Identification of selective hepatitis delta virus ribozyme inhibitors by high-throughput screening of small molecule libraries

Background & aims: Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and hepatocellular carcinoma. Hepatitis delta virus (HDV)'s tight dependency on hepatitis B virus and the host cell machinery for its life cycle limits the development of direct-acting antivirals. Thus, we aimed to identify compounds that could block HDV replication by targeting its antigenomic ribozyme. Methods: We generated stable Huh7 human hepatoma cells expressing a reporter gene (Gaussia luciferase) either downstream (Gluc-2xRz) or upstream (2xRz-Gluc) of two HDV antigenomic ribozyme sequences. We performed high-throughput screening of three small molecule libraries. The secreted luciferase was measured as a readout of ribozyme inhibition upon addition of the molecules. Each plate was considered valid when the Z factor was >0.4. Specificity and toxicity evaluations were performed for the hits with a Z-score >5 and half-maximal inhibitory concentration was calculated by performing a dose-response experiment. Results: A dose-dependent induction of luciferase expression was detected in Gluc-2xRz-transfected cells incubated with the antisense morpholino, suggesting that the catalytic activity of the ribozyme cloned downstream of the reporter gene was efficiently inhibited. Among the 6,644 compounds screened, we identified four compounds that showed a specific inhibitory effect on the HDV antigenomic ribozyme in Gluc-2xRz cells, i.e. three histone deacetylase inhibitors and the purine analogue 8-azaguanine. The latter also significantly decreased HDV replication (by 40%) in differentiated HepaRG cells six days post infection. Conclusion: Using a novel cell culture model, we identified four small molecules active against the antigenomic HDV ribozyme. These results may provide insights into the structural requirements of molecules designed for the potent and specific inhibition of HDV replication. Impact and implications: Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and the development of hepatocellular carcinoma. Despite the current development of several new compounds, there is still a need for efficient antiviral treatments specifically targeting hepatitis delta virus (HDV). This work describes a novel cell culture model that allows for the high-throughput screening of compounds able to inhibit HDV ribozymes. We identified four small molecules active against the antigenomic HDV ribozyme (the ribozyme involved in the early step of HDV replication), with the strongest activity shown by 8-azaguanine, a purine analogue. Our data may provide insights into the structural requirements of molecules designed to inhibit HDV.