Identification of novel HBV/HDV entry inhibitors by pharmacophore- and QSAR-guided virtual screening

The hepatic bile acid transporter Na+/taurocholate co-transporting polypeptide (NTCP) was identified in 2012 as the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Since then, this carrier has emerged as promising drug target for HBV/HDV virus entry inhibitors, but the synthetic peptide Hepcludex® of high molecular weight is the only approved HDV entry inhibitor so far. The present study aimed to identify small molecules as novel NTCP inhibitors with anti-viral activity. A ligand-based bioinformatic approach was used to generate and validate appropriate pharmacophore and QSAR (quantitative structure-activity relationship) models. Half-maximal inhibitory concentrations (IC50) for binding inhibition of the HBV/HDV-derived preS1 peptide (as surrogate parameter for virus binding to NTCP) were determined in NTCP-expressing HEK293 cells for 150 compounds of different chemical classes. IC₅₀ values ranged from 2 μM up to >1000 μM. The generated pharmacophore and QSAR models were used for virtual screening of drug-like chemicals from the ZINC¹⁵ database (~11 million compounds). The 20 best-performing compounds were then experimentally tested for preS1- peptide binding inhibition in NTCP-HEK293 cells. Among them, four compounds were active and revealed experimental IC50 values for preS1-peptide binding inhibition of 9, 19, 20, and 35 μM, which were comparable to the QSAR-based predictions. All these compounds also significantly inhibited in vitro HDV infection of NTCP-HepG2 cells, without showing any cytotoxicity. The bestperforming compound in all assays was ZINC000253533654. In conclusion, the present study demonstrates that virtual compound screening based on NTCP-specific pharmacophore and QSAR models can predict novel active hit compounds for the development of HBV/HDV entry inhibitors.