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1.
Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct domains, protease and helicase, that are essential for HCV proliferation. Therefore, NS3 is considered a target for anti-HCV treatment. To study RNA aptamers of the NS3 helicase domain, we carried out in vitro selection against the HCV NS3 helicase domain. RNA aptamers obtained after eight generations possessed 5' extended single-stranded regions and the conserved sequence (5'-GGA(U/C)GGAGCC-3') at stem-loop regions. Aptamer 5 showed strong inhibition of helicase activity in vitro. Deletion and mutagenesis analysis clarified that the conserved stem-loop is important and that the whole structure is needed for helicase inhibition. We compared the inhibition of helicase activity between aptamer 5 and 3'+-UTR of HCV. 相似文献
2.
Umehara T Fukuda K Nishikawa F Kohara M Hasegawa T Nishikawa S 《Journal of biochemistry》2005,137(3):339-347
The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Delta, was conjugated via an oligo(U) tract to the 3'-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity in vitro, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication in vitro. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition. 相似文献
3.
Naud J Lemke C Goudreau N Beaulieu E White PD Llinàs-Brunet M Forgione P 《Bioorganic & medicinal chemistry letters》2008,18(11):3400-3404
The design and synthesis of tripeptide-based inhibitors of the HCV NS3 protease containing a novel P2-triazole is described. Replacement of the P2 quinoline with a triazole moiety provided a versatile handle which could be expediently modified to generate a diverse series of inhibitors. Further refinement by the incorporation of an aryl-substituted triazole and replacement of the P1 acid with an acyl sulfonamide ultimately provided inhibitors with interesting cellular activity. 相似文献
4.
Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase 下载免费PDF全文
Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity. 相似文献
5.
Han W Hu Z Jiang X Wasserman ZR Decicco CP 《Bioorganic & medicinal chemistry letters》2003,13(6):1111-1114
Using a tetrapeptide-based alpha-ketoamide template, various amines and amino acids were incorporated to explore the prime side of the HCV NS3 protease catalytic site. Glycine carboxylic acid was found to be the most effective prime group. Further optimization yielded an inhibitor with IC(50) of 0.060 microM. 相似文献
6.
Srikanth Venkatraman Wanli Wu Neng-Yang Shih F. George Njoroge 《Bioorganic & medicinal chemistry letters》2009,19(16):4760-4763
Chronic hepatitis C infection is the primary cause for cirrhosis of the liver and hepatocellular carcinoma leading to liver failure and transplantation. The etiological agent hepatitis C virus produces a single positive strand RNA that is processed further with the help of NS3 serine protease to produce mature virus. Inhibition of this protease can potentially be used to develop drugs for HCV infections. Boceprevir is a ketoamide derived novel inhibitor of HCV NS3 protease that has been progressed to clinical trials and proven to be efficacious in humans. Herein, we report our efforts in identifying an aza-peptide derivative as a potential second generation compound, that lacks electrophilic ketoamide group and are potent in enzyme and replicon assay. 相似文献
7.
Chang Ho Lee Young Ju Lee Ji Hyun Kim Jong Hoon Lim Jung-Hye Kim Wonkyo Han Soo-Han Lee Gyu-Jeong Noh Seong-Wook Lee 《Journal of virology》2013,87(12):7064-7074
This study identified specific and avid RNA aptamers consisting of 2′-hydroxyl- or 2′-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2′-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2′-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2′-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2′-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach. 相似文献
8.
Nizi E Koch U Ontoria JM Marchetti A Narjes F Malancona S Matassa VG Gardelli C 《Bioorganic & medicinal chemistry letters》2004,14(9):2151-2154
The N-terminal aminoacid of phenethylamide tripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced with an alpha-hydroxy acid to obtain more 'drug like' inhibitors with low micromolar activity. The preferred S-configuration of the capping residue can be explained by molecular modeling studies. 相似文献
9.
Nizi E Koch U Ponzi S Matassa VG Gardelli C 《Bioorganic & medicinal chemistry letters》2002,12(22):3325-3328
The N-terminal aminoacid of alpha-ketotripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced with an alpha-hydroxy acid, leading to capped dipeptide inhibitors such as 20 with an IC(50) value of 3.0 microM. The importance of the lipophilic side chain interactions at S3 of the protease and the requirement of the capping residue with R configuration have been explained by molecular modeling studies. 相似文献
10.
Todd C. Appleby Robert Anderson Anna M. Pyle Xiaohong Liu John R. Somoza 《Journal of molecular biology》2011,405(5):1139-1749
The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3′ end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a “spring-loading” mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate. 相似文献
11.
A series of bicyclic pyrimidinone-based HCV NS3 protease inhibitors was synthesized via selective C8 position functionalization. Substituted phenylamides and phenylureas were preferred in the S2 binding pocket. 相似文献
12.
