Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor® 647 conjugate of staurosporine (a “tracer”) from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

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A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-014-7886-5 ◽

2014 ◽

Vol 406 (20) ◽

pp. 4889-4897 ◽

Cited By ~ 19

Author(s):

Christian Meyners ◽

Robert Wawrzinek ◽

Andreas Krämer ◽

Steffen Hinz ◽

Pablo Wessig ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescence Lifetime ◽

Binding Assay

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A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107304480 ◽

2007 ◽

Vol 12 (6) ◽

pp. 828-841 ◽

Cited By ~ 24

Author(s):

Connie S. Lebakken ◽

Hee Chol Kang ◽

Kurt W. Vogel

Keyword(s):

Fluorescence Lifetime ◽

Kinase Inhibitors ◽

Binding Assay ◽

Atp Binding ◽

Inhibitor Binding ◽

Simple Method ◽

Alexa Fluor ◽

Binding Data ◽

Wide Range ◽

Threonine Kinases

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. ( Journal of Biomolecular Screening 2007:828-841)

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