کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1175272 961794 2007 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease
چکیده انگلیسی

Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised. For the optimized substrate T-Abu-Q, with sequence Ac-Asp-Glu-Lys(TAMRA)-Glu-Glu-Abu-Ψ(COO)Ala-Ser-Lys(QSY-7)-amide, the kinetic parameters with HCV NS3 protease are Km = 30 μM, kcat = 0.6 s−1, and kcat/Km = 20,100 s−1 M−1. We show that this substrate is suitable for inhibitor analysis and mechanistic studies so long as the substrate concentration is low enough (0.5 μM) to avoid complications from high inner filter effects. The substrate is especially useful with ultra-high-density screening formats, such as microarrayed compound screening technology, because there is less spectral interference from the compounds being tested than with more traditional (EDANS/DABCYL) FRET protease substrates. The merits of the new substrate, as well as potential applications of this FRET pair to other protease substrates, are discussed.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 368, Issue 2, 15 September 2007, Pages 156–167
نویسندگان
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