HCV NS2-NS3 Protease Inhibitor Screening

NS2-NS3-FLAG (JFH-1 isolate, genotype 2a) was purified, and proteolysis reactions were performed as previously described (Shaw et al., 2015 (link)). Compounds were added to reactions prior to addition of NS2-NS3-FLAG by dilution from DMSO stocks (final DMSO 0.75%). Reactions were incubated at room temperature for 16 h, terminated by addition of Laemmli buffer and the NS3-FLAG proteolysis product quantified by western blot using M2 anti-FLAG monoclonal antibody (Sigma Aldrich) and IRDye 680RD Donkey anti-Mouse (LI-COR Biosciences) on an Odyssey imager (LI-COR). NS3-FLAG was normalised to DMSO control and a non-linear regression fitted with Prism 6 (GraphPad) to determine 50% effective concentration (EC₅₀).

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Shaw J., Harris M, & Fishwick C.W. (2015). Identification of a lead like inhibitor of the hepatitis C virus non-structural NS2 autoprotease. Antiviral Research, 124, 54-60.

Publication 2015

M2 anti-FLAG monoclonal antibody IRDye 680RD Donkey anti-Mouse Odyssey imager Prism 6

Addition Anti antibody Dilution Dmso Donkey Genotype Laemmli buffer Mouse Prism Proteolysis Western blot

Corresponding Organization : University of Leeds

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Variable analysis

independent variables

Compounds added to reactions prior to addition of NS2-NS3-FLAG

dependent variables

NS3-FLAG proteolysis product quantified by western blot

control variables

Final DMSO concentration at 0.75%
Reactions incubated at room temperature for 16 h

controls

DMSO control

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