Zaki Monawar Eisa 《Saudi Journal of Biological Sciences》2011,18(2):195-200
Hepatitis C virus (HCV) causes serious infections in the liver which may lead to liver cirrhosis and hepatocellular carcinoma. Non structural 3 (NS3) protein is one of the most important proteins of the virus which has protease and helicase activities. Protease activity has a crucial role in the replication and persistence of the virus. Site directed mutation was carried out in the protease region of one NS3 and another site directed mutation in the helicase region of another NS3. The expression of both mutated NS3 was compared with wild NS3. Expression of the three different NS3 types was confirmed by in situ staining and western blotting using an anti-NS3 antibody and correlated with a reduced antiviral response after treatment with interferon-α. Mutation analysis showed that the NS3 protease activity andnot the NS3 helicase was essential for the inhibition of the interferon-α response. 相似文献
13.
Hepatitis C virus (HCV) NS3 protease is the key enzyme for its maturation. Three hypotheses have been advanced in the literature to demonstrate the mechanism of the activation of the HCV NS3 protease. A virus-encoded protein NS4A and substrate are proposed to be involved in the activation of the HCV NS3 protease. However, the three hypotheses are not completely consistent with one another. Multiple molecular dynamics simulations were performed on various NS3 protease systems: free NS3 protease, NS3/4A, NS3/inhibitor, and NS3/4A/inhibitor complexes, to further unravel the mechanism of the activation of the NS3 protease. Simulation results suggest that the binding of NS4A induces a classic serine protease conformation of the catalytic triad of the NS3 protease. NS4A rearranges the secondary structure of both the N-terminus and catalytic site of the NS3 protease, reduces the mobility of the global structure of the NS3 protease, especially the catalytic site, and provides a rigid and tight structure, except for the S1 pocket, for the binding and hydrolysis of substrates. The binding of substrate also contributes to the activation of the NS3 protease by an induced-fit of the classic serine protease catalytic triad. However, the global structure of the NS3 protease is still loose and highly flexible without stable secondary structural elements, such as helix α0 at the N-terminus and helix α1 and β-sheet E1-F1 at the catalytic site. The structure of the NS3 protease without NS4A is not suitable for the binding and hydrolysis of substrates. 相似文献
14.
Sperandio D Gangloff AR Litvak J Goldsmith R Hataye JM Wang VR Shelton EJ Elrod K Janc JW Clark JM Rice K Weinheimer S Yeung KS Meanwell NA Hernandez D Staab AJ Venables BL Spencer JR 《Bioorganic & medicinal chemistry letters》2002,12(21):3129-3133
Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes. 相似文献
15.
Peptide-based alpha-ketoamides, alpha-ketoesters and alpha-diketones were designed, synthesized and evaluated against HCV NS3 protease. Alpha-ketoamides have the highest affinity among the three classes, with 8 being the most potent inhibitor with an IC50 of 340 nM. 相似文献
16.
17.
NS3H, the helicase domain of HCV NS3, possesses RNA-stimulated ATPase and ATP hydrolysis-dependent dsRNA unwinding activities. Here, the ability of NS3H to facilitate RNA structural rearrangement is studied using relatively long RNA strands as the model substrates. NS3H promotes intermolecular annealing, resolves three-stranded RNA duplexes, and assists dsRNA and ssRNA inter-conversions to establish a steady state among RNA structures. NS3H facilitates RNA structure conversions in a mode distinct from an ATP-independent RNA chaperone. These findings expand the known function of HCV NS3 helicase and reveal a role for viral helicase in assisting RNA structure conversions during virus life cycle. 相似文献
18.
P1 Phenethyl peptide boronic acid inhibitors of HCV NS3 protease 总被引:1,自引:0,他引:1
Priestley ES De Lucca I Ghavimi B Erickson-Viitanen S Decicco CP 《Bioorganic & medicinal chemistry letters》2002,12(21):3199-3202
A series of peptide boronic acids containing extended, hydrophobic P1 residues was prepared to probe the shallow, hydrophobic S1 region of HCV NS3 protease. The p-trifluoromethylphenethyl P1 substituent was identified as optimal with respect to inhibitor potency for NS3 and selectivity against elastase and chymotrypsin. 相似文献
19.
Olaf Isken Thomas Walther Luis Wong-Dilworth Dirk Rehders Lars Redecke Norbert Tautz 《PLoS pathogens》2022,18(6)
Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3. 相似文献
20.
Zhang X Schmitt AC Jiang W Wasserman Z Decicco CP 《Bioorganic & medicinal chemistry letters》2003,13(6):1157-1160
Starting from a hexapeptide boronic acid lead, 3-amino bicyclic pyrazinones as novel beta-sheet dipeptide mimetics have been designed and synthesized. Side-chain manipulation of this scaffold generated a series of potent, nonpeptidic inhibitors of HCV NS3 protease. 相似文献