January 15, 2017 (Vol. 37, No. 2)

GEN Interviewed Six Experts to Explore Kinase Assays

Efficient kinase assays and precision medicine go hand in hand. As precision medicine becomes more of a reality, researchers are taking a closer look at the role of kinase assays. As an integral aspect of kinase inhibitor therapeutics and with over 35 kinase inhibitors already approved for cancer therapy, kinase assays have a significant role in realizing personalized treatments in cancer. For this seminal roundup, GEN interviewed six experts to explore the many ways that kinase assays are being used now and what is being done to improve their use for cancer and other diseases. Find out how you can get the most out of these assays and advance your research.


GEN’ expert panel

GEN: What role do you envision kinase assays fulfilling within the growing precision medicine space?

Dr. Ferrer: Kinase assays will sit alongside the development of kinase inhibitor therapies. Despite the success of several kinase inhibitors as cancer therapies (imatinib, crizotinib, and trastuzumab for example), these are only effective in a subset of patients. One size doesn’t fit all. To overcome this, we need to better understand how patients respond to treatments, and kinase assays will likely form the downstream readout of therapeutic compounds.

The power of validated biomarkers and specific kinase assays is that they help to stratify patients according to their specific responses to kinase modulators. This has great potential for realizing personalized treatments in cancer.

Dr. Haack: Precision medicine is affecting all aspects of the drug development process and in-depth profiling of kinase activities is needed to fully understand the mechanisms of action, off-target effects, and patient heterogeneity in signaling and kinase activity.

For example, mutations leading to aberrant activation of multiple kinases are most often the drivers of tumorigenesis. Understanding the kinases driving the individual patient’s tumor may lead to insights into the correct combination of small-molecule inhibitors required to overcome tumor progression. Such improved understanding derived from accurate and multiplexed kinase assays will help to optimally match patients to therapies.

Dr. King: Over 35 kinase inhibitors have been approved for cancer therapy alone. While precision medicine’s value has been demonstrated by drugs like dabrafenib, unfortunately, resistance mechanisms are inevitable. Mutations often arise to overcome drug binding, effectively presenting “new” targets. Given the number of these drugs in mainstream use, kinase assays will thus fulfil an important role in drug discovery for these “new” targets going forward.

Furthermore, being able to profile a new drug candidate against an extensive kinome panel permits the generation of more selective drugs. This improves the potential for use in combination therapies to address other, off-target resistance mechanisms.

Dr. Bossé: Kinases are molecular switches involved in the etiology of multicausal diseases such as cancer. Kinase inhibitors used in clinics are either selective (single-kinase) or may affect multiple kinases that are often part of different pathways (multi-kinase). Profiling lead compounds for their functional activity, potency and selectivity on a wide array of kinases, in a cellular format if possible, allows for the design of drugs with optimal therapeutic properties and limited side-effects.

There is growing evidence that considering each patient/tumor individually and using either a single multi-kinase inhibitor treatment or a combination of single kinase inhibitors are effective strategies to treat cancer.

Dr. Goueli: Although FDA-approved drugs for cancer show early success during first-line treatment, resistance ensues due to the development of resistant mutations, which necessitates the need for second-, third-, etc., generation drugs to address the drug insensitivity. Thus, the use of gene sequencing of the targeted kinase using patient tumor tissue or liquid biopsy will be a common practice at the doctor’s office to identify the newly developed mutation(s). This can serve as a guide for the development and design of future drugs using biochemical, biophysical, and phenotypic assays to address the lack of sensitivity of the tumor to existing drugs.

Dr. Tang: Currently, precision medicine predominantly relies on a patient’s genomic information. However, the complexity of the genome and environmental effects makes identifying disease drivers a challenge. Additional information, such as protein expression and activity, is required to tailor therapy.

Protein kinases are one of the most important regulators involved in almost all disease processes, and their activities can be analyzed by a variety of methods (e.g., the RayBio Human RTK Array which identifies phosphorylation of 58 different RTKs and 13 closely related proteins). Characterizing kinase-mediated signaling and/or abnormalities will enable the prediction of genetic variant functionality and will become a key part of precision medicine research strategies.

Dr. Hu: Precision medicine is personalized medicine used to treat specific disease based on person’s genetic information. This targeted therapy is the foundation of precision medicine. Different from standard chemotherapy, targeted therapy uses drugs to more precisely identify and attack cancer cells with little damage to normal cells. Kinase inhibitors play the most important role in targeted therapy. Thus, efficient kinase assays will help to promote the development of precision medicine.


GEN: Advances in immunotherapy have dominated the news in recent months, providing many patients with treatment options they never had previously. What kinds of kinase assays exist that are advantageous to investigators working on creating new immunotherapy compounds?

Dr. Ferrer: There are essentially two types of kinase assays: those that screen the kinome and those that assay kinase activity. Kinome screening uses high-throughput assays to measure interactions between kinases and potentially therapeutic compounds. This makes them ideal for drug prescreening phases.

Kinase activity assays come into play once a compound has been identified as kinase modulator. These smaller scale assays are frequently overlooked, but are necessary to examine functionality in detail.

Modulation of immune checkpoints by tumors is still a new field, and we can expect to see kinase assays playing an essential role in its progression.

Dr. Haack: Small-molecule kinase inhibitors to targets such as ALK5, RON, and CSF1 are specifically developed as immunotherapies. In addition, many of the existing targeted therapies using kinase inhibitors are being explored in combination therapies with checkpoint inhibitors or other immunotherapies.

The complexity of the tumor microenvironment on one hand and the chances for increased side effects and combinatorial toxicities with combination therapies on the other hand will demand the development of highly specific and validated kinase drugs. Proteomic technologies such as those based on mass spectrometry could provide multiplexed profiles and interrogate the activation status in cells or even tissues.

Dr. King: Recent advances in immunotherapy invoke the potential for targeting signal transduction pathways associated with immune checkpoint regulation. Development of new macromolecular inhibitors would require receptor binding and cellular assays.

However, potentially synergistic small-molecule kinase inhibitors would still require conventional enzyme assays. Signaling pathways via Csk and Cbl are known to play significant roles in T-cell receptor regulation, suggesting downstream kinases that may be of interest for inhibition or selectivity profiling. Interestingly, PTPN22 plays a role in T-cell regulation and may be an attractive drug target, which raises the possibility for a departure into the realm of phosphatase assays.

Dr. Bossé: Immunotherapy consists of generating antibodies specific to cell surface markers (namely receptor tyrosine kinases, RTK) or developing molecules inhibiting the interaction between cell surface immune checkpoints binding partners (e.g., PD1/PDL1). Therefore, assays to measure RTK activity and that of downstream effectors of immune checkpoints components (i.e., cytosolic kinases and phosphatases) are essential to investigators working in immunotherapy.

Measuring RTK activity can be done using either biochemical or cellular assay formats. Characterizing downstream effectors of cell surface immune checkpoint components is essentially achieved using functional cellular assays (e.g., SHP2, PI3K for PD1).

Dr. Goueli: Cellular immune responses to specific epitopes of highly expressed and/or mutated targets, such as EGFR, MET, etc., have been reported for different types of cancers. The observation of increased PD-L1 expression in mutated EGFR lead to the use of PD1 antibody treatment, resulting in improved survival of mice with lung mutated EGFR driven tumors. This increased expression of PD-L1 was shown to be regulated by downstream kinase signaling pathways, and thus kinase assays that monitor the activity of the relevant signaling kinases before and after treatment will help in dissecting the pathway(s) involved in immunotherapeutic response. These can be biochemical using the newly generated mutant protein if available, or cell-based assays and high-content screening of the downstream effector markers using selective antibodies.

Dr. Tang: Kinases are known to play pivotal roles in the resolution of an immune response and could therefore serve as relevant therapeutic intervention points. Phospho-specific antibody-based methods, such as Western blot and ELISA, are gold standards for detecting protein phosphorylation, and are commonly used to validate drug candidates that are identified by biochemical kinase assays.

Recent advances in automation makes high-throughput, large-scale Western blot possible. In addition, phosphorylation antibody arrays are rapid, sensitive, and economical tools for the simultaneous detection of phosphorylation of multiple proteins in certain pathways (e.g., the RayBio human AAH-AKT array). These assays can help to better define the selectivity profile of immunotherapy compound candidates.

Dr. Hu: Immunotherapy is emerging as a promising therapy for both blood and solid tumors. One of the big challenges in immunotherapy is that tumors can evade the immune attack by suppressing the immune cell function. Thus, boosting the immune cell function against hostile tumor microenvironment is part of the mission of cancer immunotherapy. Cell-based kinase assays could be used to select or screen compounds, which can boost the immune function. The iONCscan and PathHunter® Kinase Cell Based Assay from Discover X are good platform for this purpose.


GEN: Is there any new technology on the horizon you believe will have a dramatic impact on kinase assay development?

Dr. Ferrer: Technologies that allow us to investigate the effects of small-molecule kinase inhibitors or downstream kinase activity in a high-throughput, multiplex context will have the most impact. Identifying treatment sensitivities and resistances will also help advance personalized medicines.

New multiplexing technologies, like our Firefly™ platform, can report the levels of a wide range of molecules, raising the possibility of activity assays in conjunction with current protein and nucleic acid capabilities.

Dr. Haack: One of the most promising assay formats being developed today for cell-based kinase assays is immuno-affinity enrichment with multiple reaction monitoring (iMRM). Drug discovery relies on both the interrogation of pathway signaling and on screening small molecules or biologics for activity. iMRM will radically change both of those processes by providing a quantitative multiplex assay. As user access increases, this technique has the potential for replacing many other commonly run assays.

Dr. King: Information-rich readouts from high-content analysis, cellular mass spectrometry, and systems pharmacology approaches enable interrogation of kinase activity in a more physiologically relevant environment. Advances in these technologies, using cell extracts before and after compound treatment, are making this more of a reality. The off-target effects of inhibitors are a problem, and being able to identify these within a cell in a high-throughput manner, using phosphoproteomic approaches, would also be a significant advancement for the field.

Dr. Bossé: In 2015, Hart et al. showed that high-resolution CRISPR-Cas9 screens revealed specific cancer liabilities, namely cell-type-specific dependencies for specific RTKs. These findings will significantly influence the development of assays for the RTKs identified and/or their subcellular downstream effectors (namely Ser/Thr kinases). The emerging use of 3D cell and tissue culture to create more biologically relevant models will also have a significant impact on kinase assay development.

Dr. Goueli: Biochemical assays in a high-throughput format remain primary tools in developing new drugs. Because of the frequent emergence of new mutations of the targets, screening for new drugs will depend on the nature of that target. If the mutated target is available in purified form, it can be screened for developing new drug using similar strategy. However, some targets form homo- and heterodimers in the cell; and hence in situ interrogation will be necessary since the conformation of the targets (active vs. inactive state) determines whether the drug is efficacious.

Dr. Tang: Phosphorylation antibody array and automated Western blot are both emerging technologies that are reliable and high-throughput. Phosphorylation antibody array can simultaneously detect phosphorylation of multiple proteins that are in related pathways. It is a rapid and convenient tool for studying kinases and inhibitors in a large, cell signaling network context. Automated Western blot uses a capillary-based immunoassay platform that integrates and automates the entire protein separation and detection process. It not only runs large-scale samples, but also provides quantitative results.

Dr. Hu: The interaction between tumor and immune system is complex. To create a cell-based assay platform mimicking the tumor microenvironment is of paramount importance for drug discovery.

Based on my personal view, I think the 3D high-throughput cell-based kinase assay is the most needed advance in technology. 




























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