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 rate, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated, 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 (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.

It has been demonstrated that the carboxyl terminus of microbody enzymes functions as a targeting signal to microbodies in higher plants. We have examined an ability of 24 carboxy-terminal amino acid sequences to facilitate the transport of a cytosolic passenger protein, beta-glucuronidase, into microbodies in green cotyledonary cells of transgenic Arabidopsis. Immunoelectron microscopic analysis revealed that carboxy-terminal tripeptide sequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as a microbody-targeting signal, although tripeptides with proline at the first amino acid position and isoleucine at the carboxyl terminus show weak targeting efficiencies. All known microbody enzymes that are synthesized in a form similar in size to the mature molecule, except catalase, contain one of these tripeptide sequences at their carboxyl terminus.

Malate synthase is a glyoxysome-specific enzyme. The carboxy-terminal tripeptide of the enzyme is Ser-Arg-Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy-terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of beta-glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeptides with S, A or P at the -3 position, K or R at the -2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.

Cyclothiazomycinis a novel renin inhibitor produced by Streptomyces sp. NR0516. It was isolated from fermentation broth by extraction with butyl alcohol, QAE-Toyopearl column chromatography and preparative HPLC.Cyclothiazomycin, which was determined to be a unique polythiazole-containing bicyclic peptide, exhibited inhibitory activity against human plasma renin with IC50 being 1.7/xm.The renin angiotensin system is implicated in several forms of hypertension. As a highly specific aspartic protease, renin cleaves angiotensinogen to form angiotensin I. This metabolic intermediate is further cleaved by angiotensin converting enzyme (ACE) to yield angiotensin II, which causes vasoconstriction and stimulates secretion of aldosterone and catecholamine leading to elevation of blood pressure. In this cascade reaction, renin is a rate-limiting enzyme1'2). Therefore, renin inhibitors are expected to the most effective in preventing these disorders.

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