High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

被引：18

作者：

Tani, Hidenori ^{[1
,2
]}

Akimitsu, Nobuyoshi ^{[3
]}

Fujita, Osamu ^{[1
,2
]}

Matsuda, Yasuyoshi ^{[1
,2
]}

Miyata, Ryo ^{[1
]}

Tsuneda, Satoshi ^{[2
]}

Igarashi, Masayuki ^{[4
]}

Sekiguchi, Yuji ^{[1
]}

Noda, Naohiro ^{[1
,2
]}

机构：

[1] Natl Inst Adv Ind Sci & Technol, Inst Biol Resources & Funct, Tsukuba, Ibaraki 3058566, Japan

[2] Waseda Univ, Dept Life Sci & Med Biosci, Shinjuku Ku, Tokyo 1628480, Japan

[3] Univ Tokyo, Radioisotope Ctr, Tokyo 1130032, Japan

[4] Microbial Chem Res Ctr, Shinagawa Ku, Tokyo 1410021, Japan

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2009年 / 379卷 / 04期

关键词：

Hepatitis C virus; NS3; helicase; Fluorescence quenching; Photoinduced electron transfer; BODIPY FL; RNA HELICASE; NS3; PROTEIN; QUANTITATIVE DETECTION; ENZYMATIC-ACTIVITIES; BINDING ACTIVITY; DNA HELICASE; PROBE; DOMAIN;

D O I：

10.1016/j.bbrc.2009.01.020

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the Screening for NS3 helicase inhibitors from cell extracts Of Microorganisms, and found several cell extracts containing potential inhibitors. (C) 2009 Elsevier Inc. All rights reserved.

引用

页码：1054 / 1059

页数：6

共 25 条

[1]

Baker Danial E, 2003, Rev Gastroenterol Disord, V3, P93

[2] Direct fluorometric measurement of hepatitis C virus helicase activity [J].

Boguszewska-Chachulska, AM ;

Krawczyk, M ;

Stankiewicz, A ;

Gozdek, A ;

Haenni, AL ;

Strokovskaya, L .

FEBS LETTERS, 2004, 567 (2-3) :253-258

[3] Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus [J].

Borowski, P ;

Kuehl, R ;

Mueller, O ;

Hwang, LH ;

zur Wiesch, JS ;

Schmitz, H .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1999, 266 (03) :715-723

[4] ISOLATION OF A CDNA CLONE DERIVED FROM A BLOOD-BORNE NON-A, NON-B VIRAL-HEPATITIS GENOME [J].

CHOO, QL ;

KUO, G ;

WEINER, AJ ;

OVERBY, LR ;

BRADLEY, DW ;

HOUGHTON, M .

SCIENCE, 1989, 244 (4902) :359-362

[5] The challenge of developing a vaccine against hepatitis C virus [J].

Forns, X ;

Bukh, J ;

Purcell, RH .

JOURNAL OF HEPATOLOGY, 2002, 37 (05) :684-695

[6] Side effects of therapy of hepatitis C and their management [J].

Fried, MW .

HEPATOLOGY, 2002, 36 (05) :S237-S244

[7] Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus [J].

Gallinari, P ;

Brennan, D ;

Nardi, C ;

Brunetti, M ;

Tomei, L ;

Steinkühler, C ;

De Francesco, R .

JOURNAL OF VIROLOGY, 1998, 72 (08) :6758-6769

[8] The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication [J].

Gu, BH ;

Liu, CB ;

Lin-Goerke, J ;

Maley, DR ;

Gutshall, LL ;

Feltenberger, CA ;

Del Vecchio, AM .

JOURNAL OF VIROLOGY, 2000, 74 (04) :1794-1800

[9] DNA helicase activity of the hepatitis C virus nonstructural protein 3 [J].

Gwack, Y ;

Kim, DW ;

Han, JH ;

Choe, J .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1997, 250 (01) :47-54

[10] Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein [J].

Gwack, Y ;

Kim, DW ;

Han, JH ;

Choe, J .

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1996, 225 (02) :654-659

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