Identification of Marine-Derived Compounds with Potent Antiviral Activity in a Recombinant HBV cccDNA Cell Model

Hepatitis B virus (HBV) infection continues to pose a significant threat to global public health. Current therapies, including nucleoside analogs and interferon-α, suppress viral replication but fail to eliminate covalently closed circular DNA (cccDNA) - a viral minichromosome essential for HBV persistence. Targeting cccDNA is critical for achieving a functional cure, yet few effective inhibitors have been reported. Building on our previously established HepG2-HBV/loxP cell model, we applied a high-throughput screening workflow to identify cccDNA-targeting agents from marine natural compounds. Screening a marine-derived natural product library identified five compounds that reduced hepatitis B surface antigen (HBsAg) secretion without notable cytotoxicity. Further validation in HepDES19 cells via hepatitis B e antigen (HBeAg) assays further narrowed candidates to two leads: W-609 (austinol) and KK8 (asperfatsine C), with IC₅₀ values of 2.29 ± 0.41 and 2.97 ± 1.96 nM, respectively (compared to RG7834's IC₅₀ of 2.00 ± 0.16 nM). Dose-response studies confirmed their anti-cccDNA activity: austinol exhibited IC₅₀ values of 69.24 nM in HepG2-HBV/loxP cells and 2.23 μM in HepDES19 cells, whereas asperfatsine C showed IC₅₀ values of 503.10 nM in HepG2-HBV/loxP cells but 10.66 μM in HepDES19 cells. Both compounds are derived from marine sources, exhibiting distinct structural features: austinol is characterized by a meroterpenoid scaffold incorporating a pentacyclic nucleus with a 6/6/6/6/5 ring system, while asperfatsine C contains an isoechinulin-type diketopiperazine skeleton with a long-chain fatty acid moiety. This study systematically screened a collection of marine-derived compounds using a recombinant cccDNA-based cellular model, leading to the identification of several promising anti-HBV inhibitors that facilitate drug development for chronic hepatitis B.