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

Anne Marie Dougherty,^1, Haitao Guo,^2, Gael Westby,¹ Yuanjie Liu,² Ender Simsek,² Ju-Tao Guo,² Anand Mehta,² Pamela Norton,² Baohua Gu,² Timothy Block,^1,2 and Andrea Cuconati^1*

Institute for Hepatitis and Virus Research, Hepatitis B Foundation, Doylestown, Pennsylvania 18902,¹ Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, Pennsylvania 18902²

Received 23 April 2007/ Returned for modification 6 June 2007/ Accepted 24 August 2007

The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals are thought to play a role in suppressing the 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. By 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 a 50% effective concentration of approximately 1.5 µM in a secondary, HBV-expressing cell line, with a concentration that exhibited 50% cytotoxicity of >50 µM. The equilibrium concentration of HBF-0259 in aqueous solution at physiological pH was 15 to 16 µM; the selective index was thus >9. As intended by our screening paradigm, HBF-0259 is a selective, potent inhibitor of secretion of both subviral and DNA-containing viral particles, while the secretion of -1-acid glycoprotein and -1-antitrypsin was unaffected. The HBV e antigen, which is not a constituent of HBV particles, was also unaffected, suggesting that the 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 those 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 the 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.

Published ahead of print on 17 September 2007.

These authors contributed equally.