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New HCV Drug Candidate for Treatment
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"Team finds way to stop hepatitis-C virus from multiplying"
".....using replicon cells, we identified a previously unknown small-molecule HCV replication inhibitor with the potential to be a clinical drug candidate for the treatment of HCV....
....The luciferase activities of the HCV replicon cells showed that replication was suppressed by NA255 in a dose-dependent manner with a mean of 50% inhibitory concentration (IC50) of 2 nM.....These results indicate that NA255 is a potent small-molecule inhibitor of HCV replication......in our preliminary in vitro primary hepatocyte infection system, NA255 suppressed HCV replication more continuously than IFN-a....
.....Strain F1476, a producer strain of NA255, is a filamentous fungus that was isolated from fallen leaves collected in Kamakura, Japan"
To clarify the mode of action of NA255, we first evaluated inhibitory activity against the viral enzymes that are essential for HCV replication. NA255 did not significantly inhibit NS3 serine protease, NS3 helicase or NS5B RNA polymerase in vitro. In addition, there were no changes after treatment with NA255 in the expression levels of interferon (IFN)-induced antiviral response genes such as RNA-dependent protein kinase9, 2'-5'-oligoadenylate synthetase10 or RNase L11 (data not shown), suggesting that NA255 could be an anti-HCV drug with a previously unknown mode of action.
10/17/2005
The Asahi Shimbun
A Japanese research team said it has found a method that prevents the hepatitis-C virus from multiplying.
The method deals with cells infected with the virus, not the virus itself, meaning that drugs could be developed to stop the multiplication process while preventing the virus from becoming resistant, the researchers said.
It is still unknown how hepatitis-C virus (HCV) multiplies once inside infected cells.
But researchers know that once the virus enters the cell, it develops a platform for multiplication by combining itself with certain lipid, an organic compound.
Using the lipid, the team at Chugai Pharmaceutical Co., led by Masayuki Sudo, pinpointed the platform inside cells where the HCV had combined itself with the lipid.
Without the platform, the HCV is unable to duplicate itself, the researchers said.
Using human liver cells, the team added a substance to the lipid that prevented it from combining with the HCV. Thus, the platform for multiplication could not synthesize, the researchers said.
"If we can target the mechanism of virus-infected cells, it could prompt the development of more effective drugs," Sudo said.
The team's report will be published on the Web site of the U.S. science journal Nature Chemical Biology on Monday.
An estimated 1 million to 2 million patients in Japan are infected with the hepatitis-C virus.
"HCV is troublesome because of its many mutations," said Takaji Wakita, senior researcher at Tokyo Metropolitan Institute for Neuroscience, said. "If we can target something that is contained in the cells, we may be able to come up with drugs that would prevent the virus from developing resistance. We need to make sure of the side effects, including the possibility that the treatment could affect other cells."(IHT/Asahi: October 17,2005)
"These data suggest that NA255 inhibits the interaction of HCV-NS5B with lipid rafts through inhibition of sphingolipid biosynthesis and that this association is involved in HCV replicon replication because an active HCV replication complex is present in Golgi-derived DRM fractions"
Letter
Nature Chemical Biology
Published online: 16 October 2005
"Host sphingolipid biosynthesis as a target for hepatitis C virus therapy"
Hiroshi Sakamoto1, Koichi Okamoto1, Masahiro Aoki1, Hideyuki Kato1, Asao Katsume1, Atsunori Ohta1, Takuo Tsukuda1, Nobuo Shimma1, Yuko Aoki1, Mikio Arisawa1, Michinori Kohara2 and Masayuki Sudoh1
ABSTRACT
An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate1, 2, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated3, and it is possible that inhibition of host proteins affords a therapeutic strategy for treatment of HCV infection. Using an HCV subgenomic replicon cell culture system, here we have identified, from a secondary fungal metabolite, a lipophilic long-chain base compound, NA255 (Compound 1), a previously unknown small-molecule HCV replication inhibitor. NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase, and it disrupts the association among HCV nonstructural (NS) viral proteins on the lipid rafts. Furthermore, we found that NS5B protein has a sphingolipid-binding motif in its molecular structure and that the domain was able to directly interact with sphingomyelin. Thus, NA255 is a new anti-HCV replication inhibitor that targets host lipid rafts, suggesting that inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of HCV infection.
