Comparison of LanthaScreen Eu Kinase Binding Assay and Surface Plasmon Resonance Method in Elucidating the Binding Kinetics of Focal Adhesion Kinase Inhibitors

Abstract

An understanding of the dynamics of drug-target interactions is important in the drug discovery process. Information related to the binding kinetics of a drug toward its target or off-target aids in determining the efficacy or toxicity of a drug. Biophysical techniques such as surface plasmon resonance (SPR) have been available for over 20 years, but have been predominantly utilized to characterize protein-protein interactions. With improvements in instrument sensitivity and data analysis software, interactions between proteins (such as kinases) and small molecules have been successfully evaluated. More recently, the LanthaScreen Eu kinase binding assay for characterizing kinase inhibitors has been described. This assay monitors displacement of an Alexa Fluor 647-labeled tracer from the ATP-binding site of an epitope-tagged kinase by a test compound. Such behavior results in a decrease in time-resolved fluorescence energy transfer signal. In this report, a side-by-side comparison of the LanthaScreen Eu kinase binding assay and the SPR method was performed using inhibitors of focal adhesion kinase. The two methods yielded comparable results and identified compounds with time-dependent inhibition and relatively slow dissociation.

Binding kinetics in drug discovery ndash A current perspective

“…The kinetic resolution of TR-FRET readout is higher as compared to radioligand binding but lower as compared to SPR and the data obtained are comparable to other technologies (77,121,122). Many publications using TR-FRET for quantification of binding kinetic rates are based on the displacement assay format (59,89,90,95,122,124) but -as commented above -the method has been recently used for true competitive binding kinetics experiments (77). Among the advantages of this application (compared to displacement assays) are the absence of pre-incubation step and delay times between sample mixing and reading, which allows association and dissociation rates to be measured in the same experiment and a more reliable analysis of compound with fast association and © 1996-2017 dissociation kinetics (see end of section 4.1.2 and (77)).…”

“…TR-FRET unites the simplicity and speed of a homogenous assay format with excellent signal-to-noise ratios, low protein demands and high sensitivity and is a powerful tool for high-throughput applications. The kinetic resolution of TR-FRET readout is higher as compared to radioligand binding but lower as compared to SPR and the data obtained are comparable to other technologies (77,121,122). Many publications using TR-FRET for quantification of binding kinetic rates are based on the displacement assay format (59,89,90,95,122,124) but -as commented above -the method has been recently used for true competitive binding kinetics experiments (77).…”

“…Initially developed for batch microtiter platebased formats, recent literature shows that microfluidic setups can regulate and eliminate rebinding by removal of unbound compounds to study both, the effect of a drug on its target with und without rebinding to estimate residence times (141). II) The newest electro-optical sensor which can been applied to quantify small molecule binding kinetics, is based on grating coupled interferometry (GCI) (122,142,143). This technique promises to increase the kinetic resolution and sensitivity of biosensors while decreasing the costs of materials.…”

Proteome-wide drug screening using mass spectrometric imaging of bead-arrays

“…Conversely, a variety of effective label-free methods exist which can measure drug-protein interactions. Two of the most widely used proximity based assays are fluorescence resonance energy transfer (FRET) (where the unlabeled drug competes for a labeled compound in the protein binding site) and surface plasmon resonance (SPR) 18 . However, these assays are not ideally suited for proteome-wide screening.…”

“…FRET type assays generally require some a priori knowledge of binding or target activation mechanisms and are not easily generalized to the entire proteome ( e.g. fluorescent tracers are used at the ATP binding site in time-resolved fluorescence resonance energy transfer [TR-FRET] based LanthaScreen® kinase assays 18 ). Furthermore, such assays utilize microtiter plates, which although suitable for high throughput screening (HTS), remain impractical for routine proteome-wide screening.…”