Serine palmitoyltransferase inhibitor suppresses HCV replication in a mouse model

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Abstract

Serine palmitoyltransferase (SPT) is a first-step enzyme in the sphingolipid biosynthetic pathway. Myriocin is an inhibitor of SPT and suppresses replication of the hepatitis C virus (HCV) replicon. However, it is still unknown whether this SPT inhibitor suppresses HCV replication in vivo. We investigated the anti-HCV effect of myriocin against intact HCV using chimeric mice with humanized liver infected with HCV genotype 1a or 1b. We administered myriocin into HCV infected chimeric mice and succeeded in reducing the HCV RNA levels in serum and liver to 1/10–1/100 of the levels prior to the 8 day treatment. Furthermore, combined treatment with pegylated interferon reduced the HCV RNA levels to less than 1/1000 of the control levels. We strongly suggest that suppression of SPT reduces HCV replication, and therefore that the SPT inhibitor is potentially a novel drug in the treatment of HCV infection.

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Materials and methods

Inhibition assay of replication in HCV replicon cells by myriocin. Myriocin (Sigma, St. Louis, MO, USA) was added in the growth medium of HCV subgenomic replicon cells FLR3-1 (genotype 1b, Con-1; [12]) at a final concentration of 0.2, 1.0, 3.9, 15.6 or 62.5 nM. After 72 h incubation, we performed luciferase assays using the Bright-Glo luciferase assay kit (Promega, Madison, WI, USA).

Measurement of cell viability using the Tetra Color One (WST-8) assay. Myriocin was added to FLR3-1 cells as

Anti-HCV effect of the SPT inhibitor, myriocin

We examined the anti-HCV effect and cell toxicity of myriocin in the HCV subgenomic replicon cells FLR3-1. Luciferase activity was greatly decreased by myriocin in a dose-dependent manner without affecting cell viability (Fig. 2A) or cell growth (data not shown). The maximum inhibition rate was about 80% in the presence of over 62.5 nM myriocin (Fig. 2A), while the 50% inhibitory concentration (IC50) was about 5.8 nM (Table 1). Reduction of NS3 protein, which plays a key role in HCV replication,

Discussion

In the present study, the SPT inhibitor myriocin was shown to inhibit replication of intact HCV in vivo. We initially investigated the fundamental inhibitory effects and mechanisms of myriocin against replication of the HCV replicon and found that inhibition of HCV replicon replication is compatible with a decrease in ceramide and sphingomyelin in the cells. The inhibitory effect of myriocin on replication of the HCV replicon differed slightly from that on de novo biosynthesis of ceramide and

Acknowledgments

The authors thank PhenixBio Co., Ltd. for maintenance of the humanized-liver mice. We thank for I. Kusanagi for technical assistance and F. Ford for editorial assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation of Japan; and the Ministry of Health, Labor and Welfare of Japan.

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