Journal of Biomolecular Screening

 

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Journal of Biomolecular Screening, Vol. 7, No. 2, 149-154 (2002)
DOI: 10.1177/108705710200700208

A Collaborative Screening Program for the Discovery of Inhibitors of HCV NS2/3 cis-Cleaving Protease Activity

Mike Whitney

Aurora Biosciences Corp., San Diego, CA

Jeffrey H. Stack

Aurora Biosciences Corp., San Diego, CA

Paul L. Darke

Merck Research Laboratories, West Point, PA

Wei Zheng

Merck Research Laboratories, West Point, PA

Joe Terzo

Merck Research Laboratories, West Point, PA

Jim Inglese

Merck Research Laboratories, West Point, PA

Berta Strulovicil

Merck Research Laboratories, West Point, PA

Lawrence C. Kuo

Merck Research Laboratories, West Point, PA

Brian A. Pollok

Aurora Biosciences Corp., San Diego, CA

This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ß-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.


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