Another challenge with lipid kinases is ensuring that in vitro inhibitory compounds are able to effectively hit the target inside cells. A current trend in pharmaceutical research is to test the activity of a compound within a cell early in the development process. This approach can yield preliminary penetration and toxicity information in addition to simple efficacy.
Echelon has developed cell-based assays for class I PI3 kinases (whose in vivo product is PIP3) called PIP mass assays, which measure the mass or amount of PIP3 in a complex biological sample. These assays report pro-growth/migration input through cellular PIP3 levels, which represents the combined activity of kinases (e.g., PI3K) and phosphatases (e.g., PTEN).
Since levels of PI3K activity (and PIP3) in quiescent cells are typically low, the assay is performed by activating the pathway with a growth factor, stopping the reaction, and harvesting cells. PIP3 is quantified by either lipid overlay (i.e., PIP-Strip) or competitive ELISA after extraction and purification steps. Figure 2 shows PIP3 levels in mouse fibroblasts and a matched pair of human breast cancer cell lines. The assay detected elevated PIP3 levels in activated cells; the increase was blocked with the PI3K inhibitor Wortmannin.
The biological importance of lipids as signal-transduction messengers is rapidly emerging. Recent research has shown altered lipid pathways to be important in disease states. Historically, these lipids presented unique challenges due to difficulties with synthesis and handling. New technologies and modern assays are making these difficult molecules easier to investigate.