Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DNS3 with a binding constant of about 10 nm and inhibit approximately 90% of the protease activity of DNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DNS3 with a K i < 100 nm in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.Keywords: HCV; RNA aptamer; SELEX; NS3 serine protease.Hepatitis C virus (HCV) is the major etiological agent of posttransfusion non-A, non-B hepatitis (reviewed in [1]). Chronic HCV infection is a serious disease throughout the world and can also develop into chronic hepatitis, liver cirrhosis or hepatocellular carcinoma [2]. Although the number of HCV carriers has increased to about 300 million worldwide, the principle drugs against HCV are all based on interferon and effective antiviral drugs have not yet been developed.HCV has a single, positive-stranded RNA genome approximately 9.5 kb in length. The genetic organization of HCV is similar to that of flaviviruses and pestiviruses and HCV has been classified as a new genus of the family Flaviviridae. HCV proteins include the core (C), envelope (E), and nonstructural (NS) groups of proteins. The proteins are synthesized as a polyprotein of approximately 3000 amino acids in the order NH 2 ±C±E1±E2±p7±NS2±NS3±NS4A±NS4B±NS5A±NS5B± COOH [3±5]. Mature viral structural and nonstructural proteins are produced from the polyprotein by a series of cotranslational and post-translational cleavages mediated by host signal peptidases [6±8] and two viral proteases, NS2-3 [9,10] and NS3 [11±14]. The NS2-3 protease activity is zinc-dependent and it cleaves at the NS2/NS3 junction. The protease activity of NS3 lies in the N-terminal third of the polypeptide. The NS3 protease is a trypsin-like serine protease which is essential for processing many of the nonstructural proteins of HCV, including NS3, NS4A, NS4...

Hepatitis C virus (HCV) is a single-stranded RNA virus and its genome is translated into a single large polyprotein. The viral-encoded NS3 protein possesses protease, nucleoside triphosphatase, and helicase activities. Since these activities appear to be important for viral replication, efforts are being made to identify compounds that might inhibit the enzymatic activities of NS3 and serve as potential anti-HCV agents. We used a genetic selection strategy in vitro to isolate, from a pool of completely random RNA (120 random bases), those RNA aptamers that could bind to NS3. After six cycles of selection and amplification, 14% of the pooled RNAs could bind specifically to the NS3 protein. When the aptamers in the pool (cycle 6) were analyzed for binding and inhibition of the proteolytic activity of NS3 with the NS5A/NS5B peptide as substrate (S1), two aptamers, designated G6-16 and G6-19 RNA, were found to inhibit NS3 in vitro. Kinetic studies of the inhibition revealed that the aptamer G6-16 inhibited the NS3 protease with an inhibitory constant (Ki) of 3 microM. We also analyzed aptamers G6-16 and G6-19 for their action with a longer protein substrate (amino acid region 2203-2506) and found that these aptamers efficiently inhibited the proteolytic activity of NS3. In addition, both G6-16 and G6-19 aptamers were found to inhibit the helicase activity of NS3. Since these aptamers possesses dual inhibitory function for NS3, they could prove to be useful as anti-HCV drug leads.

Alpha-melanocyte-stimulating hormone (a-MSH) is a neuropeptide expressed in pituitary and brain that is known to regulate energy balance, appetite control, and neuroimmune functions. The biosynthesis of a-MSH requires proteolytic processing of the proopiomelanocortin (POMC) precursor. Therefore, this study investigated the in vivo role of the prohormone convertase 2 (PC2) processing enzyme for production of a-MSH in PC2-deficient mice. Specific detection of a-MSH utilized radioimmunoassay (RIA) that does not crossreact with the POMC precursor, and which does not crossreact with other adrenocorticotropin hormone (ACTH) and b-endorphin peptide products derived from POMC. a-MSH in PC2-deficient mice was essentially obliterated in pituitary, hypothalamus, cortex, and other brain regions (collectively), compared to wild-type controls. These results demonstrate the critical requirement of PC2 for the production of a-MSH. The absence of a-MSH was accompanied by accumulation of ACTH, ACTH-containing imtermediates, and POMC precursor. ACTH was increased in pituitary and hypothalamus of PC2-deficient mice, evaluated by RIA and reversed-phase high pressure liquid chromatography (RP-HPLC). Accumulation of ACTH demonstrates its role as a PC2 substrate that can be converted for a-MSH production. Further analyses of POMC-derived intermediates in pituitary, conducted by denaturing western blot conditions, showed accumulation of ACTH-containing intermediates in pituitaries of PC2-deficient mice, which implicate participation of such intermediates as PC2 substrates. Moreover, accumulation of POMC was observed in PC2-deficient mice by western blots with anti-ACTH and anti-b-endorphin. In addition, increased b-endorphin 1)31 was observed in pituitary and hypothalamus of PC2-deficient mice, suggesting b-endorphin 1)31 as a substrate for PC2 in these tissues. Overall, these studies demonstrated that the PC2 processing enzyme is critical for the in vivo production of a-MSH in pituitary and brain. Keywords: alpha-melanocyte-stimulating hormone, brain, pituitary, prohormone convertase, proopiomelanocortin. The neuropeptide a-MSH (melanocyte-stimulating hormone) is derived from its POMC (proopiomelanocortin) precursor (Roberts et al. 1979;Chang et al. 1980) by proteolytic processing within neuroendocrine secretory vesicles of pituitary and brain (Steiner et al. 1992;Hook et al. 1994;Cawley et al. 1998;Seidah et al. 1999). Specific proteolytic processing of POMC at multibasic residues generates distinct neuropeptide products that consist of a-MSH, ACTH (adrenocorticotropin hormone), and b-endorphin. These POMC products are abundant in pituitary and brain where they are secreted for the regulation of pituitary and brain functions that include energy balance, appetite control, and neuroimmune functions (Ichiyama et al. 2000;Williams et al. 2001;Pritchard et al. 2002;Zimanyi and Pelleymounter 2003 These authors contributed equally to this study.Abbreviations used: ACTH, adrenocorticotropin hormone; a-MSH, amelanocyte-stimulating horm...

Histone deacetylase (HDAC) inhibitors are showing promise as treatment for a variety of human cancers, but their precise mechanism of action has not been elucidated. We examined the effects of the HDAC inhibitor butyrate on colon cancer cells, focusing on its effect on the cell cycle promoter cyclin B(1). In HT-29 cells, sodium butyrate-mediated growth inhibition is associated with a marked decrease in cyclin B(1) mRNA levels. The decrease in cyclin B(1) occurred in a delayed fashion (at 24 h), is completely blocked by concomitant treatment with protein synthesis inhibitors, and appears to be dependent on changes in transcription. Cyclin B(1) repression is linked to the differentiation process in colon cancer cells, not merely with growth arrest. The mechanism of cyclin B(1) repression by butyrate requires prolonged histone hyperacetylation and is at least partly dependent on p21 expression. In fact, p21/WAF-1 appears to directly repress a minimal cyclin B(1) promoter (-90 bp), a process that can be mediated by the amino-terminal portion of the p21 protein. These findings highlight key molecular mechanisms by which HDAC inhibitors mediate their beneficial effects on human cancer cells.