Structure of Sch 68631: A new hepatitis C virus proteinase inhibitor from Streptomyces sp.

Tetrahedron Letters

Volumn 37, Issue 40, 1996, Pages 7229-7232

  Chu, Min ^a   Mierzwa, Ronald ^a   Truumees, Imbi ^a   King, Arthur ^a   Patel, Mahesh ^a   Berrie, Raymond ^a   Hart, Andrea ^a   Butkiewicz, Nancy ^a   DasMahapatra, Bimalendu ^a   Chan, Tze Ming ^a   Puar, Mohindar S ^a  

^a MERCK RESEARCH LABORATORIES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTIVIRUS AGENT; PROTEINASE INHIBITOR; SCH 68631; SERINE PROTEINASE; UNCLASSIFIED DRUG;

ARTICLE; DRUG ISOLATION; DRUG STRUCTURE; ENZYME INHIBITION; HEPATITIS C VIRUS; HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; MASS SPECTROMETRY; NUCLEAR MAGNETIC RESONANCE; STREPTOMYCES; ULTRAVIOLET SPECTROPHOTOMETRY;

HEPATITIS C VIRUS; STREPTOMYCES; STREPTOMYCES SP.;

EID: 16044365570     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/0040-4039(96)01626-7     Document Type: Article

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16. 15. Cell-free Translation Assay for HCV NS3 Protease. Translated substrate was prepared as follows: The plasmid pTS102 was linearized with EcoRl and transcribed with T7 RNA polymerase (Promega) to produce RNA encoding HCV polyprotin ΔNS5A/Δ5B from amino acid residue 2312-2621 to produce RNA encoding HCV polyprotein ΔNS4A/Δ4B from a residue 1693-1903). The in vitro transcribed RNA was translated in rabbit reticulocyte lysates (Promega) in the presence of {35S}-methionine. Translation reactions wre terminated by adding DNAse-free RNAse (Boehringer Mannheim) and cyclohexamide (Sigma) to 10 ug/ml followed by incubation at 3O°C for 15 min. Standard protease assays were initiated by the addition of 35 nM (10 nM) partially purified HCV protease to 2 ul {35S}-labeled translated substrate in a 20 ul volume containing 10 mM Tris pH 7.5, 120 mM NaCl, 5 mM DTT, 0.5% EDTA, 0.1% Tween 20, and 12% glycerol followed by incubation at 3O°C for 30 min. Inhibitors were added to standard assay mixtures prior to incubation, and appropriate solvents were also added to standard assay mixes as controls. Cleavage reactions were terminated by addition of an equal volume of 2X Laemmli sample buffer, and boiling 3 min. Cleavage products were analyzed by SDS/ 15% PAGE gel electrophoresis and autoradiography.