A high throughput assay of the hepatitis C virus nonstructural protein 3 serine proteinase.
A simple assay was developed based on intramolecular fluorescence resonance energy transfer for detection of the activity of hepatitis C virus (HCV) serine proteinase. Two quenched-fluorogenic substrates, (7-methoxycoumarin-4-yl)acetyl (Mca) Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-(2,4-dinitrophenyl, Dnp) Lys (Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Lys[Dnp], QF-1) and Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Lys(Dnp)-Arg-Arg (QF-2), which derived from the NS5A/5B junction of the HCV polyprotein, were designed. Kinetic studies revealed that QF-1 and QF-2 had high affinity for a recombinant enzyme which is a fusion protein of maltose binding protein and almost entire nonstructural protein (MBP-NS3), with Km values comparable to that of longer substrate based on the same cleavage site. QF-1 and QF-2 were cleaved by MBP-NS3 efficiently with kcat values of 7.5 and 4.2 min(-1), respectively. QF-2 was also found to be a good substrate of deltaNS3 which contained serine proteinase part of NS3 with kcat value of 4.3 min(-1). The cleavage reaction is detected continuously by the elevation of the fluorescence due to release from quenching. The fluorescence of the substrates increases in proportion to progress of the cleavage reaction under the standard conditions. This method was applied for screening of HCV serine protease inhibitors using a fluorescence multiwell plate reader. A group of natural occurring products, flavonoids, was subjected to be screened. Two flavonoids out of 25 were found to inhibit the enzyme moderately at a concentration of 100 microM. The data agreed with those obtained by high-performance liquid chromatography (HPLC). This method is suited to sensitive quantitation of the enzyme reaction as well as the high throughput analysis of the inhibitors.
Kakiuchi
N
N
National Institute of Bioscience and Human Technology, AIST, MITI, Tsukuba, Japan. kakiuchi@nibh.go.jp
Nishikawa
S
S
Hattori
M
M
Shimotohno
K
K
D000595:Amino Acid Sequence; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases