Development Of An Insect-Cell-Based Assay For Detection Of Kinase Inhibition Using Nf-Kappa B-Inducing Kinase As A Paradigm

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 it cell-based assay format using NIK [NF-kappa B (nuclear factor kappa B)-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 I kappa B (inhibitor of NF-kappa B) kinase-1. The assay Was robust, yielding it 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-kappa B 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 case of use, amenability to scale-up, protein expression levels and the flexibility to express it number of proteins by infecting with numerous baculoviruses.