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

• Published in: Nature biotechnology Vol. 26; no. 9; pp. 1019 - 1027
• Main Authors: Gerber, Doron, Maerkl, Sebastian J, Sklan, Ella H, Quake, Stephen R, Elazar, Menashe, Bryson, Paul D, Glenn, Jeffrey S, Einav, Shirit
• Format: Journal Article
• Language: English
• Published: New York, NY Nature Publishing Group 01.09.2008
• Subjects: Amino Acid Sequence; Analysis; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral agents; Benzimidazoles - pharmacology; Binding sites; Biological and medical sciences; Biotechnology; DNA sequencing; Drug therapy; Fundamental and applied biological sciences. Psychology; Genome, Viral; Health. Pharmaceutical industry; Hepacivirus - metabolism; Hepatitis; Hepatitis C; Hepatitis C - metabolism; Hepatitis C - therapy; Hepatitis C - virology; Hepatitis C Antibodies - metabolism; Industrial applications and implications. Economical aspects; Inhibitory Concentration 50; Kinetics; Medical sciences; Microfluidic Analytical Techniques; Microfluidics; Miscellaneous; Molecular Sequence Data; Nucleotide sequencing; Pharmaceutical industry; Pharmacology; Pharmacology. Drug treatments; Product development; Protein Binding; Protein Structure, Tertiary; Research methodology; Ribonucleic acid; RNA; Viral Nonstructural Proteins - chemistry; Virus Replication; Switzerland; High throughput screening; Transmembrane protein; Fluid mechanics; RNA; Infection; Virus; Target; Analysis method; Viral disease; Affinity; Replication; Inhibitor; Flaviviridae; Hepatitis C virus; Hepacivirus; Microfluidics; Viral hepatitis C
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt.1490

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 approximately 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.