title: Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives.
abstract: The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, two benzo-1,2,4-thiadiazine compounds were shown to be potent, highly specific inhibitors of the genotype 1b HCV RdRp containing a carboxyl-terminal 21 residue truncation (delta21 HCV RdRp) (Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J., Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates, A. T., Earnshaw, D. L., Casper, D. J., Kaura, A., Baker, A., Greenwood, C., Gutshall, L. L., Maley, D., DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah, Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem. 277, 38322-38327). Compound 4 (C(21)H(21)N(3)O(4)S) reduces viral replication by virtue of its direct interaction with the viral polymerase rather than by nonspecific titration of nucleic acid template. In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of delta21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNA-bound form of delta21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.
authors.affiliation: Department of Virology, The Metabolic and Viral Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Bloomington, Indiana 47405, USA.
authors.forename: Baohua
authors.initials: B
authors.lastname: Gu
authors.forename: Victor K
authors.initials: VK
authors.lastname: Johnston
authors.forename: Lester L
authors.initials: LL
authors.lastname: Gutshall
authors.forename: Tammy T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Richard R
authors.initials: RR
authors.lastname: Gontarek
authors.forename: Michael G
authors.initials: MG
authors.lastname: Darcy
authors.forename: Rosanna
authors.initials: R
authors.lastname: Tedesco
authors.forename: Dashyant
authors.initials: D
authors.lastname: Dhanak
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Duffy
authors.forename: C Cheng
authors.initials: CC
authors.lastname: Kao
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sarisky
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis / Q000187:drug effects*; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors; D013829:Thiadiazines / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D012367:RNA, Viral; D013829:Thiadiazines; D012324:RNA-Dependent RNA Polymerase