Standardizing the Format
In order to address some of the challenges of kinase assays (combining relevant biology with HT, reducing costs and assay development times), researchers at Bayer Healthcare’s lead discovery screening department have adapted a platform approach. “We standardize the assay format (in our case, TR-FRET) and work mainly with generic reagents. We separate the project-specific part (kinase substrate, product-specific antibody) from the costly TR-FRET part,” states Ulf Boemer, Ph.D., HT coordinator.
Dr. Boemer explains that instead of directly labeling the substrate with cross-linked allophycocyanin (XL) and the antibody with an EU-Chelate, “we use biotinylated substrate and streptavidin-XL and an unlabeled antibody with ProtG-Eu-chelate.” This allows his group to use the same TR-FRET reagents for all assays.
This has several advantages including lower costs (no beads or radioactivity), higher sensitivity with low nanomolar concentration of products, and miniaturization. However, TR-FRET does require a suitable antibody and lacks sufficient sensitivity for cell-based assays, but it is good for biochemical assays. Dr. Boemer says the field is evolving toward more cell-based kinase screening and a broader mode of action characterization.
“Normally a full-deck HTS against a kinase results in several thousand hits and only a selection of the compounds can be characterized in more detail due to the limited throughput of secondary assays. To avoid missing less potent hit clusters with interesting properties, we run broader characterization (ATP-competition) in HT-mode with all hits. This gives you a broader data basis for the selection of compounds for the lower throughput assays.”
Another key point is that the company bundles all its efforts for different projects in one assay center. Every new compound that is tested for any of the different kinase projects is tested against all active kinase assays. “We try to combine fast primary assay support for cost-effective profiling to get cross-fertilization to see if certain motifs appear on another kinase project. This is important,” Dr. Boemer explains.
There is also a growing trend to identify allosteric modulators to set up assays to bind inactive forms of kinase. This increases the chances of selectivity and generates IP. “The kinase IP field is crowded, and it’s difficult to obtain. That’s why we always screen the full library and don’t focus on specific kinases.”