title: Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation.
abstract: The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K(d)s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC(50)=5±2μM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer St., Milwaukee, WI 53211, United States.
authors.forename: Jean
authors.initials: J
authors.lastname: Ndjomou
authors.forename: Rajesh
authors.initials: R
authors.lastname: Kolli
authors.forename: Sourav
authors.initials: S
authors.lastname: Mukherjee
authors.forename: William R
authors.initials: WR
authors.lastname: Shadrick
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Hanson
authors.forename: Noreena L
authors.initials: NL
authors.lastname: Sweeney
authors.forename: Diana
authors.initials: D
authors.lastname: Bartczak
authors.forename: Kelin
authors.initials: K
authors.lastname: Li
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Frankowski
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Schoenen
authors.forename: David N
authors.initials: DN
authors.lastname: Frick
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004265:DNA Helicases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D016174:Hepacivirus / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D004265:DNA Helicases; D004791:Enzyme Inhibitors; D012324:RNA-Dependent RNA Polymerase; D012697:Serine Endopeptidases; D013844:Thiazoles; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C035646:primuline