title: Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.
abstract: The current standard of care for hepatitis C virus (HCV)-infected patients consists of lengthy treatment with interferon and ribavirin. To increase the effectiveness of HCV therapy, future regimens will incorporate multiple direct-acting antiviral (DAA) drugs. Recently, the HCV-encoded NS5A protein has emerged as a promising DAA target. Compounds targeting NS5A exhibit remarkable potency in vitro and demonstrate early clinical promise, suggesting that NS5A inhibitors could feature in future DAA combination therapies. Since the mechanisms through which these molecules operate are unknown, we have used NS5A inhibitors as tools to investigate their modes of action. Analysis of replicon-containing cells revealed dramatic phenotypic alterations in NS5A localization following treatment with NS5A inhibitors; NS5A was redistributed from the endoplasmic reticulum to lipid droplets. The NS5A relocalization did not occur in cells treated with other classes of HCV inhibitors, and NS5A-targeting molecules did not cause similar alterations in the localization of other HCV-encoded proteins. Time course analysis of the redistribution of NS5A revealed that the transfer of protein to lipid droplets was concomitant with the onset of inhibition, as judged by the kinetic profiles for these compounds. Furthermore, analysis of the kinetic profile of inhibition for a panel of test molecules permitted the separation of compounds into different kinetic classes based on their modes of action. Results from this approach suggested that NS5A inhibitors perturbed the function of new replication complexes, rather than acting on preformed complexes. Taken together, our data reveal novel biological consequences of NS5A inhibition, which may help enable the development of future assay platforms for the identification of new and/or different NS5A inhibitors.
authors.affiliation: Pfizer Global Research & Development, Sandwich, Kent CT13 9NJ, United Kingdom. paul.targett-adams@pfizer.com
authors.forename: Paul
authors.initials: P
authors.lastname: Targett-Adams
authors.forename: Emily J S
authors.initials: EJ
authors.lastname: Graham
authors.forename: Jenny
authors.initials: J
authors.lastname: Middleton
authors.forename: Amy
authors.initials: A
authors.lastname: Palmer
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Shaw
authors.forename: Helen
authors.initials: H
authors.lastname: Lavender
authors.forename: Philip
authors.initials: P
authors.lastname: Brain
authors.forename: Thien Duc
authors.initials: TD
authors.lastname: Tran
authors.forename: Lyn H
authors.initials: LH
authors.lastname: Jones
authors.forename: Florian
authors.initials: F
authors.lastname: Wakenhut
authors.forename: Blanda
authors.initials: B
authors.lastname: Stammen
authors.forename: David
authors.initials: D
authors.lastname: Pryde
authors.forename: Chris
authors.initials: C
authors.lastname: Pickford
authors.forename: Mike
authors.initials: M
authors.lastname: Westby
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D002219:Carbamates; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics; D022781:Hepatocytes / Q000648:ultrastructure / Q000821:virology; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008055:Lipids; D018613:Microscopy, Confocal; D008958:Models, Molecular; D011759:Pyrrolidines; D012093:Replicon; D054852:Small Molecule Libraries; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D007093:Imidazoles; D008055:Lipids; D011759:Pyrrolidines; D054852:Small Molecule Libraries; D014633:Valine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir