Identification of D4 as a novel antiviral compound inhibiting hepatitis B virus surface antigen via TMEM40 upregulation.

Chronic hepatitis B virus (HBV) infection poses a significant global public health challenge. Current therapies rarely achieve a functional cure, defined as the clearance of hepatitis B surface antigen (HBsAg) and fulfillment of other criteria. A critical unmet need is the development of agents that effectively suppress HBsAg production. Notably, serum HBsAg in patients predominantly originates from subviral particles (SVPs). To identify compounds inhibiting SVP production, we previously developed a cell model (HepG2-S-HiBiT) secreting HiBiT-tagged HBsAg, enabling high-throughput screening. Using this model, we screened a library comprising more than 5000 compounds and identified a hit compound (Compound 2) that potently inhibited HBsAg production. Subsequent structural optimization yielded a lead derivative, designated D4. In vitro cellular assays confirmed that D4 suppresses HBsAg production through a transcription-dependent mechanism. Transcriptomic profiling further revealed that Transmembrane Protein 40 (TMEM40) is significantly upregulated following D4 exposure. Further mechanistic investigations established that TMEM40 exerts anti-HBV activity via activation of the JAK-STAT signaling pathway. Collectively, our findings demonstrate that D4 represents a promising lead scaffold for the development of novel anti-HBV therapeutics, exerting its antiviral effects by upregulating TMEM40 and subsequently activating the JAK-STAT pathway to suppress viral transcription.