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

Abstract

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 M in a secondary, HBV-expressing cell line, with a concentration that exhibited 50% cytotoxicity of >50 M. The equilibrium concentration of HBF-0259 in aqueous solution at physiological pH was 15 to 16 M; 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.

Prediction of HBF-0259 interactions with hepatitis B Virus receptors and surface antigen secretory factors

“…The interaction energy of HBF-0259 to PreS1 and PreS2 [1][2][3][4][5][6][7][8][9][10][11] were lower comparing to other predicted receptors. HBF-0259 obviously interacts more strongly with SCCA1.…”

“…HBF-0259, (7-(2-Chloro-6-fluorophenyl)-5-(4-chlorophenyl)-4, 5, 6, 7-tetrahydro-tetrazolo [1, 5-a] pyrimidine), could act as an inhibitor of HBsAg secretion which is proved to be efficient in certain in vitro studies [4]. Moreover, the triazole derivates of this compound have similar anti-HBV effects [38,39].…”

“…Amino acids involved in interaction site of HBF-0259 were analyzed for conservancies among target molecules including, SCCA1, FNIII 1 , preS2 [1][2][3][4][5][6][7][8][9][10][11] , NTCP 84-165 , FTL, ASGPRCRD, preS1 . The results showed no conserved sequences in any of mentioned receptors ( Table 2).…”

“…Moreover, the triazole derivates of this compound have similar anti-HBV effects [38,39]. Anti-HBV potential of this non-toxic compound are hypothesized to be result of HBsAg inhibition through direct interaction with the viral or host molecules [4,38,42]. Cyclophilin A (CypA), Annexin II, and Ras-Associated Protein 7 (Rab7) are known cellular components involved in HBsAg secretion (Table 1).…”

“…In this study, in order to predict the mechanism(s) of action by which HBF-0259 decreases HBsAg secretion, we predict the interaction of HBF-0259 with suggested HBV receptors, preS regions of HBsAg (preS1 and preS2 [1][2][3][4][5][6][7][8][9][10][11] ) and cellular HBsAg secreting factors by computational approaches (Table 1).…”