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Antimicrob. Agents Chemother. doi:10.1128/AAC.00541-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Substituted Tetrahydro-Tetrazolo-Pyrimidine is a Specific and Novel Inhibitor of Hepatitis B Virus Surface Antigen Secretion

Institute for Hepatitis and Virus Research, Hepatitis B Foundation, Doylestown, PA 18902; Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, PA, USA 18902

	Abstract

The high levels of hepatitis B surface antigen (HBsAg)-bearing subviral particles in the serum of chronically-infected individuals are thought to play a role in suppressing hepatitis B virus (HBV)-specific immune response. Current therapeutics are not directed at reducing this viral antigenemia, thus our group has focused on identifying inhibitors of HBsAg secretion. Using the HBV-expressing cell line HepG2.2.15, high throughput screening of an 80,288-compound synthetic small molecule library identified HBF-0259, an aromatically-substituted tetrahydro-tetrazolo-(1, 5-a)-pyrimidine. Following resynthesis, HBF-0259 had an EC₅₀ of approximately 1.5 µM in a secondary, HBV expressing cell line, with a CC₅₀ of >50. Equilibrium concentration of HBF-0259 in aqueous solution at physiological pH is 15-16 µM; SI was thus >9. As intended by our screening paradigm, HBF-0259 is a selective, potent inhibitor of secretion of both subviral and DNA-containing HBsAg viral particles, while secretion of -1-acid glycoprotein and -1 antitrypsin was unaffected. HBV e antigen (HBeAg), which is not a constituent of HBV particles, was also unaffected, suggesting that secretion of particles bearing HBV structural glycoproteins is targeted directly. Inhibitory activity was also confirmed by transfection of HBsAg, indicating that the action of the compound is independent of other viral proteins. HBF-0259 had no effect on HBV DNA synthesis, demonstrating that inhibition is independent of viral genomic replication. Finally, HBF-0259 had little or no effect on cell-to-cell spread of two unrelated viruses, suggesting that it is a specific inhibitor of secretion of HBsAg. Possible mechanisms of action and the implications for its development are discussed.