The Anti-Hepatitis C Agent Nitazoxanide Induces Phosphorylation of Eukaryotic Initiation Factor 2α Via Protein Kinase Activated by Double-Stranded RNA Activation
Received 22 September 2008; accepted 23 July 2009. published online 06 August 2009.
Background & Aims
New therapies are needed to treat patients infected with hepatitis C virus (HCV), a major worldwide cause of chronic liver disease. Nitazoxanide (NTZ), originally used to treat cryptosporidiosis infection, recently was shown to have unexpected antiviral activity in the HCV replicon system and in chronically infected patients. A pilot clinical study suggested that NTZ can augment the antiviral effect of interferon (IFN), although the molecular basis for its effect was unknown.
Methods
We analyzed the effects of NTZ on the regulation of eukaryotic initiation factor-2α (eIF2α) and its IFN-induced kinase, protein kinase activated by double-stranded RNA (PKR), in cells that support HCV RNA replication and in vitro biochemical assays.
Results
NTZ increased eIF2α phosphorylation, a modification known to mediate host cell antiviral defenses. The addition of IFN to cell cultures increased NTZ-induced eIF2α phosphorylation. NTZ also increased PKR phosphorylation. In vitro, NTZ promoted PKR autophosphorylation, a key step in activating PKR's kinase activity for eIF2α. Finally, NTZ-induced eIF2α phosphorylation was reduced in the presence of specific inhibitors of PKR autophosphorylation.
Conclusions
An important mechanism of NTZ's action involves activation of PKR, a key kinase that regulates the cell's innate antiviral response. These observations could explain the clinical antiviral effect of NTZ. NTZ might represent a new class of small molecules capable of potentiating and recapitulating important antiviral effects of IFN.
⁎Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Palo Alto, California
‡Department of Structural Biology, Stanford University School of Medicine, Palo Alto, California
§The Romark Institute for Medical Research, Tampa, Florida
∥Veterans Administration Medical Center, Palo Alto, California
Reprint requests Address requests for reprints to: Jeffrey S. Glenn, MD, PhD, Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford University, 269 Campus Drive, Stanford, California 94305-5187. fax: (650) 723-3032
S.A.M. is currently at the Department of Chemistry, University of Manitoba, Winnipeg, MB, Canada.
M.E. and M.L. contributed equally to this article.
Conflicts of interest The authors disclose the following: Dr Glenn is the recipient of grant funding from, and is a consultant to, Romark Laboratories; Dr Rossignol is an employee of Romark Laboratories, LC. The remaining authors disclose no conflicts.
Funding This work was supported by a grant from the Romark Institute of Medical Research and a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research (J.S.G.), and National Institutes of Health grants AI47365 and GM078346 (J.D.P.).
ABSTRACT
The Anti-Hepatitis C Agent Nitazoxanide Induces Phosphorylation of Eukaryotic Initiation Factor 2α Via Protein Kinase Activated by Double-Stranded RNA Activation