A novel high throughput screening assay for HCV NS3 serine protease inhibitors.
Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.
Berdichevsky
Yevgeny
Y
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202, Tel-Aviv University, 69978, Ramat Aviv, Israel.
Zemel
Romy
R
Bachmatov
Larisa
L
Abramovich
Alex
A
Koren
Ruth
R
Sathiyamoorthy
Peramachi
P
Golan-Goldhirsh
Avi
A
Tur-Kaspa
Ran
R
Benhar
Itai
I
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000302:isolation & purification / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D005470:Fluorometry; D049452:Green Fluorescent Proteins; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010946:Plants, Medicinal / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus