Disruption of HBx-DDB1 by NTZ: New Mechanistic Insight Into an Old Drug With Broad Anti-infective Activities.

Hepatitis B virus (HBV) infection leads to chronic hepatitis B (CHB) in approximately 250 million people worldwide, putting them at high risk for developing cirrhosis and hepatocellular carcinoma. HBV is a partially double-stranded DNA virus that belongs to the Hepadnaviridae family. After entry into host cells, the viral genome is transported into the nucleus and converted to a covalently closed circular DNA (cccDNA), which serves as the template for all HBV viral RNAs. Currently available HBV antiviral drugs inhibit the reverse transcription of HBV pregenomic RNA but fail to suppress the established cccDNA reservoir in infected hepatocytes, resulting in viral rebound after therapy. In addition, HBV surface antigen hepatitis B surface antigen expressed from the cccDNA maintains immune tolerance to prevent induction of antibodies to hepatitis B surface antigen, which are critical for a functional cure of CHB in patients.

The HBV regulatory protein hepatitis B virus X protein (HBx) is critical for HBV gene expression from the episomal cccDNA template via its interaction with cellular proteins. The best-characterized HBx binding partner is the damage-specific DNA binding protein 1 (DDB1). This binding is essential for HBx-enhanced HBV replication. The DNA repair factor DDB1 functions as a linker protein for the assembly of a number of Cullin 4-ROC1/RING E3 ubiquitin ligase. DDB1 bridges CUL4 to individual DDB1-binding WD40 proteins (or DDB Cullin-associated factors), which in turn recruit substrates to the CUL4–ROC1 catalytic core for subsequent ubiquitination and degradation. A structural study has shown that HBx contains an H-box that is shared by several DDB1-binding WD40 proteins and directly binds to DDB1, suggesting that HBx functions as a DDB Cullin-associated factor to retarget the DDB1–Cullin 4-ROC1/RING E3 ubiquitin ligase to a new host factor. Two recent reports have shown that the HBx-DDB1-CUL4-ROC1 E3 ligase complex binds and degrades the structural maintenance of chromosomes (SMC) complex proteins SMC5/6 to enhance HBV replication.2, 3

It is clear that disruption of the HBx/DDB1 interaction provides a strategy to develop novel therapeutics that inhibit HBV cccDNA activity. However, multiple efforts have failed to identify viable small molecules that can efficiently disrupt the interaction in vivo. Sekiba et al developed an elegant high-throughput screening assay to efficiently identify inhibitors of the HBx/DDB1 interaction. A novel split luciferase assay based on HBx–DDB1 interaction was used for screening compounds. The investigators reported that nitazoxanide (NTZ), a thiazolide anti-infective agent that has been approved by the Food and Drug Administration for treating multiple viruses and parasites, efficiently inhibits the HBx–DDB1 protein interaction. Consistently, NTZ significantly prevents HBx-induced SMC5 protein degradation and suppresses HBx-dependent HBV cccDNA gene expression. Importantly, NTZ also inhibits HBV replication in human primary hepatocytes infected with HBV.

These results indicate that NTZ inhibits HBV cccDNA activity and may provide a new therapeutic agent with the potential for a functional cure for CHB. In combination with current anti-HBV inhibitors nucleos(t)ide analogs/reverse transcriptase inhibitors in CHB patients, it may achieve inhibition of HBV gene expression from the NUC-resistant cccDNA. The clearance of hepatitis B surface antigen in CHB may reverse its associated immune tolerance and help to induce antibodies to hepatitis B surface antigen for a functional cure.

One major limitation of current HBV therapies is the inability to target cccDNA. HBx has evolved to bind DDB1 and counteract SMC5/6 activity to facilitate HBV replication. It will be important to determine how NTZ inhibits HBx/DDB1 interaction. The reported NZT activity in suppressing HBV replication, however, is relatively moderate. It will require further optimization to develop into a clinically efficacious anti-HBV drug. NZT originally was discovered in the 1980s and is used for treating various helminthic and protozoal infections; and it also is effective in treating a number of viral infections. The antiprotozoal activity of NZT is believed to be owing to interference with the pyruvate-ferredoxin oxidoreductase enzyme-dependent electron transfer reaction, which is essential to anaerobic energy metabolism. It will be of interest to determine whether NTZ inhibits other viruses via similar DDB1-related mechanisms, including viruses with episomal DNA genomes.