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Integration of Adenylate Kinase 1 with Its Peptide Conformational Imprint. Int J Mol Sci 2022; 23:ijms23126521. [PMID: 35742970 PMCID: PMC9223553 DOI: 10.3390/ijms23126521] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Revised: 06/09/2022] [Accepted: 06/09/2022] [Indexed: 02/01/2023] Open
Abstract
In the present study, molecularly imprinted polymers (MIPs) were used as a tool to grasp a targeted α-helix or β-sheet of protein. During the fabrication of the hinge-mediated MIPs, elegant cavities took shape in a special solvent on quartz crystal microbalance (QCM) chips. The cavities, which were complementary to the protein secondary structure, acted as a peptide conformational imprint (PCI) for adenylate kinase 1 (AK1). We established a promising strategy to examine the binding affinities of human AK1 in conformational dynamics using the peptide-imprinting method. Moreover, when bound to AK1, PCIs are able to gain stability and tend to maintain higher catalytic activities than free AK1. Such designed fixations not only act on hinges as accelerators; some are also inhibitors. One example of PCI inhibition of AK1 catalytic activity takes place when PCI integrates with an AK19-23 β-sheet. In addition, conformation ties, a general MIP method derived from random-coil AK1133-144 in buffer/acetonitrile, are also inhibitors. The inhibition may be due to the need for this peptide to execute conformational transition during catalysis.
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Study of membrane deformations induced by Hepatitis C protein NS4B and its terminal amphipathic peptides. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2020; 1863:183537. [PMID: 33383025 DOI: 10.1016/j.bbamem.2020.183537] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 12/13/2019] [Revised: 11/27/2020] [Accepted: 12/13/2020] [Indexed: 11/21/2022]
Abstract
Many viruses destabilize cellular membranous compartments to form their replication complexes, but the mechanism(s) underlying membrane perturbation remains unknown. Expression in eukaryotic cells of NS4B, a protein of the hepatitis C virus (HCV), alters membranous complexes and induces structures similar to the so-called membranous web that appears crucial to the formation of the HCV replication complex. As over-expression of the protein is lethal to both prokaryotic and eukaryotic cells, NS4B was produced in large quantities in a "cell-free" system in the presence of detergent, after which it was inserted into lipid membranes. X-ray diffraction revealed that NS4B modifies the phase diagram of synthetic lipid aqueous phases considerably, perturbing the transition temperature and cooperativity. Cryo-electron microscopy demonstrated that NS4B introduces significant disorder in the synthetic membrane as well as discontinuities that could be interpreted as due to the formation of pores and membrane merging events. C- and N-terminal fragments of NS4B are both able to destabilize liposomes. While most NS4B amphipathic peptides perforate membranes, one NS4B peptide induces membrane fusion. Cryo-electron microscopy reveals a particular structure that can be interpreted as arising from hemi-fusion-like events. Amphipathic domains are present in many proteins, and if exposed to the aqueous cytoplasmic medium are sufficient to destabilize membranes in order to form viral replication complexes. These domains have important functions in the viral replication cycle, and thus represent potential targets for the development of anti-viral molecules.
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Jirasko V, Lakomek N, Penzel S, Fogeron M, Bartenschlager R, Meier BH, Böckmann A. Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus. Chembiochem 2020; 21:1453-1460. [PMID: 31850615 PMCID: PMC7318649 DOI: 10.1002/cbic.201900765] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2019] [Indexed: 01/01/2023]
Abstract
Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D 1 H-detected 1 H,15 N and 3D 1 H,13 C,15 N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.
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Affiliation(s)
- Vlastimil Jirasko
- ETH ZürichPhysical ChemistryVladimir-Prelog Weg 28093ZürichSwitzerland
| | | | - Susanne Penzel
- ETH ZürichPhysical ChemistryVladimir-Prelog Weg 28093ZürichSwitzerland
| | - Marie‐Laure Fogeron
- Institut de Biologie et Chimie des ProteinesMMSBLabex EcofectUMR 5086 CNRSUniversité de Lyon7 passage du Vercors69367LyonFrance
| | - Ralf Bartenschlager
- Department of Infectious DiseasesMolecular VirologyHeidelberg UniversityIm Neuenheimer Feld 34569120HeidelbergGermany
- Division of Virus-Associated Carcinogenesis (Germany)Cancer Research Center (DKFZ)Im Neuenheimer Feld 24269120HeidelbergGermany
| | - Beat H. Meier
- ETH ZürichPhysical ChemistryVladimir-Prelog Weg 28093ZürichSwitzerland
| | - Anja Böckmann
- Institut de Biologie et Chimie des ProteinesMMSBLabex EcofectUMR 5086 CNRSUniversité de Lyon7 passage du Vercors69367LyonFrance
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Preclinical evaluation of Amphihevir, a first-in-class clinical Hepatitis C virus NS4B inhibitor. Antimicrob Agents Chemother 2019:AAC.01237-19. [PMID: 31527036 DOI: 10.1128/aac.01237-19] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Amphihevir, a benzofuran derivative, is the first reported NS4B inhibitor that has advanced to clinical trials (currently in Phase Ib). Here, we report the results of a preclinical study of its potency, toxicity, selectivity, DMPK, and safety profiles. Amphihevir displayed good antiviral activities against genotype 1a (EC50=0.34 nM) and genotype 1b (EC50=1.97 nM) replicons and evident cytotoxicity in twelve strains of cell lines derived from animals and humans. Amphihevir was found to be inactive against other viruses, human kinases, and GPCRs, which implies its good selectivity. A 9-day long-term treatment of genotype 1b replicon with Amphihevir resulted in a 3.8 Log10 decline of the hepatitis C viral RNA at a concentration of 25×EC90 Drug resistance screening showed that mutations occurred at H94, F98, and V105 of NS4B, which mediated the resistance to Amphihevir. This result suggests that NS4B is the main target of Amphihevir. There was no cross-resistances between Amphihevir and NS5A, NS3/4A, and NS5B inhibitors, suggesting that Amphihevir on combination of other anti- hepatitis C virus drugs could treat hepatitis C, as proven by studies of Amphihevir and other hepatitis C virus inhibitors. Pharmacokinetic studies demonstrated that Amphihevir has good oral bioavailability and appropriate T1/2 in rats and dogs, thereby supporting its use once per day. Finally, Amphihevir showed good safety profiles in rats and dogs. The results shed light on the use of Amphihevir as a potential treatment option for chronic hepatitis C patients.
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Jiang XH, Xie YT, Cai YP, Ren J, Ma T. Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway. BMC Microbiol 2017; 17:124. [PMID: 28545480 PMCID: PMC5445264 DOI: 10.1186/s12866-017-1032-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Accepted: 05/15/2017] [Indexed: 12/21/2022] Open
Abstract
Background Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Results Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of β-catenin (F = 165.26, P < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression (P < 0.01); and promoted proliferation of Huh7 cells (P < 0.01 or P < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of β-catenin (F = 66.54, P < 0.01) and the Wnt3a-induced proliferation of LO2 cells (P < 0.01 or P < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05). Conclusions HCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-1032-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Xiao-Hua Jiang
- Department of Infectious Diseases, the First Affiliated Hospital of the University of South China, Hengyang, 421001, China
| | - Yu-Tao Xie
- Department of Infectious Diseases, Xiangya Hospital of Central South University, Changsha, 410087, China.
| | - Ya-Ping Cai
- Department of Epidemiology and Health Statistics, the University of South China, Hengyang, 421001, China
| | - Jing Ren
- Department of Infectious Diseases, the First Affiliated Hospital of the University of South China, Hengyang, 421001, China
| | - Tao Ma
- Department of Infectious Diseases, the First Affiliated Hospital of the University of South China, Hengyang, 421001, China
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PTC725, an NS4B-Targeting Compound, Inhibits a Hepatitis C Virus Genotype 3 Replicon, as Predicted by Genome Sequence Analysis and Determined Experimentally. Antimicrob Agents Chemother 2016; 60:7060-7066. [PMID: 27620477 PMCID: PMC5118984 DOI: 10.1128/aac.01272-16] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2016] [Accepted: 09/01/2016] [Indexed: 01/03/2023] Open
Abstract
PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of ∼5 nM. De novo resistance selection identified mutations encoding amino acid substitutions mapping to the first predicted transmembrane region of NS4B, a finding consistent with results for PTC725 and other NS4B-targeting compounds against HCV gt1. This is the first report of the activity of an NS4B targeting compound against HCV gt3. In addition, we have identified previously unreported amino acid substitutions selected by PTC725 treatment which further demonstrate that these compounds target the NS4B first transmembrane region.
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Glab-Ampai K, Malik AA, Chulanetra M, Thanongsaksrikul J, Thueng-In K, Srimanote P, Tongtawe P, Chaicumpa W. Inhibition of HCV replication by humanized-single domain transbodies to NS4B. Biochem Biophys Res Commun 2016; 476:654-664. [PMID: 27240954 DOI: 10.1016/j.bbrc.2016.05.109] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2016] [Accepted: 05/21/2016] [Indexed: 12/21/2022]
Abstract
NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti-viral immunity. Thus, NS4B is an attractive target of a novel anti-HCV agent. In this study, humanized-nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN), for making them cell penetrable (become transbodies). Human hepatic (Huh7) cells transfected with HCV JFH1-RNA that were treated with transbodies from four Escherichia coli clones (PEN-VHH7, PEN-VHH9, PEN-VH33, and PEN-VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated-transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNβ and IL-28b. The transbodies have high potential for testing further as a novel anti-HCV agent, either alone, adjunct of existing anti-HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.
