Technical study

Upstate, in collaboration with Cisbio, have made available two universal screening formats using the sensitive homogenous assay format of HTRF, for lipid and serine/threonine kinase (STKs). All four isoforms of PI3 kinase use the same set of reagents of which PI3?, PI3? and PI3??are available in Upstate’s KinaseProfiler service, and all four as reagent bundles. The principle of the assay is illustrated in Figure 1. A detection complex is formed by the addition of biotinylated-PIP3, GST-GRP1 (pleckstrin homology domain that binds PIP3), anti-GST antibody labelled with Eu3+ Cryptate and Streptavidin-XLent. The amount of signal is inversely proportional to the amount of PIP3 generated by the lipid kinase as it competes with biotinylated PIP3 for binding to GRP1.

This assay format was used to investigate the inhibition profile of literature lipid kinase inhibitors. Representative data are shown in Table 1 and are in good agreement with published values.

An advantage of HTRF technology is the ability to perform kinase reactions at any concentration of ATP. An example of this is shown in Figure 2.

The universal STK HTRF assay format exploits the use of a proprietary monoclonal antibody that selectively recognises a phosphorylated serine residue within a common C-terminal epitope. The principle of the assay is shown in Figure 3. Variation at the N-terminal allows different STKs to be accessed. FRET occurs between the phosphorylated peptide and streptavidin-XL665. The amount of signal is proportional to the activity of the STK. In collaboration with Cisbio, an initial selection of 70 kinases has been validated using one of three biotinylated peptides. It is likely by variation at the N-terminus that this list will grow to in excess of 100 STKs.

 

Therefore, Upstate and Cisbio have strived to minimise assay development time and cost by offering universal formats that operate on a common set of reagents where the only variable is the kinase.

Figure 1: PI3 kinase HTRF lipid kinase assay

Figure 2: Inhibition of PI3? lipid kinase by curcumin
Inhibition by curcumin was shown to be ATP competitive. The IC50 value shifted from 5.3 + 0.8 ?M @ 10 ?M to 10.7 + 1.2 ?M @ 100 ?M ATP (KM[app])

Figure 3: Principle of the HTRF universal STK assay

Table 1: Inhibition data of Lipid Kinases

Inhibitor	PI3 Kinase Isoform
LY294002	2.0 + 0.6 M	1.8 + 0.3 M	1.4 + 0.5 M	17 + 3 M
Wortmannin	24 + 6 nM	21 + 5 nM	15 + 5 nM	16 + 3 nM
D000*	> 100 M	57 + 11 M	1.6 + 0.8 M	40 + 10 M

 

Data shown are the mean IC50 value + SD (n=6) at KM (app) ATP
D000 – ? specific inhibitor (Cancer Res. (2003) 63, 1667-1675)