Abstract
Chronic hepatitis B (CHB) is a global health problem caused by human hepatitis B virus (HBV). Current treatment with interferons and nucleos(t)ide analogs (NAs) can cause population tolerance and drug resistance. Therefore, new antiviral drugs, especially those targeting host factors, are urgently needed. Here, we identified Evans blue as a new HBV inhibitor by screening an FDA drug library using Huh7DhNTCP cells and confirmed the antiviral activity in primary human hepatocytes and human sodium taurocholate cotransporting polypeptide (hNTCP)-transfected porcine primary hepatocytes. Our efficacy study showed that Evans blue has an IC50 of 2 μM against HBV infection in Huh7DhNTCP cells, and no apparent toxicity at up to 1000 μM. The IC50 of Evans blue against HBV in primary human hepatocytes was approximately 5 μM. Mechanism studies revealed that Evans blue has a dual anti-HBV effect. It inhibits both the binding of viral preS1 to host cells through the host factor NTCP and the virus capsid assembly by targeting the host factor BK channel. The KD of the direct interaction between Evans blue and NTCP is 8.82E-8 M. Evans blue can suppress capsid assembly at micromolar concentrations by reducing the cytosolic calcium ion concentration. Since the antiviral effects on HBV binding and assembly are both achieved through targeting host factors, Evans blue inhibits the infection of nucleos(t)ide analog drug-resistant HBV strains in Huh7DhNTCP cells. Taken together, our results suggest that Evans blue may be a promising anti-HBV drug candidate in the classes of both entry and assembly inhibitors.
Highlights
Hepatitis B virus (HBV) infection is a global health threat that is estimated to cause chronic hepatitis and hepatocellular carcinoma (HCC) in over 240 million people worldwide (Ott et al, 2012)
We found that micromolar concentrations of Evans blue dramatically reduced the levels of HBeAg (Figure 1C) and HBV DNA (Figure 1D)
These results indicate that Evans blue is a highly selective HBV inhibitor with a selectivity index (SI) (CC50/IC50) of at least 500 in Huh7DhNTCP cells
Summary
Hepatitis B virus (HBV) infection is a global health threat that is estimated to cause chronic hepatitis and hepatocellular carcinoma (HCC) in over 240 million people worldwide (Ott et al, 2012). Hepatitis B virus studies are limited by a lack of reliable in vitro infection cell models, including liver cells, many of which cannot support HBV infection due to a lack of HBV. Evans Blue Inhibits HBV specific receptors or co-receptors (Glebe and Urban, 2007). The identification of HBV receptors is critical. Several cellular proteins have been suspected but not confirmed as HBV receptor molecules. Sodium taurocholate cotransporting polypeptide (NTCP), a hepatic membrane transporter involved in bile acid uptake, has been reported as an HBV receptor. Exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to HBV infections (Yan et al, 2012)
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