High-throughput cell-based screening for hepatitis C virus NS3/4A protease inhibitors.
Hepatitis C virus (HCV) encodes a viral protease, nonstructural (NS)3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(delta4AB)SEAP, a reporter consisting of enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (delta4AB), and secreted alkaline phosphatase (SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293EEG(delta4AB)SEAP-NS3/4A, which constitutively expresses EG(delta4AB)SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor >0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.
Lee
Jin-Ching
JC
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli County, Taiwan, Republic of China.
Yu
Ming-Chen
MC
Lien
Tzu-Wen
TW
Chang
Ching-Fung
CF
Hsu
John T-A
JT
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000998:Antiviral Agents / Q000494:pharmacology; D002219:Carbamates / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects* / Q000254:growth & development; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D047028:Macrocyclic Compounds / Q000494:pharmacology; D010957:Plasmids; D011804:Quinolines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods; D015842:Serine Proteinase Inhibitors / Q000032:analysis / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000469:Alkaline Phosphatase; D000998:Antiviral Agents; D002219:Carbamates; D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D047028:Macrocyclic Compounds; D011804:Quinolines; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D013844:Thiazoles; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C479555:BILN 2061; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C485184:enhanced green fluorescent protein