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MESH Headings
- Antibodies, Monoclonal, Humanized/administration & dosage
- Antibodies, Monoclonal, Humanized/chemistry
- Antibodies, Monoclonal, Humanized/genetics
- Antibodies, Viral/administration & dosage
- Antibodies, Viral/chemistry
- Antibodies, Viral/genetics
- Antiviral Agents/administration & dosage
- Antiviral Agents/chemistry
- Carrier Proteins/administration & dosage
- Carrier Proteins/chemistry
- Carrier Proteins/genetics
- Cell Line
- Cell Surface Display Techniques
- Cell-Penetrating Peptides/administration & dosage
- Cell-Penetrating Peptides/chemistry
- Cell-Penetrating Peptides/genetics
- Computer Simulation
- Hepacivirus/genetics
- Hepacivirus/immunology
- Hepacivirus/physiology
- Humans
- Immunity, Innate/genetics
- Models, Molecular
- Recombinant Proteins/chemistry
- Recombinant Proteins/genetics
- Recombinant Proteins/immunology
- Single-Domain Antibodies/administration & dosage
- Single-Domain Antibodies/chemistry
- Single-Domain Antibodies/genetics
- Transfection
- Viral Nonstructural Proteins/genetics
- Viral Nonstructural Proteins/immunology
- Viral Nonstructural Proteins/physiology
- Virus Replication/genetics
- Virus Replication/immunology
- Virus Replication/physiology
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Affiliation(s)
- Kittirat Glab-Ampai
- Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
| | - Aijaz Ahmad Malik
- Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
| | - Monrat Chulanetra
- Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
| | - Jeeraphong Thanongsaksrikul
- Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand
| | - Kanyarat Thueng-In
- Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; School of Pathology, Institute of Medicine, Suranaree University of Technology, Nakhonratchaseema Province 30000, Thailand
| | - Potjanee Srimanote
- Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand
| | - Pongsri Tongtawe
- Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand
| | - Wanpen Chaicumpa
- Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand.
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8
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Dual ATPase and GTPase activity of the replication-associated protein (Rep) of beak and feather disease virus. Virus Res 2016; 213:149-161. [DOI: 10.1016/j.virusres.2015.12.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2015] [Revised: 12/01/2015] [Accepted: 12/01/2015] [Indexed: 11/18/2022]
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9
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Savardashtaki A, Sharifi Z, Hamzehlou S, Farajollahi MM. Analysis of Immumoreactivity of Heterologously Expressed Non-structural Protein 4B (NS4B) from Hepatitis C Virus (HCV) Genotype 1a. IRANIAN JOURNAL OF BIOTECHNOLOGY 2015; 13:32-37. [PMID: 28959307 DOI: 10.15171/ijb.1321] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
BACKGROUND Detection of hepatitis C virus specific antibodies is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and has been widely used in commercial Enzyme Immunoassays (EIA). Additionally, NS4B, a key protein in the virus replication, can be an alternative target for antiviral therapy. OBJECTIVES Development of a new method for high-level expression and purification of NS4B coding region was the aim of the report. MATERIALS AND METHODS Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. coli BL21. Induction was performed by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. Immunoreactivity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzymelinked immunosorbent assay (ELISA). RESULTS The recombinant NS4B protein was expressed and its immunoreactivity was confirmed by ELISA and western blotting. CONCLUSIONS The directional TOPO cloning provides an efficient and easy platform for heterologous expression of immunoreactive HCV NS4B.
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Affiliation(s)
- Amir Savardashtaki
- Cellular and Molecular Research Center, Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Zohreh Sharifi
- Department of Virology, Iranian Blood Transfusion Organization, Tehran, Iran
| | - Sepideh Hamzehlou
- Cellular and Molecular Research Center, Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Mohammad M Farajollahi
- Cellular and Molecular Research Center, Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
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10
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Cannalire R, Barreca ML, Manfroni G, Cecchetti V. A Journey around the Medicinal Chemistry of Hepatitis C Virus Inhibitors Targeting NS4B: From Target to Preclinical Drug Candidates. J Med Chem 2015; 59:16-41. [PMID: 26241789 DOI: 10.1021/acs.jmedchem.5b00825] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) infection is a global health burden with an estimated 130-170 million chronically infected individuals and is the cause of serious liver diseases such as cirrhosis and hepatocellular carcinoma. HCV NS4B protein represents a validated target for the identification of new drugs to be added to the combination regimen recently approved. During the last years, NS4B has thus been the object of impressive medicinal chemistry efforts, which led to the identification of promising preclinical candidates. In this context, the present review aims to discuss research published on NS4B functional inhibitors focusing the attention on hit identification, hit-to-lead optimization, ADME profile evaluation, and the structure-activity relationship data raised for each compound family taken into account. The information delivered in this review will be a useful and valuable tool for those medicinal chemists dealing with research programs focused on NS4B and aimed at the identification of innovative anti-HCV compounds.
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Affiliation(s)
- Rolando Cannalire
- Department of Pharmaceutical Sciences, Università degli Studi di Perugia , Via A. Fabretti, 48-06123 Perugia, Italy
| | - Maria Letizia Barreca
- Department of Pharmaceutical Sciences, Università degli Studi di Perugia , Via A. Fabretti, 48-06123 Perugia, Italy
| | - Giuseppe Manfroni
- Department of Pharmaceutical Sciences, Università degli Studi di Perugia , Via A. Fabretti, 48-06123 Perugia, Italy
| | - Violetta Cecchetti
- Department of Pharmaceutical Sciences, Università degli Studi di Perugia , Via A. Fabretti, 48-06123 Perugia, Italy
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11
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Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein. Antimicrob Agents Chemother 2015; 59:6539-50. [PMID: 26259798 DOI: 10.1128/aac.00813-15] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2015] [Accepted: 07/22/2015] [Indexed: 12/12/2022] Open
Abstract
The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.
