cccDNA) minichromosome can persist and sustain viral transcription, creating a need for scalable, reporter readouts that facilitate early discovery of cccDNA-modulating agents. Here, we developed two complementary hepatocyte HiBiT reporter models: a replication-competent HBV reporter in HepaRG cells (HepaRG-Hibit16), in which a secreted split-NanoLuc HiBiT signal is linked to cccDNA-associated expression, and a Cre/Lox-based recombinant cccDNA (rcccDNA) reporter in HepG2 cells (HepG2-Rccc1a) that rapidly generates rcccDNA with a matched HiBiT readout. Screening of 1,403 FDA-approved compounds across both models identified 13 concordant, non-cytotoxic hits. Palovarotene, a retinoic acid receptor-γ agonist, was selected as an exemplar concordant hit and reduced HBV antigens, HBV DNA, and cccDNA and inhibited HBV infection in multiple hepatocyte-based in vitro systems without overt cytotoxicity at the tested concentrations. Together, this dual-reporter strategy supports efficient cross-model triage of candidate cccDNA modulators for subsequent orthogonal validation.