ARTICLE
The majority of acute hepatitis C virus (HCV) infections become chronic; some progress toward liver cirrhosis or hepatocellular carcinoma4. Currently, viral enzyme-targeted drugs are being developed on the basis of viral nonstructural (NS) proteins-NS3/4A serine protease and NS5B RNA-dependent RNA polymerase-and are currently under clinical investigation for the treatment of HCV infection5, 6. However, resistance to antiviral agents directly targeting viral enzymes is a major factor limiting the efficacy of therapies against many retroviruses or RNA viruses owing to the error-prone nature of the viral reverse transcriptases or RNA-dependent RNA polymerases7. As these HCV-specific inhibitors enter clinical trials, resistance could become a major problem in patients treated with drugs targeting viral proteins. Currently, an HCV subgenomic replicon cell culture system is used as the cell-based model to study HCV replication and host-cell interactions8. It provides a useful tool for HCV drug development as well as clarification of the mechanisms of HCV RNA replication. The replicon cell line #Huh-7/3-1 constitutively expresses an HCV subgenomic replicon (genotype 1b, HCV-Con1) and enables the quantification of replication levels by measuring luciferase, making it suitable for high-throughput screening of HCV replication inhibitors. Here, using replicon cells, we identified a previously unknown small-molecule HCV replication inhibitor with the potential to be a clinical drug candidate for the treatment of HCV.
To identify a lead compound inhibiting HCV replication, we performed standard cell-based high-throughput screening using natural product libraries derived from microbial and fungal metabolites. We selected several hits that showed HCV replication inhibitory activity without host cellular toxicity. The most active extracts derived from Fusarium sp. led to the isolation of NA255 (Compound 1; Fig. 1a). The luciferase activities of the HCV replicon cells showed that replication was suppressed by NA255 in a dose-dependent manner with a mean of 50% inhibitory concentration (IC50) of 2 nM (Fig. 1b). In addition, levels of the replicon RNA significantly decreased after treatment with NA255 according to northern blot analysis (Fig. 1c). NA255 had no effect on host cell viability, as measured by the WST-8 assay (Fig. 1b, IC50 > 50 M), total cell counts using the Trypan Blue exclusion test, or host cell cycle progression from flow-cytometry analysis (Supplementary Fig. 1 online). We detected HCV-NS3 protein, which includes the protease and helicase domains, in HCV replicon cells using immunostaining analysis. In the absence of NA255 treatment, the NS3 protein was mainly localized in the perinuclear region (Fig. 1d, upper panel). After treatment with NA255 for 96 h, NS3 protein disappeared substantially (Fig. 1d, lower panel). Western blot analysis showed that NA255 treatment resulted in reduced levels of viral proteins such as NS3, NS5A and NS5B over time (Fig. 1e). These results indicate that NA255 is a potent small-molecule inhibitor of HCV replication.
To clarify the mode of action of NA255, we first evaluated inhibitory activity against the viral enzymes that are essential for HCV replication. NA255 did not significantly inhibit NS3 serine protease, NS3 helicase or NS5B RNA polymerase in vitro. In addition, there were no changes after treatment with NA255 in the expression levels of interferon (IFN)-induced antiviral response genes such as RNA-dependent protein kinase9, 2'-5'-oligoadenylate synthetase10 or RNase L11 (data not shown), suggesting that NA255 could be an anti-HCV drug with a previously unknown mode of action.
