Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis

Shirit Einav, Doron Gerber, Paul D. Bryson, Ella H. Sklan, Menashe Elazar, Sebastian J. Maerkl, Jeffrey S. Glenn, Stephen R. Quake

Research output: Contribution to journalArticlepeer-review

214 Scopus citations

Abstract

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3′ terminus of the negative strand of the viral genome with a dissociation constant (K d) of ∼3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.

Original languageEnglish
Pages (from-to)1019-1027
Number of pages9
JournalNature Biotechnology
Volume26
Issue number9
DOIs
StatePublished - Sep 2008
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research (to J.S.G.) and National Institutes of Health (NIH) RO1 DK066793, an NIH Director’s Pioneer Award (to S.R.Q.) and NIH 1RO1 HG002644-01A1. D.G. was supported in part by the Fulbright Foundation. S.E. is a recipient of an American Liver Foundation Postdoctoral Fellowship Award. We also wish to thank the Stanford University SPARK Program, High-Throughput Bioscience Center and the Microfluidic Foundry. The plasmid FL-J6/JFH-5¢C19Rluc2AUbi that consists of the full-length HCV genome and expresses Renilla luciferase was a gift from Charles M. Rice, Rockefeller University.

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