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

Nature Biotechnology volume 26, pages 1019–1027 (2008)Cite this article

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 (Kd) 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.

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Figure 1: Protein-RNA interactions measured on microfluidic platform.
Figure 2: NS4B binds specifically to the 3′ terminus of the HCV negative-strand RNA.
Figure 3: Identification of RNA binding domains within NS4B.
Figure 4: Small-molecule screen reveals that clemizole hydrochloride inhibits RNA binding by NS4B and HCV RNA replication in cell culture.
Figure 5: Clemizol-resistant mutant.

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Acknowledgements

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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Author notes

  1. Sebastian J Maerkl

    Present address: Present address: School of Engineering, École Polytechnique Fédérale de Lausanne, Building BM 2111, Station 17, 1015 Lausanne, Switzerland.,

  2. Shirit Einav and Doron Gerber: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, 94305, California, USA

    Shirit Einav

  2. Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, CCSR 3115A, 269 Campus Drive, Stanford, 94305, California, USA

    Shirit Einav, Paul D Bryson, Ella H Sklan, Menashe Elazar & Jeffrey S Glenn

  3. Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, 94305, California, USA

    Doron Gerber, Sebastian J Maerkl & Stephen R Quake

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Correspondence to Jeffrey S Glenn or Stephen R Quake.

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Einav, S., Gerber, D., Bryson, P. et al. Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nat Biotechnol 26, 1019–1027 (2008). https://doi.org/10.1038/nbt.1490

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