From a chemical substructure search, we found that NA255 had a structure similar to that of myriocin (Compound 2; ref. 12), a selective inhibitor of serine palmitoyltransferase (SPT), the enzyme responsible for the condensation of L-serine with palmitoyl CoA to produce 3-ketodihydrosphingosine, the first step in sphingolipid biosynthesis13. We examined the effect of NA255 on SPT inhibitory activity in vitro using purified human recombinant SPT encoding two different genes, LCB1 and LCB2 (refs. 14,15). NA255 displayed potent inhibition of SPT (IC50 = 10 nM; Fig. 2a). To assess whether NA255 inhibits the de novo biosynthesis of sphingolipids in cells, replicon cells were incubated with [14C]serine in the presence of NA255. NA255 inhibited the de novo synthesis of sphingolipids such as [14C]ceramide and [14C]sphingomyelin (SM) in a dose-dependent manner, but no changes were observed in the levels of phosphatidylethanolamine (PE) and phosphatidylserine (PS; Fig. 2b). To address whether sphingolipids are required for HCV replication, we attempted knockdown by small interfering RNAs (siRNAs) using two different siRNAs (designated si246 and si633). Immunoblot analysis of extracts from siRNA-transfected replicon cells demonstrated that the LCB1-directed siRNAs effectively reduced expression of LCB1 compared with the control siRNA (Fig. 2c). Knockdown of LCB1 substantially inhibited HCV replicon replication, depending on knockdown protein levels of LCB1, and had hardly any effect on cell viability (Fig. 2d). To assess whether inhibition of HCV replicon by NA255 was dependent on sphingolipid depletion, we incubated replicon cells with C2-ceramide, a cell-permeable ceramide analog, in the presence of NA255. Treatment of cells with C2-ceramide reversed the suppression by NA255 of HCV-NS3 protein levels, in a dose-dependent manner (Fig. 2e). Also, sphinganine, a close downstream product of SPT, in combination with NA255 or myriocin, substantially cancelled the replicon inhibitory effect (Supplementary Table 1 online). To further explore the involvement of the sphingolipid biosynthesis pathway in HCV replication, we examined the effect of sphingolipid-related small molecule compounds on HCV replicon replication. HCV replication was suppressed by myriocin, a known inhibitor of SPT, and fumonisin B1 (Compound 3; ref. 16), an inhibitor of dihydroceramide synthase (Fig. 2f, upper panels). In mammalian cells, ceramide is synthesized in the endoplasmic reticulum (ER) and translocates to the Golgi compartment for conversion to SM. (1R, 3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12; Compound 4) is an inhibitor of ceramide trafficking from ER to Golgi17. HPA-12 also substantially abrogated HCV replicon replication without cell toxicity (Fig. 2f, lower left panel). These results suggested that de novo synthesis of sphingolipids is required for HCV replication after translocation to the Golgi, and that interruption of the sphingolipid biosynthesis pathway provides an approach for the development of new HCV therapies.
Recent studies have demonstrated that HCV RNA and NS proteins are associated with intracellular membranes, including ER and Golgi18, and that the majority of the active replication complexes are present in Golgi-derived detergent-resistant membrane (DRM), most likely in lipid rafts18, 19, microdomains that are enriched in sphingolipids and cholesterol20. Since sphingolipids are essential components of the lipid raft, we examined the effect of NA255 on this HCV replication complex formation. When we treated cell lysates with Nonidet P-40, a nonionic detergent, HCV-NS proteins were found in both the DRM and the detergent-sensitive membrane fractions. NA255 treatment led to a marked decrease in the ratio of NS5B proteins in DRM fractions compared with control treatment (Fig. 3). We observed a substantial relocation of NS5B at 10 nM NA255 (Supplementary Fig. 2 online). We obtained a similar result after treating replicon cells with myriocin (Supplementary Fig. 2 online). In contrast, the DRM fractions of NS3 and NS5A were not affected (Fig. 3a,b). Lipid rafts are localized not only in Golgi but also in plasma membrane. To examine the effect of NA255 on the raft-associated protein in host cell plasma membrane, we assessed the distribution of host protein caveolin-2, which is mostly localized in plasma membrane and associated with lipid rafts. Caveolin-2 was present mostly in DRM fractions and was not affected by treatment with NA255 (Fig. 3a). These data suggest that NA255 inhibits the interaction of HCV-NS5B with lipid rafts through inhibition of sphingolipid biosynthesis and that this association is involved in HCV replicon replication because an active HCV replication complex is present in Golgi-derived DRM fractions18, 19.
