A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors

Abstract

The authors present a fluorescence lifetime-based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)-binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states.

Measuring and interpreting the selectivity of protein kinase inhibitors

“…8a). The probes are often attached to fluorescent markers in order to detect them [87]. In one case, staurosporine or close analogues have been used as probes without additional markers, since excitation of the compounds at 296 nm results in emission at 378 and 396 nm [74].…”

The Neural Cell Adhesion Molecule Promotes FGFR-Dependent Phosphorylation and Membrane Targeting of the Exocyst Complex to Induce Exocytosis in Growth Cones

“…We then investigated how phosphorylation would regulate exocyst complex function by analyzing the recruitment of exocyst complex subunits to NCAMϩ/ϩ growth cone membranes from the cytosol preincubated with staurosporine, a general inhibitor of kinases (Lebakken et al, 2007). Exo70 and sec8 from the staurosporine-treated cytosol attached to the growth cone membranes with an approximately twofold lower efficiency than from untreated cytosol (Fig.…”

Development and Applications of a Broad-Coverage, TR-FRET-Based Kinase Binding Assay Platform

“…5 For example, in that format, 70 nM PCK-theta (PRKCQ) kinase was required to bind to a sufficient fraction of its tracer to develop a suitable assay, and this negatively affected the ability of the assay to properly report affinities of tight binding inhibitors. For example, the tight binding inhibitor, staurosporine, was seen to compete with tracer with an IC 50 of 40 nM, which was approximately 10-fold higher than that seen when tested in 2 different activity assays that used substantially lower concentrations of kinase.…”

“…When the same assay was reconfigured in the TR-FRET format presented here, the observed IC 50 for staurosporine was reduced to 3 nM (data not shown). 37 The ability to use a sub-K d concentration of kinase, as well as a concentration of tracer that is close to the K d for the kinase⋅tracer interaction, is particularly advantageous when using tracers that have relatively low affinity for the kinase (>100 nM K d ) and is a distinguishing feature of the TR-FRET format that we have presented when compared to binding assays performed in FLT, 5 FP, 2 or EFC 7 formats. Although the convenience of using an epitope tag for the purification or visualization of a recombinant kinase is well established, such tags could potentially alter the activity of the kinase being studied.…”