title: Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.
abstract: The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.
authors.affiliation: Department of Medicinal Chemistry, The Musculoskeletal, Microbial and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.
authors.forename: Dashyant
authors.initials: D
authors.lastname: Dhanak
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Duffy
authors.forename: Victor K
authors.initials: VK
authors.lastname: Johnston
authors.forename: Juili
authors.initials: J
authors.lastname: Lin-Goerke
authors.forename: Michael
authors.initials: M
authors.lastname: Darcy
authors.forename: Antony N
authors.initials: AN
authors.lastname: Shaw
authors.forename: Baohua
authors.initials: B
authors.lastname: Gu
authors.forename: Carol
authors.initials: C
authors.lastname: Silverman
authors.forename: Adam T
authors.initials: AT
authors.lastname: Gates
authors.forename: Michael R
authors.initials: MR
authors.lastname: Nonnemacher
authors.forename: David L
authors.initials: DL
authors.lastname: Earnshaw
authors.forename: David J
authors.initials: DJ
authors.lastname: Casper
authors.forename: Arun
authors.initials: A
authors.lastname: Kaura
authors.forename: Audrey
authors.initials: A
authors.lastname: Baker
authors.forename: Cathy
authors.initials: C
authors.lastname: Greenwood
authors.forename: Lester L
authors.initials: LL
authors.lastname: Gutshall
authors.forename: Derrick
authors.initials: D
authors.lastname: Maley
authors.forename: Alfred
authors.initials: A
authors.lastname: DelVecchio
authors.forename: Ricardo
authors.initials: R
authors.lastname: Macarron
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Hofmann
authors.forename: Zaid
authors.initials: Z
authors.lastname: Alnoah
authors.forename: Hung-Yuan
authors.initials: HY
authors.lastname: Cheng
authors.forename: George
authors.initials: G
authors.lastname: Chan
authors.forename: Sanjay
authors.initials: S
authors.lastname: Khandekar
authors.forename: Richard M
authors.initials: RM
authors.lastname: Keenan
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sarisky
mesh_terms: D002460:Cell Line; D002942:Circular Dichroism; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D015394:Molecular Structure; D011489:Protein Denaturation; D012313:RNA / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013829:Thiadiazines / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013829:Thiadiazines; D017361:Viral Nonstructural Proteins; D012313:RNA; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase