Targeting Kinases Involved in the Innate Immune Response Using the Transcreener ADP Assay

Protein kinases play an important role in innate immunity signaling, both by transducing signals from pattern recognition receptors and by mediating the downstream effects of type I interferons and cytokines.  Therapeutic modulation of these kinases is being investigated clinically to dampen the immune system in autoimmune diseases and to stimulate it for anti-viral or cancer immunotherapy.  The use of a common biochemical platform for discovering and characterizing small molecule kinase inhibitors would facilitate these efforts.  The Transcreener ADP² Kinase Assay meets this need as it relies on direct detection of ADP, so it can be applied across diverse kinases, and it has been extensively validated for kinase discovery programs since 2007.  Here, we describe how the Transcreener ADP² Kinase Assay allowed rapid assay development to enable screening and dose-response measurements for five kinases involved in innate immunity: AMP-activated kinase (AMPK), Janus kinase 1 and 3 (JAK1,3), TANK Binding Kinase 1 (TBK1), and IκB Kinase (IKK).

The Transcreener ADP² Kinase assay relies on selective immunodetection of ADP: enzymatically generated ADP displaces a fluorescent tracer from the antibody resulting in a change in fluorescent properties.  The assay has been formatted for fluorescence polarization (FP), time-resolved FRET (TR-FRET), and fluorescence intensity (FI) readouts.  A key step in assay development is optimizing the dynamic range of the assay based on the desired concentration of ATP, assuming 2-20% conversion of ATP to ADP.  After choosing an ATP concentration close to the reported Km for each kinase, the concentration of ADP² antibody was titrated to identify the EC₈₀ concentration using reaction conditions specific for each kinase.  Next, each kinase was titrated to identify the optimal concentration for producing a robust signal under initial velocity conditions.  In some cases, we also performed some optimization of acceptor substrate(s), but these few steps are all that is required to develop an assay for most kinases.  We measured Z’ values greater than 0.7 for all assays and dose-response measurements with known inhibitors yielded IC₅₀ values consistent with literature reports.  Initial assay development was done using the FP assay, but similar results were observed using the TR-FRET and FI assay.  In summary, the Transcreener ADP² assay provides a universal platform for biochemical kinase assays that should facilitate drug discovery programs targeting protein kinases involved in innate immunity.