Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity

- Characterization of inhibitors possessing sub-picomolar to sub-millimolar affinity.
- Single probe for determination of affinities of ATP- and protein/peptide substrate-competitive inhibitors.
- Graphical presentation of the Cheng-Prusoff equation aiding the selection of assay conditions.
- Method for calculating the limit of Kd determination.

High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies. High affinity (20 pM) and low background signal of the free probe supports the determination of dissociation constants of tight-binding as well as low affinity inhibitors. The calculated lowest Kd value that can be accurately determined with the method is 60 fM.We also introduce graphical presentation of the linearized Cheng-Prusoff equation and demonstrate multiple possibilities for its application (deciding upon the assay formats, calculation of the limits of Kd determination, etc.). The open toolbox (http://www.ut.ee/medchem/toolbox-fluorescence-probes) is available for creating the map of resolvable affinities if applying the competitive probes at defined assay conditions.

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