Tizoxanide (2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide) Inhibits Hepatitis C Virus Replication Through Modulation of NS5A Hyperphosphorylation

Abstract

Nitazoxanide (NTZ, Alinia&reg, Romark Laboratories, LC) is a licensed thiazolide anti-infective that is currently in clinical development for the treatment of chronic hepatitis C, but the antiviral mechanism has remained undetermined. The current investigations sought to determine the effect of tizoxanide (TIZ, active metabolite of NTZ) on intracellular HCV proteins to reveal a mechanism of action. The overall rate of reduction of HCV proteins in HCV replicon containing cell lines treated with TIZ was consistent with loss of viral RNA template. Examination of intracellular membrane preparations (where replication of HCV is localized) from HCV replicon cell lines revealed enhanced levels of hyperphosphorylated HCV NS5A (p58) peaking after 48 hours of TIZ treatment, coincident with the initial reduction of HCV RNA. The phosphorylation state of NS5A is established as a regulator of the switch from active viral genome replication to packaging and assembly; overproduction of p58 is established as being inhibitory to HCV replication. Consistent with this model, HCV RdRp activity on nascent HCV genomes in membrane preparations from TIZ-treated cultures was also reduced. Casein kinase I-alpha (CKI&alpha) is a cellular kinase responsible for hyperphosphorylation of NS5A. CKI activity in intracellular membrane preparations from TIZ-treated HCV replicon cells was approximately 2-fold higher than in untreated cells in enzymatic assays. However, TIZ had no direct effect on CKI&alpha activity in enzymatic assays, including autophosphorylation, or on another cellular kinase associated with p58 formation, PLKI, in either cellular samples or direct enzymatic assays. Conclusions: These investigations provide a mechanism-of-action for the anti-HCV activity of TIZ. The primary cellular target for TIZ remains unidentified, but is most likely a protein involved in the upstream regulation of the cellular kinase, CK1&alpha.