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Assaying Protein Kinase Activity with Radiolabeled ATP

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Evaluating PI3 kinase isoforms using Transcreener ADP assays.

Tony A Klink¹, Karen M Kleman-Leyer, Andrew Kopp

¹BellBrook Labs 5500 Nobel Drive, Suite 250 Madison, WI 53711, USA. tony.klink@bellbrooklabs.com

Journal of Biomolecular Screening

|June 21, 2008

Summary

A new assay enables flexible screening of lipid kinase inhibitors by detecting adenosine diphosphate (ADP). This method optimizes lipid substrates and accurately profiles inhibitors for phosphoinositide-3-kinase (PI3K) drug discovery.

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Area of Science:

Biochemistry
Drug Discovery
Enzyme Assays

Background:

Developing drugs targeting lipid kinases is challenging due to the lack of suitable screening assays.
Existing methods struggle to present diverse lipid substrates in optimal environments.

Purpose of the Study:

To introduce a novel assay for screening lipid kinase inhibitors.
To demonstrate the assay's flexibility in accommodating various lipid substrates.
To optimize lipid substrate presentation and assess its impact on phosphoinositide-3-kinase (PI3K) activity and inhibition.

Main Methods:

Utilized the Transcreener ADP Assay for homogeneous immunodetection of adenosine diphosphate (ADP).
Employed fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) for signal output.
Optimized dispersal of C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and profiled known inhibitors.

Main Results:

The assay successfully detected ADP, the universal product of kinase reactions, allowing flexible substrate variation.
The nonphysiological C8 lipid substrate supported the highest PI3K activity.
FP and TR-FRET assays showed excellent concordance (r(2)=0.93) in inhibitor IC(50) values.
Inhibitor rank order was consistent across C8 and C16 substrates with minor deviations.

Conclusions:

The Transcreener ADP Assay provides a robust and flexible platform for lipid kinase drug screening.
Lipid substrate choice significantly impacts PI3K activity, with C8 supporting higher activity.
The assay accurately reflects inhibitor efficacy and can detect intrinsic ATP hydrolysis, aiding in comprehensive drug profiling.