Evaluating PI3 kinase isoforms using Transcreener ADP assays.
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 Transcreener 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 (r(2)=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 PI3Kalpha isoform, and inhibitors affected PI3Kalpha intrinsic ATP hydrolysis activity similarly to lipid phosphorylation.
BellBrook Labs 5500 Nobel Drive, Suite 250 Madison, WI 53711, USA. tony.klink@bellbrooklabs.com
Tony A
TA
Klink
Karen M
KM
Kleman-Leyer
Andrew
A
Kopp
Thane A
TA
Westermeyer
Robert G
RG
Lowery
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001681:Biological Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein / Q000378:metabolism; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism
D000244:Adenosine Diphosphate; D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D019793:Fluorescein; D005456:Fluorescent Dyes; D019869:Phosphatidylinositol 3-Kinases; D020033:Protein Isoforms