To determine whether HCV protein could interact directly with sphingolipids, we searched for the sphingolipid-binding domain (SBD) in HCV-NS protein. The V3 loop of the human immunodeficiency virus (HIV)-1 surface envelope glycoprotein gp120 is an SBD that mediates the attachment of HIV-1 to plasma membrane lipid rafts21. In addition, it has been identified as a common sphingolipid-binding motif in gp120, prion protein (PrP), and -amyloid peptide22. To search for the SBD in HCV protein, we carried out structure similarity searches using a combinatorial extension program. We found the presence of an HIV-1 gp120 V3-like motif in an HCV-encoded NS5B protein (Fig. 4a,b). The V3-like domain of NS5B consists of a helix-turn-helix motif (Glu230-Gly263, Fig. 4c) formed by 34 amino acid residues, located in the finger domain of NS5B and of the same size as the V3 loop of gp120, so that superimposition is possible (Fig. 4b). It has been demonstrated that SM is associated with a peptide derived from the sphingolipid-binding motif of PrP22. To examine whether SM interacts with the SBD of NS5B (NS5B-SBD), we synthesized a peptide (fragment 231-261) derived from the putative sphingolipid-binding motif of NS5B and used surface plasmon resonance (SPR) spectroscopy. We found that SM substantially binds with the NS5B-SBD peptide in a dose-dependent manner, compared with control peptide (Fig. 4d and Supplementary Fig. 3 online). A similar result was observed with a PrP peptide under the same conditions, but it showed weak binding compared with NS5B-SBD (Fig. 4e). To confirm the binding of full-length NS5B protein to SM, we evaluated binding by ELISA using lipid-coated microplates. The intact NS5B protein effectively bound to SM, and showed some cross-reaction with galactosylceramide (Supplementary Fig. 3 online). These results indicate that NS5B has a sphingolipid-binding motif, and the domain was able to directly interact with SM.
Our present study suggests that SPT is a valuable new drug target that can be exploited for the development of HCV therapies. The blocking of SPT activity, both by small molecular weight compounds and by siRNAs, demonstrated notable antiviral effects in replicon cells. In addition, in our preliminary in vitro primary hepatocyte infection system, NA255 suppressed HCV replication more continuously than IFN- (data not shown). This anti-HCV effect is based on the disruption of host sphingolipid biosynthesis. Recently, it was reported that modulation of sphingolipid metabolism affects the susceptibility to HIV-1 infection, thereby inhibiting HIV-1 entry at the plasma membrane23. Further studies are needed to address the therapeutic potential of this attractive targeting drug, as current IFN-based HCV therapy has limitations.
