Discovery of Ziresovir as a Potent, Selective, and Orally Bioavailable Respiratory Syncytial Virus Fusion Protein Inhibitor.

Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article describes the process of RO-0529 as a potent, selective, and orally bioavailable RSV F protein inhibitor and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-0529 demonstrates single-digit nM EC50 potency against laboratory strains, as well as clinical isolates of RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced cell-cell fusion in cellular assays.

Role of the PI3K­AKT­mTOR pathway in hepatitis B virus infection and replication.

The replication of hepatitis B virus (HBV) may be modulated by a variety of cell signaling pathways, including the phosphatidylinositol 3­kinase (PI3K)­RAC­α serine/threonine­protein kinase (AKT)­serine/threonine­protein kinase mTOR (mTOR) pathway. The aim of the present study was to determine the regulatory effects of this pathway on the infection and replication of HBV. The results indicated that the HBV entry process may activate the AKT pathway, as demonstrated by AKT phosphorylation in HBV natural infection. However, inhibition of AKT phosphorylation by short­term treatment with AKT inhibitors was unable to block HBV entry, which suggested that AKT activation induced by HBV infection is not essential for viral entry process. Prolonged treatment with PI3K­AKT­mTOR pathway inhibitors markedly promoted HBV replication in HBV replicating and natural infection models. The PI3K­AKT­mTOR pathway was therefore identified to be a negative regulator of HBV replication. These inhibitors enhanced the replication and transcription of HBV in an HBx­dependent way. The results additionally indicated that a PI3K inhibitor, Ly294002, inhibited the secretion of the small surface antigen of HBV in a PI3K­AKT­independent manner. The inhibitor Ly294002 may be used as a tool for the drug development of surface antigen secretion inhibitors.

HBVcircle: A novel tool to investigate hepatitis B virus covalently closed circular DNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA. METHODS: Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI). RESULTS: We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system. CONCLUSION: Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery. LAY SUMMARY: To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.