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Journal of Biomolecular Screening
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13/6/476    most recent
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Evaluating PI3 Kinase Isoforms Using TranscreenerTM ADP Assays

Tony A. Klink

BellBrook Labs 5500 Nobel Drive, Suite 250 Madison, WI 53711, tony.klink{at}bellbrooklabs.com

Karen M. Kleman-Leyer

Andrew Kopp

Thane A. Westermeyer

Robert G. Lowery

Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The TranscreenerTM ADP Assay relies on homogeneous immunodetection of adenosine diphosphate (ADP), using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP—the invariant product of all kinase reactions—provides complete flexibility for varying lipid substrate parameters. The authors used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The nonphysiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP- and TR-FRET-based assays, and there was excellent concordance (r2 = 0.93) in the IC 50 values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. Adenosine triphosphate (ATP) hydrolysis in the absence of substrate was detected with the PI3K{alpha} isoform, and inhibitors affected PI3K{alpha} intrinsic ATP hydrolysis activity similarly to lipid phosphorylation. (Journal of Biomolecular Screening 2008:476-485)

Key Words: ADP detection • phosphoinositide 3-kinase • fluorescence polarization • time-resolved fluorescence resonance energy transfer • intrinsic ATPase activity

This version was published on July 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 6, 476-485 (2008)
DOI: 10.1177/1087057108319864
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[Abstract] [PDF]