HCV RNA replication depends on viral protein association with raft membrane18, 19. Lipid rafts are built mainly by SM, cholesterol and glycosphingolipids (GSLs). In this report, we suggested that SM is involved in HCV replication, thereby interacting with NS5B. Depletion of cellular cholesterol, another major component of lipid rafts, has recently been shown to reduce HCV RNA replication in HCV replicon cells18. We treated replicon cells with statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, and found that HCV replication was markedly disrupted by various statins including lovastatin (Compound 5) in replicon cells (Fig. 2f, lower right panel). Recent reports have demonstrated that inhibition of geranylgeranylation, rather than the synthesis of cholesterol itself, is responsible for inhibition of HCV RNA replication24, 25. To explore the involvement of GSLs in HCV replication, we evaluated replicon activity using an inhibitor of GSL biosynthesis, 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP, Compound 6). The IC50 value of PPMP for inhibition of HCV replication was 1.2 M, whereas PPMP showed cytotoxicity (IC50 = 5.3 M; only fourfold higher than its IC50 for inhibition of HCV replication). Furthermore, we found that galactosylceramide has a weak binding activity with NS5B (Supplementary Fig. 3 online). Thus, GSLs may be involved in the HCV replication process; however, further clarification of the interaction between HCV and raft components is needed. The structures of NS3 protease and NS5B polymerase have previously been determined by X-ray crystallography26, 27. Here we identified the SBD in NS5B protein but could not find the sphingolipid-binding motif in NS3, suggesting that NS3 could be indirectly associated with lipid rafts through cofactor NS4A28. In addition, inhibition of sphingolipid biosynthesis by NA255 disrupted the association of lipid rafts with NS5B, but not with NS3 or NS5A (Fig. 3b). Unlike NS3 and NS5A, NS5B seems to have a distinct mechanism of raft association. Host protein hVAP-33 has also recently been implicated in the interaction of NS5B proteins on lipid rafts and HCV replication28. Detailed descriptions of raft-protein interactions will provide new therapeutic strategies for rational drug design. Using point mutation analysis of NS5B-SBD, we are conducting ongoing studies to clarify the link between host sphingolipids and HCV replication competency.
Methods
Isolation of NA255.
Strain F1476, a producer strain of NA255, is a filamentous fungus that was isolated from fallen leaves collected in Kamakura, Japan. Strain F1476 was identified as Fusarium sp. One loopful of microorganisms obtained from a slant culture of strain F1476 was inoculated into Erlenmeyer flasks with baffles containing liquid media (2% glucose, 1.5% glycerol, 1% potato starch, 0.25% polypeptone, 0.35% yeast extract, 0.5% calcium carbonate, 0.3% sodium chloride, 0.005% zinc sulfate heptahydrate, 0.0005% copper sulfate pentahydrate, 0.0005% manganese sulfate tetrahydrate and 1% toasted soya); cultures were then incubated, shaking, at 25 C for 3 d to obtain an inoculated culture seed. This culture seed was inoculated into Erlenmeyer flasks with baffles containing solid media (40 g pressed barley, 24 ml SF1 solution (0.1% yeast extract, 0.05% sodium tartrate, 0.05% potassium dihydrogen phosphate)), followed by stationary culturing at 25 C for 11 d. n-Butanol (12.5 l) was then added to the culture, the culture was let stand overnight, and then the culture was filtered to obtain an n-butanol extract. After concentrating, the extract was suspended in 1 l of water, adjusted to pH 2 with hydrochloric acid, and was extracted with 1.1 l of ethyl acetate. The aqueous layer was extracted again with 1.1 l of ethyl acetate and combined with the first extract. Water (0.9 l) was then added to the ethyl acetate extract (2.2 l) and distributed after adjusting to pH 10 with an aqueous sodium hydroxide solution. Ethyl acetate (1 l) was again added to the resulting aqueous layer and then extracted after adjusting to pH 3 with hydrochloric acid. The resulting aqueous layer was again extracted with 1 l of ethyl acetate. The ethyl acetate extract (2 l) thus obtained was then dried over sodium sulfate followed by concentrating and drying to obtain 567 mg of crude extract. This crude extract was dissolved in methanol and repeatedly purified by HPLC (CCPP-D, MCPD-3600 System (Tosoh), CAPCELL PAK C18 (UG 80, 20 mm 250 mm, Shiseido)), using water containing 0.01% trifluoroacetic acid and acetonitrile containing 0.01% trifluoroacetic acid (15% acetonitrile to 98% acetonitrile, stepwise). NA255 was concentrated under reduced pressure to obtain 380 mg of NA255 in the form of a white powder. 1H-NMR (in methanol d-4): 0.89 (t, J = 7 Hz, 3H), 1.20-1.40 (m, 14H), 1.53 (m, 4H), 1.73 (s, 3H), 1.77 (s, 3H), 1.96 (m, 2H), 2.42 (m, 4H), 2.57 (d, J = 16.5 Hz, 1H), 2.89 (d, J = 16.5 Hz, 1H), 2.91 (dd, J = 14, 9 Hz, 1H), 3.15 (dd, J = 14, 4.5 Hz, 1H), 3.20 (d, J = 8 Hz, 1H), 4.47 (d, J = 6 Hz, 2H), 4.63 (dd, J = 9, 4.5 Hz, 1H), 5.43 (m, 1H), 5.52 (m, 2H), 6.78 (d, J = 9 Hz, 2H), 7.10 (d, J = 9 Hz, 2H); FAB-MS (m/z; positive mode; matrix m-NBA): 660 (M + H)+; FAB-MS (m/z; negative mode; matrix m-NBA): 658 (M - H)-.
