title: Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.
abstract: The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.
authors.affiliation: Department of Chemistry & Biochemistry, University of Wisconsin-Milwaukee , Milwaukee, Wisconsin 53211, United States.
authors.forename: Sourav
authors.initials: S
authors.lastname: Mukherjee
authors.forename: Warren S
authors.initials: WS
authors.lastname: Weiner
authors.forename: Chad E
authors.initials: CE
authors.lastname: Schroeder
authors.forename: Denise S
authors.initials: DS
authors.lastname: Simpson
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Hanson
authors.forename: Noreena L
authors.initials: NL
authors.lastname: Sweeney
authors.forename: Rachel K
authors.initials: RK
authors.lastname: Marvin
authors.forename: Jean
authors.initials: J
authors.lastname: Ndjomou
authors.forename: Rajesh
authors.initials: R
authors.lastname: Kolli
authors.forename: Dragan
authors.initials: D
authors.lastname: Isailovic
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Schoenen
authors.forename: David N
authors.initials: DN
authors.lastname: Frick
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism / Q000821:virology; D049109:Cell Proliferation; D024202:Electrophoretic Mobility Shift Assay; D016174:Hepacivirus / Q000187:drug effects; D006526:Hepatitis C / Q000188:drug therapy / Q000378:metabolism / Q000821:virology*; D006801:Humans; D006868:Hydrolysis; D054833:Isoindoles; D007700:Kinetics; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000821:virology; D008958:Models, Molecular; D009696:Nucleic Acids / Q000378:metabolism*; D016566:Organoselenium Compounds / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000975:Antioxidants; D000998:Antiviral Agents; D001393:Azoles; D054833:Isoindoles; D009696:Nucleic Acids; D016566:Organoselenium Compounds; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C042986:ebselen