Assays for glucosidase inhibitors with potential antiviral activities: secreted alkaline phosphatase as a surrogate marker.
As secretion of the middle (MHBs) glycoprotein of hepatitis B virus is highly dependent upon the action of the host oligosaccharide processing enzymes glucosidase I and II, drugs that inhibit this enzyme have been proposed as potential antiviral agents. To facilitate the identification of new, more effective inhibitors of MHBs secretion, an assay has been developed based on the expression of this glycoprotein alone by transfection of Huh7 hepatoma cells. The data clearly demonstrate that both mono- and di-glycosylated forms of MHBs are produced in this system and both forms are equally dependent upon glucosidase processing for secretion. In addition, inclusion of a co-transfected reporter construct that encodes secreted alkaline phosphatase (SEAP) to permit normalization of transfection revealed that the SEAP gene product was itself sensitive to glucosidase inhibition. This sensitivity also was observed in HepG2 human hepatoma cells. Thus, measuring SEAP secretion may be another method for evaluating glucosidase inhibition. In addition, this finding has important implications for the use of a SEAP reporter in screens of potential antiviral agents.
Norton
Pamela A
PA
Jefferson Center for Biomedical Research, Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Doylestown, PA 18901, USA. pamela.norton@drexel.edu
Conyers
Bertha
B
Gong
Qiaoke
Q
Steel
Laura F
LF
Block
Timothy M
TM
Mehta
Anand S
AS
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000998:Antiviral Agents / Q000494:pharmacology*; D015415:Biomarkers; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005959:Glucosidases / Q000037:antagonists & inhibitors*; D006801:Humans
D000998:Antiviral Agents; D015415:Biomarkers; D004791:Enzyme Inhibitors; D000469:Alkaline Phosphatase; D005959:Glucosidases