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12
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Zmurko J, Neyts J, Dallmeier K. Flaviviral NS4b, chameleon and jack-in-the-box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention. Rev Med Virol 2015; 25:205-23. [PMID: 25828437 PMCID: PMC4864441 DOI: 10.1002/rmv.1835] [Citation(s) in RCA: 87] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2014] [Revised: 02/16/2015] [Accepted: 02/17/2015] [Indexed: 12/27/2022]
Abstract
Dengue virus and other flaviviruses such as the yellow fever, West Nile, and Japanese encephalitis viruses are emerging vector-borne human pathogens that affect annually more than 100 million individuals and that may cause debilitating and potentially fatal hemorrhagic and encephalitic diseases. Currently, there are no specific antiviral drugs for the treatment of flavivirus-associated disease. A better understanding of the flavivirus-host interactions during the different events of the flaviviral life cycle may be essential when developing novel antiviral strategies. The flaviviral non-structural protein 4b (NS4b) appears to play an important role in flaviviral replication by facilitating the formation of the viral replication complexes and in counteracting innate immune responses such as the following: (i) type I IFN signaling; (ii) RNA interference; (iii) formation of stress granules; and (iv) the unfolded protein response. Intriguingly, NS4b has recently been shown to constitute an excellent target for the selective inhibition of flavivirus replication. We here review the current knowledge on NS4b.
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Affiliation(s)
- Joanna Zmurko
- KU Leuven, Rega Institute for Medical Research, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy
| | - Johan Neyts
- KU Leuven, Rega Institute for Medical Research, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy
| | - Kai Dallmeier
- KU Leuven, Rega Institute for Medical Research, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy
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13
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Ashworth Briggs EL, Gomes RGB, Elhussein M, Collier W, Findlow IS, Khalid S, McCormick CJ, Williamson PTF. Interaction between the NS4B amphipathic helix, AH2, and charged lipid headgroups alters membrane morphology and AH2 oligomeric state--Implications for the Hepatitis C virus life cycle. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2015; 1848:1671-7. [PMID: 25944559 PMCID: PMC4768108 DOI: 10.1016/j.bbamem.2015.04.015] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/07/2014] [Revised: 02/27/2015] [Accepted: 04/25/2015] [Indexed: 01/27/2023]
Abstract
The non-structural protein 4B (NS4B) from Hepatitis C virus (HCV) plays a pivotal role in the remodelling of the host cell's membranes, required for the formation of the viral replication complex where genome synthesis occurs. NS4B is an integral membrane protein that possesses a number of domains vital for viral replication. Structural and biophysical studies have revealed that one of these, the second amphipathic N-terminal helix (AH2), plays a key role in these remodelling events. However, there is still limited understanding of the mechanism through which AH2 promotes these changes. Here we report on solid-state NMR and molecular dynamics studies that demonstrate that AH2 promotes the clustering of negatively charged lipids within the bilayer, a process that reduces the strain within the bilayer facilitating the remodelling of the lipid bilayer. Furthermore, the presence of negatively charged lipids within the bilayer appears to promote the disassociation of AH2 oligomers, highlighting a potential role for lipid recruitment in regulating NS protein interactions.
Changes in membrane morphology studied by 2H and 31P Solid-state NMR. Bilayer charge influences the oligomeric state of the amphipathic helix AH2 from NS4B. Interaction of AH2 with charged lipid membranes reduces strain within bilayer. AH2 from NS4B is involved in membrane remodelling and membranous web formation. Lipid bilayer/NS4B interactions may regulate Hepatitis C virus lifecycle.
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Affiliation(s)
- Esther L Ashworth Briggs
- Centre for Biological Sciences/Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK
| | - Rafael G B Gomes
- Centre for Biological Sciences/Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK; School of Medicine, University of Southampton, Southampton SO16 6YD, UK
| | - Malaz Elhussein
- School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK
| | - William Collier
- Centre for Biological Sciences/Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK
| | - I Stuart Findlow
- Centre for Biological Sciences/Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK
| | - Syma Khalid
- School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK
| | - Chris J McCormick
- School of Medicine, University of Southampton, Southampton SO16 6YD, UK.
| | - Philip T F Williamson
- Centre for Biological Sciences/Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK.
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Jiang XH, Xie YT, Jiang B, Tang MY, Ma T, Peng H. Inhibition of expression of hepatitis C virus 1b genotype core and NS4B genes in HepG2 cells using artificial microRNAs. Mol Med Rep 2015; 12:1905-13. [PMID: 25847260 DOI: 10.3892/mmr.2015.3571] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2014] [Accepted: 02/05/2015] [Indexed: 11/06/2022] Open
Abstract
The present study aimed to evaluate the silencing effect of artificial microRNAs (amiRNAs) against the hepatitis C virus (HCV) 1b (HCV1b) genotype core (C) and non-structural protein 4B (NS4B) genes. pDsRed-monomer-Core and pDsRed-monomer-NS4B plasmids, containing the target genes were constructed. A total of eight artificial micro RNA (amiRNA)-expressing plasmids, namely, pmiRE-C-mi1 to -mi4 and pmiRE-NS4B-mi1 to -mi4, were designed and constructed to interfere with various sites of the core and NS4B genes, and the amiRNA interfering plasmid and the corresponding target gene-expressing plasmid were co-transfected into HepG2 cells. At 48 h after transfection, HCV core and NS4B gene expression levels were detected using fluorescence microscopy, flow cytometry, reverse transcription quantitative polymerase chain reaction and western blot analysis. Fluorescence microscopy revealed that the target gene-transfected cells expressed red fluorescent protein, whereas the interfering plasmid-transfected cells exhibited expression of green fluorescent protein. The percentage of red fluorescent proteins and mean fluorescence intensity, as well as protein expression levels of the core and NS4B genes within the cells, which were co-transfected by the amiRNA interfering plasmid and the target gene, were significantly decreased. The results of the present study confirmed that amiRNAs may effectively and specifically inhibit the expression of HCV1b core and NS4B genes in HepG2 cells, potentially providing a novel therapeutic strategy for the treatment of HCV.
