A Serine Palmitoyltransferase Inhibitor Blocks Hepatitis C Virus Replication in Human Hepatocytes

Background & AimsHost cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice.MethodsWe tested the ability of NA808 to inhibit SPT’s enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors.ResultsNA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors.ConclusionsThe SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors

Novel oral SPT inhibitor CH5169356 inhibits hepatic stellate cell activation and ameliorates hepatic fibrosis in mouse models of non‐alcoholic steatohepatitis (NASH)

Abstract Ceramide is a central molecule of sphingolipid metabolism and is involved in the development of non‐alcoholic fatty liver disease (NAFLD) and non‐alcoholic steatohepatitis (NASH). It has already been reported that the inhibition of serine palmitoyltransferase (SPT), the rate‐limiting enzyme in the sphingolipid biosynthetic pathway, has an inhibitory effect on hepatic lipidosis, but its effect on severe hepatic fibrosis is not clear. In this study, we examined whether a SPT inhibitor could suppress the activation of hepatic stellate cells (HSC) and ameliorate the progression of NASH. Effects on sphingolipid metabolism and HSC activation marker genes by NA808, a SPT inhibitor, were evaluated in an immortalized HSC cell line (E14C12). NA808 decreased sphingolipid synthesis and the expression of α‐smooth muscle actin (α‐SMA) and collagen 1A1 mRNA in HSC. We identified a novel oral SPT inhibitor, CH5169356, which is a prodrug of NA808. CH5169356 was administered in the Ath+HF model, a NASH mouse model with liver fibrosis induced by atherogenic and high‐fat content diets. CH5169356 showed a significant decrease in the expression of α‐SMA and collagen 1A1 mRNA in the liver and an inhibition of liver fibrosis progression. CH5169356 was also evaluated in a Stelic animal model (STAM), a NASH mouse model induced through a different mechanism than that of the Ath+HF model, and showed a significant anti‐fibrotic effect. In conclusion, CH5169356 could inhibit the progression of hepatic fibrosis in the pathogenesis of NASH by suppressing HSC activation, suggesting that CH5169356 would be a potential oral NASH therapeutic agent

Pathogenesis of Hepatitis C Virus Infection in Tupaia belangeri▿†

The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

<div><p>Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (<em>SGMS1</em> and <em>2</em>) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.</p> </div

Effects of NA808 treatment on sphingomyelin (SM) and ceramide (total and individual molecular species).

<p>(<b>A, B</b>) Relative ratio of total ceramide (<b>A</b>) and SM (<b>B</b>) in uninfected mice (white, n = 4), HCV genotype 1a-infected mice (black, n = 5), and HCV-infected mice treated with NA808 for 14 days (dark gray, 5 mg/kg, n = 4; light gray, 10 mg/kg, n = 3). *<i>p</i><0.05 and **<i>p</i><0.01 compared with HCV-infected mice. (<b>C</b>) SM levels (bars) and HCV RNA levels (black arrowhead) in the livers of mice treated for 14 days with NA808 (5 or 10 mg/kg/day) and untreated chimeric mice. (<b>D, E</b>) Relative intensities of individual ceramide molecular species (<b>D</b>) and individual SM molecular species (<b>E</b>) in uninfected mice (white, n = 3), HCV-infected mice (black, n = 3), and HCV-infected mice treated with NA808 for 14 days (dark gray, 5 mg/kg, n = 2; light gray, 10 mg/kg, n = 1). In all cases, error bars indicate SDs.</p

HCV alters sphingolipid metabolism.

<p>(<b>A, B</b>) Time-course studies of humanized chimeric mice inoculated with human serum samples positive for HCV genotype 1a (<b>A</b>) or 2a (<b>B</b>). (<b>C</b>) mRNA expression of <i>SGMS1</i> and <i>SGMS2</i> in uninfected (white, n = 5) and HCV genotype 1a-infected (black, n = 7) chimeric mice. (<b>D, E</b>) Effects of HCV infection on hepatocyte SM and ceramide levels in humanized chimeric mice. Relative intensity of total ceramide (<b>D</b>) and total shingomyelin (SM) (<b>E</b>) in uninfected mouse hepatocytes (white bar, n = 4), HCV genotype 1a-infected mouse hepatocytes (black bar, n = 5), and HCV genotype 2a-infected mouse hepatocytes (dark gray bar, n = 3). (<b>F</b>) Mass spectrum of SM in Bligh & Dyer extracts of a human hepatocyte cell line (HuH-7 K4). (<b>G, H</b>) Effects of HCV infection on hepatocyte SM and ceramide levels in humanized chimeric mice. Relative intensity of individual ceramide molecular species (<b>G</b>) and individual SM molecular species (<b>H</b>) in uninfected mouse hepatocytes (white bar, n = 3), HCV genotype 1a-infected mouse hepatocytes (black bar, n = 3), and HCV genotype 2a-infected mouse hepatocytes (dark gray bar, n = 3). In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01 compared with uninfected hepatocytes.</p

Relationship between the SGMS genes and HCV infection.

<p>(<b>A, B</b>) The correlation between SGMS1/2 and liver HCV-RNA of HCV infected humanized chimeric mice (n = 7). (<b>C</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on HCV in HCV-infected cells. (<b>D</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on the expression of SGMS1/2 mRNA measured by RTD-PCR. (<b>E</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM each) measured by RTD-PCR. (<b>F</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM) on HCV replication in FLR 3-1. In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01.</p