The ~600 kinases that make up the human kinome comprise a group of exciting druggable targets that can be modulated using small molecule or monoclonal antibody approaches.
There are currently 15 FDA-approved kinase inhibitor drugs, yet there remains much uncapped potential, as the biological function of ~90% of the kinome is unknown.
Methods have been designed for improving small molecule type I kinase inhibitors, which target the ATP binding pocket common to all kinases in their active form, as well as type II inhibitors, which bind to the same area as type I molecules, but also to an additional allosteric binding pocket in the inactive DFG motif conformation of the kinase. Novel assays have identified kinase targets, improved the function of certain kinase inhibitors, and led to the discovery of new inhibitor classes to accelerate drug discovery.
Sheraz Gul, Ph.D., vp and head of biology at European ScreeningPort, indicates that he has had a long-standing interest in kinases, particularly NF-κB inducing kinase (NIK). NIK is known to phosphorylate IKK-α at Ser-176, the same site where IKK-α autophosphorylates.
Dr. Gul has noticed a few discrepancies over the years with regard to reported NIK biochemical assays: various suppliers who sell recombinantly expressed and purified NIK have shown that it is catalytically active in biochemical assays when using a number of substrates (peptide and the generic kinase substrate myelin basic protein), however not when using IKK-α protein or a peptide derived from it as substrates.
Using such non-IKK-α derived substrates is somewhat of a concern, as myelin basic protein will undergo phosphorylation by most kinases. Additionally, it is possible that a highly active kinase partner bound to NIK may be responsible for the observed phosphorylation of non-IKK-α-derived substrates in biochemical assays.
In light of the intriguing aspects of how physiologically relevant NIK activity arises, Dr. Gul concludes that, rather than developing a biochemical assay, a cell-based assay to search for NIK inhibitors that prevent IKK-α phosphorylation would more effective. However, cell-based assays do have their disadvantages, primarily that most hits often exhibit cytotoxicity.
His research team began by performing a dual transfection assay of catalytically inactive full-length IKK-α and full-length NIK in insect cells, and have translated this to a HEK293 cell-based system and performed proof-of-concept screens against compound libraries with both assays. The screen that utilized the former assay yielded a sizeable number of hits, some of which were shown to prevent p52 translocation into the nucleus from the cytoplasm, as this would be the downstream effect of inhibiting NIK.
As an alternative to the dual transfection assay, Dr. Gul’s research team made the surprising finding that batches of cells that were separately transfected with NIK and catalytically inactive IKK-α, which were mixed together upon cell lysis, did not result in the catalytically inactive IKK-α undergoing phosphorylation, indicating that its NIK- mediated phosphorylation can only occur in a native cellular environment. This was all the more reason to prioritize cell-based assays over biochemical assays when searching for NIK inhibitors that prevent IKK-α phosphorylation.
“NIK is clearly an unusual kinase, as there have been no reports of a biochemical assay being developed that specifically makes use of its well-known substrate, IKK-α (or relevant kinase inactive mutant),” Dr. Gul says.
“It is not necessary that every kinase assay be cell-based, but for those that have characteristics like NIK, you will get many hits in a biochemical assay whose activities will not translate to a cell-based assay. Therefore, in order to mitigate the risk of this all-too-common scenario, it should be best practice to use a panel of both biochemical and cell-based assays as early as possible.”