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) s erine proteinase. Two quenched-fluorogenic substrates, (7-methoxycoumarin-4 -yl) acetyl (Mca) Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-(2,4-dinitrophenyl, D np) Lys (Mca-Asp-Asp-IIe-Val-Pro-Cys-Ser-Met-Ser-Lys[Dnp], QF-1) and Mca-As p-Asp-Ile-Val-Pro-Cys-Ser-Met-Lys(Dnp)-Arg-Arg (QF-2), which derived from t he 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 whic h is a fusion protein of maltose binding protein and almost entire nonstruc tural protein (MBP-NS3), with K-m values comparable to that of longer subst rate 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 wa s also found to be a good substrate of Delta NS3 which contained serine pro teinase part of NS3 with kcat value of 4.3 min(-1). The cleavage reaction i s 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 m ethod was applied for screening of HCV serine protease inhibitors using a f luorescence multiwell plate reader. A group of natural occurring products, flavonoids, was subjected to be screened. Two flavonoids out of 25 were fou nd to inhibit the enzyme moderately at a concentration of 100 mu M. The dat a agreed with those obtained by high-performance liquid chromatography (HPL C). This method is suited to sensitive quantitation of the enzyme reaction as well as the high throughput analysis of the inhibitors. (C) 1999 Elsevie r Science B.V. All rights reserved.