A2342 is a novel small-molecule, orally available, selective inhibitor of hepatic transporter Na+/taurocholate co-transporting polypeptide (NTCP). NTCP mediates uptake of bile acids into hepatocytes and acts as a host receptor for hepatitis B and D viruses (HBV/HDV). The preS1 domain of the large envelope protein of HBV/HDV is essential for binding of these virus particles to NTCP. We previously demonstrated that A2342 lowers HBV DNA viral load in infected human hepatocytes in vitro and also attenuates HBV viral load and HBV antigens in infected humanized mice in vivo. Here, we aim to provide more detail on the NTCP inhibitory mechanism.
Human NTCP-expressing HEK293 cells were used to evaluate inhibition of 1 µM [3H]taurocholic acid transport and 5 nM [3H]preS1 peptide binding as a surrogate parameter for virus binding. Human NTCP-expressing HepG2 cells were used to evaluate entry inhibition of HDV and HBV via immunostaining against hepatitis D antigen (HDAg) and hepatitis B core antigen (HBcAg), respectively, and measuring hepatitis B e antigen (HBeAg) levels in supernatant of HBV-infected cells. Cryopreserved human hepatocytes infected with a clinical isolate of HBV were used for infection experiments; HBV DNA levels in supernatant were analysed. The peptides preS1 (genotype D) or tenofovir disoproxil fumarate (TDF) were used as positive controls.
A2342 inhibited [3H]taurocholic acid uptake (half-maximal inhibitory concentration [IC50], 186 nM) and [3H]preS1 peptide binding (IC50, 149 nM) in a concentration-dependent manner. In infection experiments, A2342 (range, 100−400 nmol/L) inhibited numbers of HBcAg-positive cells by 75%−90% and HDAg-positive cells by 40%−60%. HBeAg levels in supernatant were reduced with A2342 by 83–87% at all concentrations. In cryopreserved human hepatocytes, A2342 reduced HBV DNA levels in a concentration-dependent manner when given 18 h before infection. A2342 was not effective when given 18 h post-infection. Additive antiviral effects were evident when A2342 was given together with TDF. A2342 was devoid of cellular toxicity in hepatocytes and cell lines.
A2342 inhibits NTCP-mediated bile acid uptake and HBV preS1 peptide binding in similar manners and blocks entry of HBV and HDV. These data further support A2342 as the first orally bioavailable molecule able to inhibit HBV/HDV entry via NTCP in the nanomolar range.
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