May 1, 2009 (Vol. 29, No. 9)

Firms Use Myriad Strategies to Identify and Characterize Kinase Inhibitors

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.” 

Antibody-Free ADP Detection Assay

Although there are many commercial kinase assays, there remains high demand for a single assay that is applicable to both a single kinase target and all kinase targets. “Having a single assay that meets criteria for HTS and MOA will simplify and standardize assay development for kinase drug discovery. Not only will it reduce development time and costs (one assay vs. two or more), it will also make data comparison more straightforward. For example, the potency of SAR compounds can be directly compared with HTS single-shot or dose-response data,” says Hu Li, Ph.D., manager, molecular discovery research, GlaxoSmithKline.

Dr. Li’s research group recently evaluated the ADP-Glo™ assay developed by Promega as part of a beta-testing effort. This is a homogenous signal-increase assay that measures ADP production from a kinase reaction via conversion to ATP; then quantifies ATP using luciferase in the presence of luciferin. The assay provides high sensitivity at ATP concentrations from low µM to high mM because the unused ATP in the reaction is depleted prior to ADP-ATP conversion. It is antibody-free and label-free and applicable to all kinases.

“Compared with phosphopeptide detection, the ADP detection method is universal because it is applicable to both serine/theonine and tyrosine kinases and can accommodate broad substrates,” explains Dr. Li.

Since it is antibody-free, the assay’s advantages include cost-effectiveness and tolerance of high ATP concentration. It includes a step that eliminates unused ATP in the reaction before quantifying ADP produced in the kinase reaction. This significantly reduces background and increases sensitivity, further helping to improve assay robustness at lower ATP conversion while enabling initial rate determination.

“ADP-Glo may prove to meet the challenges we are facing in working with protein kinases (substrate specificity, different catalytic efficiency, different Km toward ATP) due to its uniqueness,” summarizes Dr. Li.

ProQinase’s integrated protein kinase technology platform is composed of a collection of preclinical services including a large portfolio of recombinant protein kinases.

Integrated Approach

Researchers at the Dana-Farber Cancer Institute are using an integrated approach to better understand cancer initiators and drivers. “Most of our functional genomic screens have focused on kinases because it’s a trackable number of genes for proof of principal, and kinases are important in cancer,” says William Hahn, Ph.D., associate professor of medicine, Harvard Medical School.

Dr. Hahn explains that they use either RNAi libraries to suppress or overexpress genes and then combine the resulting data to look for either mutated or amplified genes. “One of the biggest challenges with functional screening is the high false positive and negative rates. The first is really the one that challenges most people because you get a long list of genes that is laborious to go through and test.”

This is the main reason why they have adopted an integrated approach, Dr. Hahn says, in addition to making their own reagents and developing better informatics tools to study data sets in a more statistically rigorous manner. They are also working with a HT facility to optimize screening; they currently spend an average of two to eight months optimizing conditions.

His group is also focused on identifying cancer drivers because many mutated genes do not contribute to the cancer phenotype. “The key in applying these comprehensive approaches is to discover cancer genes that are really important.”

This approach has proven successful— his group was able to identify a gene (CDK8) that plays a key role in colon cancer. “We had two screens going in parallel, and when we got a list of genes that scored in both of those it was only a total of nine genes. We were able to work with our collaborators to see that CDK8 was amplified in colon cancer.”

He adds that current efforts are focusing on two main areas: expanding the scale of the screens to interrogate the entire genome and generating  larger data sets to look for gene co-dependencies (a mutation sets up a co-dependency on another gene). “The reason we’re trying to use this in cancer is because there are many oncogenes that are considered un-druggable. But, if you can find partners that were only acquired in cells within the mutation, it would allow you to develop co-targeting methods.”


The Polo-like kinase I (PLK1) plays a key role in regulating cellular proliferation; deregulation of PLK1 activity is thought to promote tumorigenesis in humans. It has a unique C-terminal non-catalytic domain called polo-box domain that is critical for proper subcellular localization and protein-protein interaction. Kyung Lee, Ph.D., senior investigator, laboratory of metabolism, National Cancer Institute at NIH, and his lab have developed a specific PLK1 assay to quantify its levels and activity.

Dr. Lee says that the impetus for developing the assay was the discovery that PLK1 efficiently phosphorylates and binds to PBIP (an important scaffold that is phosphorylated and bound to PLK1 during interphase). As the cells enter mitosis, PLK1 becomes activated via an upstream kinase, causing the PBIP1 to dissociate itself. This step is most likely important for PLK1 to phosphorylate other substrates critical for proper mitotic progression.

There are several key advantages to this assay. First, it uses a specific PLK1 substrate that can’t be phosphorylated by other cellular kinases, so unpurified lysate (only requiring one to two micrograms) containing all cellular kinases can be used. It can be performed at room temperature, unlike conventional immunoprecipitation kinase assays, and results are read by a standard ELISA reader. Higher sensitivity allows for measurement of kinase activity in small tumor biopsies from patients.

PLK1 is an attractive anticancer drug target because its overexpression correlates with aggressiveness of various types of cancers in humans. “Tumor tissues exhibit several-fold elevated levels of PLK1 expression than surrounding normal tissues,” explains Dr. Lee.  Therefore, it could be used to assess early chemotherapy response.

To demonstrate its clinical potential, Dr. Lee’s group showed that PLK1 activity substantially dropped in tumor biopsies following chemotherapy. Clinicians currently use CT/MRI four to six weeks after cessation of chemotherapy to assess tumor size. “Our assay makes it possible to assess therapeutic outcome within days, allowing much earlier evaluation for timely clinical decisions,” adds Dr. Lee.

ELISA microplates were coated with soluble GST-PBIPtide containing the T78 motif (green balls) and then incubated with total cellular lysates at 30oC for 30 minutes.
(Kyung Lee, Ph.D., NCI)

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