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Affiliation(s)
- Xiao-Hua Jiang
- Department of Infectious Diseases, Xiangya Hospital of Central South University, Changsha, Hunan 410087, P.R. China
| | - Yu-Tao Xie
- Department of Infectious Diseases, Xiangya Hospital of Central South University, Changsha, Hunan 410087, P.R. China
| | - Bo Jiang
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
| | - Meng-Ying Tang
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
| | - Tao Ma
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
| | - Hua Peng
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
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15
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Encoded library technology screening of hepatitis C virus NS4B yields a small-molecule compound series with in vitro replicon activity. Antimicrob Agents Chemother 2015; 59:3450-9. [PMID: 25824229 DOI: 10.1128/aac.00070-15] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2015] [Accepted: 03/25/2015] [Indexed: 12/11/2022] Open
Abstract
To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 μM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets.
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16
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Wang Y. Scotomas in molecular virology and epidemiology of hepatitis C virus. World J Gastroenterol 2013; 19:7910-7921. [PMID: 24307785 PMCID: PMC3848139 DOI: 10.3748/wjg.v19.i44.7910] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/25/2013] [Revised: 10/22/2013] [Accepted: 11/05/2013] [Indexed: 02/06/2023] Open
Abstract
In the 1970s, scientists learned of a new pathogen causing non-A, non-B hepatitis. Classical approaches were used to isolate and characterize this new pathogen, but it could be transmitted experimentally only to chimpanzees and progress was slow until the pathogen was identified as hepatitis C virus (HCV) in 1989. Since then, research and treatment of HCV have expanded with the development of modern biological medicine: HCV genome organization and polyprotein processing were delineated in 1993; the first three-dimensional structure of HCV nonstructural protein (NS3 serine protease) was revealed in 1996; an infectious clone of HCV complementary DNA was first constructed in 1997; interferon and ribavirin combination therapy was established in 1998 and the therapeutic strategy gradually optimized; the HCV replicon system was produced in 1999; functional HCV pseudotyped viral particles were described in 2003; and recombinant infectious HCV in tissue culture was produced successfully in 2005. Recently, tremendous advances in HCV receptor discovery, understanding the HCV lifecycle, decryption of the HCV genome and proteins, as well as new anti-HCV compounds have been reported. Because HCV is difficult to isolate and culture, researchers have had to avail themselves to the best of modern biomedical technology; some of the major achievements in HCV research have not only advanced the understanding of HCV but also promoted knowledge of virology and cellular physiology. In this review, we summarize the advancements and remaining scotomas in the molecular virology and epidemiology of HCV.
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17
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Tomohiro T, Inoguchi H, Masuda S, Hatanaka Y. Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers. Bioorg Med Chem Lett 2013; 23:5605-8. [PMID: 23999042 DOI: 10.1016/j.bmcl.2013.08.041] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2013] [Revised: 07/22/2013] [Accepted: 08/08/2013] [Indexed: 01/02/2023]
Abstract
A specific illumination approach has been developed for identification of adenosine triphosphate (ATP)-binding proteins. This strategy utilizes a tandem photoactivatable unit that consists of a diazirine group as a carbene precursor and an o-hydroxycinnamate moiety as a coumarin precursor. The photolysis of diazirine induces a specific cross-link on target proteins and is followed by photoactivation of coumarin generation with a concomitant release of the pre-installed affinity ligand. The ATP, installed with this cross-linker at the γ-position, successfully transferred a coumarin onto ATP-binding proteins using only UV-irradiation.
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Affiliation(s)
- Takenori Tomohiro
- Laboratory of Biorecognition Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
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18
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Moradpour D, Penin F. Hepatitis C virus proteins: from structure to function. Curr Top Microbiol Immunol 2013; 369:113-42. [PMID: 23463199 DOI: 10.1007/978-3-642-27340-7_5] [Citation(s) in RCA: 149] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Great progress has been made over the past years in elucidating the structure and function of the hepatitis C virus (HCV) proteins, most of which are now actively being pursued as antiviral targets. The structural proteins, which form the viral particle, include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 viroporin, the NS2 protease, the NS3-4A complex harboring protease and NTPase/RNA helicase activities, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. NS4B is a master organizer of replication complex formation while NS5A is a zinc-containing phosphoprotein involved in the regulation of HCV RNA replication versus particle production. Core to NS2 make up the assembly module while NS3 to NS5B represent the replication module (replicase). However, HCV proteins exert multiple functions during the viral life cycle, and these may be governed by different structural conformations and/or interactions with viral and/or cellular partners. Remarkably, each viral protein is anchored to intracellular membranes via specific determinants that are essential to protein function in the cell. This review summarizes current knowledge of the structure and function of the HCV proteins and highlights recent advances in the field.
