Protein kinases are at the forefront of drug discovery, representing the largest druggable class of compounds for the pharmaceutical industry with 518 kinases associated with approximately 400 diseases.
There are many approaches to measure kinase activity in order to identify and characterize kinase inhibitors. The nature of kinases is complex, however, and companies are developing their own approaches and methods to meet this challenge. These companies will be presenting their latest efforts next month at CHI’s “Protein Kinase Targets” conference to be held in Cambridge, MA.
MAP (mitogen-activated protein)-kinases play a key role in regulating various cellular activities like gene expression, mitosis, differentiation, and apoptosis. External stimuli lead to activation of MAP-kinase via a signaling cascade. “The problem is you really can’t distinguish which kinase has been blocked because any inhibition of the three or four kinases will present the same results. What we do is a direct assay where there’s only the active enzyme plus its downstream kinase inactivated target,” explains Ralph Graeser, Ph.D, group leader, drug discovery, ProQinase.
ProQinase offers an integrated protein kinase technology platform for preclinical drug development of protein kinase inhibitors in oncology and other disease areas. “We are trying to produce protein kinases that are specifically activated and tagless. If you have kinases that are up to 100% activated, your results are more reliable. Recombinant kinases require the use of tags, which range in size from around six amino acids up to 24 Kd—almost the size of a kinase. “So, it’s not the same protein anymore as it was in the cell. We were thinking it’s best to have only the kinase and not other proteins hanging around. That was the idea we had developing this program.”
Dr. Graeser says the company provides enzymes that are as close to an active kinase in a cell as possible. This saves time because the assay is more defined and doesn’t require a lot of controls in order to discover which kinase is really involved. This approach is used for initial HT screening or for profiling kinases and/or inhibitors. In addition, every new kinase being generated as a tagless version is compared to a tagged version to see if there is a large difference in purity and other characteristics.
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.”