Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm

• Published in: Biochemical journal Vol. 419; no. 1; p. 65
• Main Authors: Hassan, Namir J, Gul, Sheraz, Flett, Fiona, Hollingsworth, Edward, Dunne, Angela A, Emmons, Amanda J, Hutchinson, Jonathan P, Hibbs, Martin J, Dyos, Susan, Kitson, Jeremy D, Hiley, Emma, Rüdiger, Martin, Tew, David G, Powell, David J, Morse, Mary A
• Format: Journal Article
• Language: English
• Published: England 01-04-2009
• Subjects: Animals; Biological Assay - methods; Blotting, Western; Cell Line; Humans; I-kappa B Kinase - metabolism; Models, Biological; NF-kappa B - metabolism; NF-kappa B p52 Subunit - metabolism; NF-kappaB-Inducing Kinase; Phosphorylation; Protein Serine-Threonine Kinases - metabolism; Spodoptera
• ISSN: 1470-8728
• DOI: 10.1042/BJ20081646

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.