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Affiliation(s)
- Darius Moradpour
- Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
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19
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Roberts CD, Peat AJ. HCV Replication Inhibitors That Interact with NS4B. SUCCESSFUL STRATEGIES FOR THE DISCOVERY OF ANTIVIRAL DRUGS 2013. [DOI: 10.1039/9781849737814-00111] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
We describe the discovery, development and in vivo activity of small molecules that inhibits HCV replication via direct interaction with the viral NS4B protein. The inhibitors were identified through a phenotypic, cell based, high throughput screen using the HCV subgenomic replicon. Compounds were then optimized to extremely high potency and pharmacokinetics. Mechanistic data generated suggests a hypothesis wherein the compounds described function by binding to NS4B, preventing the formation of the characteristic HCV induced sub‐cellular membranous web required for viral replication. Finally, in vivo proof of mechanism was established by employing a chimeric “humanized” mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo.
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Affiliation(s)
- Christopher D. Roberts
- GlaxoSmithKline Research & Development Infectious Diseases Therapeutic Area Unit, 5 Moore Drive, Research Triangle Park, NC 27709 USA
| | - Andrew J. Peat
- GlaxoSmithKline Research & Development Infectious Diseases Therapeutic Area Unit, 5 Moore Drive, Research Triangle Park, NC 27709 USA
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20
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The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 2013; 11:482-96. [PMID: 23748342 DOI: 10.1038/nrmicro3046] [Citation(s) in RCA: 276] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.
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21
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Identification of PTC725, an orally bioavailable small molecule that selectively targets the hepatitis C Virus NS4B protein. Antimicrob Agents Chemother 2013; 57:3250-61. [PMID: 23629699 DOI: 10.1128/aac.00527-13] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.
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22
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Gu M, Rice CM. Structures of hepatitis C virus nonstructural proteins required for replicase assembly and function. Curr Opin Virol 2013; 3:129-36. [PMID: 23601958 DOI: 10.1016/j.coviro.2013.03.013] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2012] [Revised: 03/08/2013] [Accepted: 03/20/2013] [Indexed: 02/07/2023]
Abstract
Approximately 3% of the world population is infected with hepatitis C virus (HCV), causing a serious public health burden. Like other positive-strand RNA viruses, HCV assembles replicase complexes in association with cellular membranes and produces progeny RNA genomes through negative-strand intermediates. The viral proteins required for RNA replication are nonstructural (NS) proteins NS3 to NS5B. Owing to many obstacles and limitations in structural characterization of proteins and complexes with multiple transmembrane segments, attempts to understand the assembly and action of the HCV replicase complex have been challenging. Nevertheless, great progress has been made in obtaining structural information for several replicase components, providing insights into some aspects of the viral genome replication machinery.
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Affiliation(s)
- Meigang Gu
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, United States.
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23
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Kanwal N, Zaidi NUSS, Gomez MK. Non-structural protein NS4B: HCV replication web inducer. ASIAN PACIFIC JOURNAL OF TROPICAL DISEASE 2012. [DOI: 10.1016/s2222-1808(12)60111-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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24
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Li S, Yu X, Guo Y, Kong L. Interaction networks of hepatitis C virus NS4B: implications for antiviral therapy. Cell Microbiol 2012; 14:994-1002. [PMID: 22329740 DOI: 10.1111/j.1462-5822.2012.01773.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatitis C virus (HCV) is an important human pathogen infecting more than 170 million people worldwide with approximately three million new cases each year. HCV depends heavily on interactions between viral proteins and host factors for its survival and propagation. Among HCV viral proteins, the HCV non-structural protein 4B (NS4B) has been shown to mediate virus-host interactions that are essential for HCV replication and pathogenesis and emerged as the target for anti-HCV therapy. Here, we reviewed recent knowledge about the NS4B interaction networks with host factors and its possible regulatory mechanisms, which will both advance our understanding of the role of NS4B in HCV life cycle and illuminate potential viral and host therapeutic targets.
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Affiliation(s)
- Shanshan Li
- Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, USA
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25
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Charged residues in hepatitis C virus NS4B are critical for multiple NS4B functions in RNA replication. J Virol 2011; 85:8158-71. [PMID: 21680530 DOI: 10.1128/jvi.00858-11] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The nonstructural 4B (NS4B) protein of hepatitis C virus (HCV) plays a central role in the formation of the HCV replication complex. To gain insight into the role of charged residues for NS4B function in HCV RNA replication, alanine substitutions were engineered in place of 28 charged residues residing in the N- and C-terminal cytoplasmic domains of the NS4B protein of the HCV genotype 1b strain Con1. Eleven single charged-to-alanine mutants were not viable, while the remaining mutants were replication competent, albeit to differing degrees. By selecting revertants, second-site mutations were identified for one of the lethal NS4B mutations. Second-site mutations mapped to NS4B and partially suppressed the lethal replication phenotype. Further analyses showed that three NS4B mutations disrupted the formation of putative replication complexes, one mutation altered the stability of the NS4B protein, and cleavage at the NS4B/5A junction was significantly delayed by another mutation. Individual charged-to-alanine mutations did not affect interactions between the NS4B and NS3-4A proteins. A triple charged-to-alanine mutation produced a temperature-sensitive replication phenotype with no detectable RNA replication at 39°C, demonstrating that conditional mutations can be obtained by altering the charge characteristics of NS4B. Finally, NS4B mutations dispensable for efficient Con1 RNA replication were tested in the context of the chimeric genotype 2a virus, but significant defects in infectious-virus production were not detected. Taken together, these findings highlight the importance of charged residues for multiple NS4B functions in HCV RNA replication, including the formation of a functional replication complex.
