Evaluating PI3 kinase isoforms using Transcreener ADP assays

Abstract

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.

FIG. 1

The Transcreener™ ADP Assays detect ADP, the invariant product of many ATP utilizing enzymes, including lipid kinases. ADP competes with the tracer for binding to the monoclonal ADP antibody. In fluorescence polarization (FP) detection, high polarization is observed when the ADP AlexaFluor® 633 tracer is bound to the ADP antibody. ADP displaces the far-red tracer resulting in low polarization. In time-resolved fluorescence resonance energy transfer (TR-FRET) detection, energy transfer from the terbium to the fluorescein (FAM) is observed when the ADP FAM tracer is bound to the ADP-antibody-Tb resulting in a high FAM₅₂₀/Tb₄₉₅ ratio. ADP disrupts TR-FRET by displacing the tracer from the antibody, resulting in a low ratio.

FIG. 2

Effect of lipid substrate side chain length and preparation technique on PI3α kinase ADP production using time-resolved fluorescence resonance energy transfer (TR-FRET). Raw data for the two-fold serial dilution of PI3Kα (n = 2) is shown with error bars with PI(4,5)P₂ C8 (30 μM), PI(4,5)P₂ C16 (30 μM), and without substrate. The C16 lipid substrate was prepared using either sonication alone, sonication followed by freeze/thawing, or sonication in the presence of phosphatidylserine. Enzyme reactions were stopped after incubation for 1.5 hr at 30°C. EC₅₀ values for the C8 lipid (sonication only method), C16 lipid (sonication and freeze/thaw method), C16 lipid (sonication only method), C16 lipid (sonication in presence of phosphatidylserine), and without substrate were 15.6, 47.8, 299, 1069, 4765 ng/mL, respectively.

FIG 3

Influence on PI(4,5)P₂ substrate concentration on PI3α kinase ADP production determined using (A) time-resolved fluorescence resonance energy transfer (TR-FRET) and (B) fluorescence polarization (FP). Raw data are shown with error bars for C8 and C16 lipid substrate (n = 2) titrations. The PI(4,5)P₂ C8 and C16 lipid substrates were prepared using the freeze/thaw method and serially titrated (n = 2) before adding a constant enzyme concentration (2.4 nM).

FIG 4

Comparison of the effect of the presence and absence of lipid substrate on the ADP/ATP standard curves. Time-resolved fluorescence resonance energy transfer (TR-FRET; Fig. 4A) and fluorescence polarization (FP; Fig. 4B) data (n = 4) are shown with error bars for C8, C16 lipid substrate and without substrate. The data show that assay signal is proportional to concentration ADP. The PI(4,5)P₂ C8 and C16 lipid substrates were prepared using the freeze/thaw method.

FIG. 5

Comparison of inhibition curves between fluorescence polarization (FP) ADP detection and time-resolved fluorescence resonance energy transfer (TR-FRET) ADP detection for PI3Kα kinase and intrinsic ATPase activities. PI3Kα sensitivity to six known lipid kinase inhibitors (Wortmannin, PI 103, PI3Kγ inhibitor, LY 294002, PI3KγII inhibitor, and Quercetin) is shown with error bars for the 20-point dose dependency curves (n = 2). (A) Using TR-FRET, ADP detection in the PI3Kα (0.36 nM)/PI(4,5)P₂ C16 lipid reaction progressed to 4.7% ATP conversion (Z’ = 0.82). (B) Using FP, ADP detection in the PI3Kα (1.0 nM)/PI(4,5)P₂ C16 lipid reaction progressed to 10.3% ATP conversion (Z’ = 0.89). (C) Using FP, ADP detection in the PI3Kα (28.2 nM) intrinsic ATPase reaction progressed to 4.1% ATP conversion (Z’ = 0.82). Polarization values were converted to percent inhibition using an ATP/ADP standard curve and Graphpad PRISM software.

FIG. 6

Correlation of IC₅₀ values measured by time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) ADP detection. PI3K isoform inhibitor potency IC₅₀ data for six inhibitors (Wortmannin, PI 103, PI3Kγ inhibitor, LY 294002, PI3KγII inhibitor, and Quercetin) determined using TR-FRET and FP detection methods was plotted in a scattergraph with the line equivalence shown using Graphpad PRISM software. A significant correlation (r² = 0.93) was obtained for the comparison.