SPT assay.
To examine the effect of NA255 on SPT inhibitory activity, we prepared human recombinant SPT encoding two different genes, LCB1 and LCB2. These cDNAs were generated from a liver cDNA library by RT-PCR. Human embryonic kidney (HEK) 293 cells were transiently transfected with His-tagged LCB1 and LCB2 in a dual expression vector, pBudCE4.1 (Invitrogen), for 72 h and then harvested. The cells were lysed in lysis buffer (50 mM NaH2PO4, 30 mM NaCl, 10 mM imidazole and 0.1% sucrose monolaurate) and sonicated 30 times with short pulses. After centrifugation at 1,200g for 5 min, the supernatant was incubated with Ni-NTA agarose (QIAGEN), washed with wash buffer (50 mM NaH2PO4, 30 mM NaCl, 20 mM imidazole and 0.1% sucrose monolaurate) and eluted with elution buffer (50 mM NaH2PO4, 30 mM NaCl, 250 mM imidazole and 0.1% sucrose monolaurate). The eluted solution was concentrated by using Amicon Ultra-4 (Millipore) and exchanged into buffer (10 mM HEPES buffer, pH 7.4, containing 250 mM sucrose and 0.1% sucrose monolaurate). The purified SPT fraction was stored at -20 C until use. Purified human SPT was added to 0.1 ml of a reaction mixture containing 200 mM HEPES buffer (pH 8.0), 5 mM EDTA, 10 mM DTT, 0.05 mM pyridoxal 5-phosphate, 0.2 mM palmitoyl-CoA, 0.1 mM L-serine and 1 Ci [3H]serine (Amersham) in the presence of NA255. After a 15-min incubation at 37 C, the reaction was stopped by the addition of 0.25 ml of 0.5 M NH4OH and 0.75 ml of chloroform/methanol (1:2, v/v). The products were extracted and the organic phases were then washed twice with water, followed by measurement of the radioactivity by lipid scintillation counting.
Detection of cellular sphingolipids.
Cells were incubated for 18 h with [14C]serine (0.2 Ci ml-1) in Opti-MEM (Gibco BRL) and washed with PBS after treatment with NA255. After cells were lysed with 0.3 ml of 0.1% SDS, and the lysate was suspended by pipetting. Total lipids were extracted with 0.9 ml of chloroform/methanol (1:2 v/v), and then 0.3 ml chloroform and 0.3 ml PBS were added and mixed well. The extracts were spotted on Silica Gel 60 thin-layer chromatography (TLC) plates (Merck) and chromatographed with chloroform/methanol (10:1, v/v) or methyl acetate/1-propanol/chloroform/methanol/ 0.25% KCl (25:25:25:10:9, v/v). Radioactive spots were evaluated using a bio-imager (BAS 1000, Fuji Photo Film).
Accession codes.
Protein Data Bank accession codes: 1QUV, HCV-NS5B; 1CE4, HIV-1 gp120 V3 loop peptide. International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Japan) accession code: FERM BP-8920, strain F1476, Fusarium sp.
Additional methods are available as Supplementary Methods online.
Note: Supplementary information is available on the Nature Chemical Biology website.
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