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26
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Conserved GXXXG- and S/T-like motifs in the transmembrane domains of NS4B protein are required for hepatitis C virus replication. J Virol 2011; 85:6464-79. [PMID: 21507970 DOI: 10.1128/jvi.02298-10] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.
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27
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Masuda S, Tomohiro T, Hatanaka Y. Rapidly photoactivatable ATP probes for specific labeling of tropomyosin within the actomyosin protein complex. Bioorg Med Chem Lett 2011; 21:2252-4. [PMID: 21421313 DOI: 10.1016/j.bmcl.2011.02.113] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2010] [Revised: 02/24/2011] [Accepted: 02/26/2011] [Indexed: 10/18/2022]
Abstract
Tropomyosin-specific photoaffinity adenosine triphosphate (ATP) probes have been first developed, in which a diazirine moiety is incorporated into the γ-phosphate group as a rapidly carbene-generating photophore. These probes clearly labeled tropomyosin in the presence of other actomyosin components, that is, myosin, actin, and troponins. The specific labeling of tropomyosin was easily identified by selective trapping of the photo-incorporated ATP probe on Fe(3+)-immobilized metal ion affinity chromatography (IMAC) beads. The characteristic nature of tropomyosin-specific photocross-linking was further confirmed with a biotin-carrying derivative of the ATP probe. These data suggest that the tropomyosin on the actin filament assembly is located in close proximity to the ATP binding cavity of myosin.
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Affiliation(s)
- Souta Masuda
- Laboratory of Biorecognition Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
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28
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Rai R, Deval J. New opportunities in anti-hepatitis C virus drug discovery: targeting NS4B. Antiviral Res 2011; 90:93-101. [PMID: 21295075 DOI: 10.1016/j.antiviral.2011.01.009] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2010] [Revised: 01/24/2011] [Accepted: 01/26/2011] [Indexed: 10/18/2022]
Abstract
Current therapy for chronic hepatitis C virus (HCV) infection constitutes a combination of pegylated interferon alfa-2a or alpha-2b and ribavirin. Although successful for many patient populations, this regimen has numerous limitations, including non-response, relapse, poor tolerability and long duration of treatment. To address these shortcomings, new small molecule agents are advancing in clinical development. Most of the current clinical candidates act by directly inhibiting key enzymes in the viral life-cycle: the NS5B polymerase, or the NS3/4A protease. Less well-studied, the non-structural 4B (NS4B) protein has recently emerged as an alternative target for Direct-acting Antiviral Agents (DAAs). NS4B is a 27-kDa membrane protein that is primarily involved in the formation of membrane vesicles--also named membranous web--used as scaffold for the assembly of the HCV replication complex. In addition, NS4B contains NTPase and RNA binding activities, as well as anti-apoptotic properties. This review summarizes the current understanding of the structure and functions of NS4B, an essential component of the replication machinery of HCV. In this literature and patent review, we report the recent developments in anti-NS4B drug discovery. These advances open the possibility for future combination therapies with other DAAs.
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Affiliation(s)
- Roopa Rai
- Alios BioPharma, 260 E. Grand Ave., South San Francisco, CA 94080, USA.
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29
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Identification of an NTPase motif in classical swine fever virus NS4B protein. Virology 2011; 411:41-9. [PMID: 21236462 DOI: 10.1016/j.virol.2010.12.028] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2010] [Revised: 11/15/2010] [Accepted: 12/16/2010] [Indexed: 11/20/2022]
Abstract
Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.
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30
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Dvory-Sobol H, Pang PS, Glenn JS. The Future of HCV Therapy: NS4B as an Antiviral Target. Viruses 2010; 2:2481-2492. [PMID: 21157574 PMCID: PMC3000685 DOI: 10.3390/v2112481] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2010] [Revised: 09/28/2010] [Accepted: 10/13/2010] [Indexed: 12/18/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infection is a major worldwide cause of liver disease, including cirrhosis and hepatocellular carcinoma. It is estimated that more than 170 million individuals are infected with HCV, with three to four million new cases each year. The current standard of care, combination treatment with interferon and ribavirin, eradicates the virus in only about 50% of chronically infected patients. Notably, neither of these drugs directly target HCV. Many new antiviral therapies that specifically target hepatitis C (e.g. NS3 protease or NS5B polymerase inhibitors) are therefore in development, with a significant number having advanced into clinical trials. The nonstructural 4B (NS4B) protein, is among the least characterized of the HCV structural and nonstructural proteins and has been subjected to few pharmacological studies. NS4B is an integral membrane protein with at least four predicted transmembrane (TM) domains. A variety of functions have been postulated for NS4B, such as the ability to induce the membranous web replication platform, RNA binding and NTPase activity. This review summarizes potential targets within the nonstructural protein NS4B, with a focus on novel classes of NS4B inhibitors.
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Affiliation(s)
- Hadas Dvory-Sobol
- Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, CCSR 3115A, 269 Campus Drive, Palo Alto, CA, USA; E-Mails: (H.D.-S.); (P.S.P.)
| | - Philip S. Pang
- Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, CCSR 3115A, 269 Campus Drive, Palo Alto, CA, USA; E-Mails: (H.D.-S.); (P.S.P.)
- Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA, USA
| | - Jeffrey S. Glenn
- Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, CCSR 3115A, 269 Campus Drive, Palo Alto, CA, USA; E-Mails: (H.D.-S.); (P.S.P.)
- Veterans Administration Medical Center, 3801 Miranda Avenue, Palo Alto, CA, USA
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31
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Lemon SM, McKeating JA, Pietschmann T, Frick DN, Glenn JS, Tellinghuisen TL, Symons J, Furman PA. Development of novel therapies for hepatitis C. Antiviral Res 2010; 86:79-92. [PMID: 20417376 DOI: 10.1016/j.antiviral.2010.02.003] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2010] [Revised: 01/30/2010] [Accepted: 02/01/2010] [Indexed: 10/19/2022]
Abstract
The current standard of care for the treatment of hepatitis C virus (HCV) infection is a combination of pegylated IFN and ribavirin (Peg-IFN/RBV). Because of the adverse effects associated with both IFN and ribavirin and because Peg-IFN/RBV provides only about a 45-50% sustained virological response (SVR, undetectable HCV RNA for greater than 24 weeks after cessation of therapy) in genotype 1-infected individuals, there is a need for more potent anti-HCV compounds with fewer adverse effects. The twenty-first International Conference on Antiviral Research held in May 2009 in Miami Beach, Florida, featured a special session focused on novel targets for HCV therapy. The session included presentations by world-renowned experts in HCV virology and covered a diverse array of potential targets for the development of new classes of HCV therapies. This review contains concise summaries of discussed topics that included the innate immune response, virus entry, the NS2 protease, the NS3 helicase, NS4B, and NS5A. Each presenter discussed the current knowledge of these targets and provided examples of recent scientific breakthroughs that are enhancing our understanding of these targets. As our understanding of the role of these novel anti-HCV targets increases so will our ability to discover new, more safe and effective anti-HCV therapies.
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Affiliation(s)
- Stanley M Lemon
- Center for Hepatitis Research, Institute for Human Infections and Immunity, University of Texas Medical Branch at Galveston, Galveston, TX, USA
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Gouttenoire J, Penin F, Moradpour D. Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory. Rev Med Virol 2010; 20:117-29. [DOI: 10.1002/rmv.640] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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Abstract
NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.
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Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein. Virology 2009; 393:68-83. [PMID: 19703698 DOI: 10.1016/j.virol.2009.07.033] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2009] [Revised: 06/08/2009] [Accepted: 07/27/2009] [Indexed: 12/21/2022]
Abstract
During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.
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An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association. J Virol 2009; 83:11378-84. [PMID: 19692468 DOI: 10.1128/jvi.01122-09] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has been only poorly characterized to date. It is believed to comprise a cytosolic N-terminal part, a central part harboring four transmembrane passages, and a cytosolic C-terminal part. Here, we describe an amphipathic alpha-helix at the C terminus of NS4B (amino acid residues 229 to 253) that mediates membrane association and is involved in the formation of a functional HCV replication complex.
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Shiryaev SA, Chernov AV, Aleshin AE, Shiryaeva TN, Strongin AY. NS4A regulates the ATPase activity of the NS3 helicase: a novel cofactor role of the non-structural protein NS4A from West Nile virus. J Gen Virol 2009; 90:2081-5. [PMID: 19474250 DOI: 10.1099/vir.0.012864-0] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Using constructs that encode the individual West Nile virus (WNV) NS3helicase (NS3hel) and NS3hel linked to the hydrophilic, N-terminal 1-50 sequence of NS4A, we demonstrated that the presence of NS4A allows NS3hel to conserve energy in the course of oligonucleotide substrate unwinding. Using NS4A mutants, we also determined that the C-terminal acidic EELPD/E motif of NS4A, which appears to be functionally similar to the acidic EFDEMEE motif of hepatitis C virus (HCV) NS4A, is essential for regulating the ATPase activity of NS3hel. We concluded that, similar to HCV NS4A, NS4A of WNV acts as a cofactor for NS3hel and allows helicase to sustain the unwinding rate of the viral RNA under conditions of ATP deficiency.
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Dzeja P, Terzic A. Adenylate kinase and AMP signaling networks: metabolic monitoring, signal communication and body energy sensing. Int J Mol Sci 2009; 10:1729-1772. [PMID: 19468337 PMCID: PMC2680645 DOI: 10.3390/ijms10041729] [Citation(s) in RCA: 328] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2009] [Revised: 03/26/2009] [Accepted: 04/02/2009] [Indexed: 12/20/2022] Open
Abstract
Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network.
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Affiliation(s)
- Petras Dzeja
- Division of Cardiovascular Disease, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA; E-Mail:
(A.T.)
| | - Andre Terzic
- Division of Cardiovascular Disease, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA; E-Mail:
(A.T.)
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Meanwell NA, Kadow JF, Scola PM. Chapter 20 Progress towards the Discovery and Development of Specifically Targeted Inhibitors of Hepatitis C Virus. ANNUAL REPORTS IN MEDICINAL CHEMISTRY 2009. [DOI: 10.1016/s0065-7743(09)04420-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/08/2023]
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