Many scientists are familiar with lipids as hydrophobic molecules that form biological cell membranes. However, relatively few scientists appreciate the fundamental role lipids play as signaling molecules in essential cellular processes. These include cell division, cell migration, cell energy/metabolism, and apoptosis.
Of particular note, abnormalities in phosphoinositides and phosphoinositide 3-kinase (PI3K) pathways are involved in cellular pathology leading to proliferative, metabolic, and inflammatory disorders.
Given the emerging biological importance of these pathways, there is intense interest and activity by academic and industrial scientists in understanding the roles of these lipids and their potential targets for the treatment of, for example, cancer, diabetes, and heart disease.
Lipid Assay Challenges and Solutions
Lipids and phosphoinositides are difficult for many biologists to work with when using traditional methods. Without the use of radioactive isotopes, which have additional challenges, traditional methods require organic solvents, laborious extractions, and insensitive detection and quantification.
Compared to polypeptides and nucleotide strands, lipid monomers are less homogeneous. Lipids don’t form covalently linked polymers and are not generated from templates. Perhaps most important, lipids are not soluble in aqueous solutions.
Echelon Biosciences (www.echelon-inc.com) is working to span the gap between synthetic lipid chemistry and signal-transduction biology. The company’s assays offer cell biologists and screening scientists access to the expanding world of biological lipid research.
To present workable formats, lipids are synthesized by coupling accurate inositol head groups via phosphoglycerol to short-chain fatty acids, which are water-soluble and amenable to homogeneous assays. These soluble analogs retain important protein- and enzyme-binding properties and allow the addition of biotin or fluors, which facilitates the capture and/or detection of specific lipids.
Formats and Methodology
Many nonradioactive PI3K assay types are commercially available, including ELISA, fluorescence polarization (FP), luminescent (AlphaScreen™), and time-resolved fluorescence (TRueFRET™). ELISA formats are routinely performed in many labs but are low-throughput and heterogenous. Conversely, FP, AlphaScreen, and TRueFRET formats are high-throughput and homogenous. All assays are robust (Z´ values >0.5) and these latter assays are compatible with automated liquid handling.
All PI3K activity assays are competitive, measuring lipid products of individual enzymatic reactions. A typical lipid assay protocol has two primary steps to conduct this measurement. First, active enzyme is incubated with short-chain lipid substrate along with ATP in an optimized buffer. Phosphorylated lipid product accumulates as the reaction proceeds. Second, in the detection step, the enzymatic reactions are terminated and the lipid product quantified.
Finding In Vitro Inhibitors
One of the first PI3K assays validated was a collaborative development project with PerkinElmer Life and Analytical Sciences(www.perkinelmer.com). This project resulted in a sensitive AlphaScreen assay that detects PIP3 produced in vitro by PI3KPa. Utilizing this assay with recombinant enzyme and di-C8 PI(4,5)P2 substrate, IC50 values were measured for two known PI3K inhibitors. Wortmannin showed nearly 1,000 times greater inhibition of PI3Ka than LY-294002 (Figure 1). This assay was used to screen a targeted small molecule chemical library that yielded several candidate lead structures.
TRue-FRET has similar sensitivities to AlphaScreen with the added benefits of lower fluorescent background and less potential compound interference. PI3K TRue-FRET utilizes Lumiphore terbium chelates that offer a bright signal, resulting in lower consumption of reagents and reduced assay cost.
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.