title: Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm.
abstract: Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-kappaB (nuclear factor kappaB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IkappaB (inhibitor of NF-kappaB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z' value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-kappaB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.
authors.affiliation: Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. namir.j.hassan@gsk.com
authors.forename: Namir J
authors.initials: NJ
authors.lastname: Hassan
authors.forename: Sheraz
authors.initials: S
authors.lastname: Gul
authors.forename: Fiona
authors.initials: F
authors.lastname: Flett
authors.forename: Edward
authors.initials: E
authors.lastname: Hollingsworth
authors.forename: Angela A
authors.initials: AA
authors.lastname: Dunne
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Emmons
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Hutchinson
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Hibbs
authors.forename: Susan
authors.initials: S
authors.lastname: Dyos
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Kitson
authors.forename: Emma
authors.initials: E
authors.lastname: Hiley
authors.forename: Martin
authors.initials: M
authors.lastname: Rüdiger
authors.forename: David G
authors.initials: DG
authors.lastname: Tew
authors.forename: David J
authors.initials: DJ
authors.lastname: Powell
authors.forename: Mary A
authors.initials: MA
authors.lastname: Morse
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D052003:NF-kappa B p52 Subunit / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018411:Spodoptera; D000097782:NF-kappaB-Inducing Kinase
chemical_list: D051550:I-kappa B Kinase; D016328:NF-kappa B; D052003:NF-kappa B p52 Subunit; D017346:Protein Serine-Threonine Kinases; D000097782:NF-kappaB-Inducing Kinase