A collaborative screening program for the discovery of inhibitors of HCV NS2/3 cis-cleaving protease activity |
| |
Authors: | Whitney Mike Stack Jeffrey H Darke Paul L Zheng Wei Terzo Joe Inglese Jim Strulovici Berta Kuo Lawrence C Pollock Brian A |
| |
Affiliation: | Aurora Biosciences Corp. San Diego, CA 92121, USA. info@aurorabio.com |
| |
Abstract: | 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 ,B-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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|