Building kinase switches

Design and development of small molecule kinase inhibitors is a challenging task. A chemical genetics approach modulating kinase function is finding its way into pharma drug discovery programs via the use of innovative new mouse models.

“Taconic is now commercializing an innovative tool that addresses some of the current challenges in kinase target selection and compound profiling.”

Protein kinases play a key role in transmitting messages and signals that eventually reach the cell nucleus and elicit a functional response. Kinases constitute one of the largest and most functionally diverse gene families. Over 500 human protein kinases have been discovered, constituting approximately 2% of the human genome. Deregulation of protein kinase activity can be a major cause of disease. Over 150 protein kinases are currently associated with various human diseases such as rheumatoid arthritis, cardiovascular, neurological disorders and asthma. Since protein kinases regulate both cellular differentiation and proliferation, their roles in cancer are of significant interest to both basic research and pharmaceutical companies. Within the last 12 years, a large number of protein kinase inhibitors have been approved by the FDA and EMEA for the treatment of various cancers.

Protein kinase drug discovery is a major area in the pharmaceutical industry with an estimated one fourth to one third of drug discovery programs targeting protein kinases. However, identification of the relevant kinase targets of small molecule inhibitors is still a significant challenge in order to analyse their therapeutic potential for a given disease. 

While the binding of a small molecule kinase inhibitor to multiple targets is likely of therapeutic benefit, non-specific binding to other kinases does not allow assessing the biological role of the target kinase and hampers the identification of the actual on-target effect of the inhibitor. Taconic is now commercialising an innovative tool that addresses some of the current challenges in kinase target selection and compound profiling. This technology platform is called KinaseSwitchTM. Use of the KinaseSwitchTM technology allows the specific inhibition and pharmacological regulation of only one kinase. Thereby, KinaseSwitchTM provides insights into the biological function of the target kinase and can be used to assess specificity of pre-clinical drug candidates. 

This innovative technology is using chemical genetics and has been developed in the laboratory of Prof. Kevan Shokat (UCSF; Senior Scientific Consultant to TaconicArtemis). The approach utilizes genetics to insert silent active site mutations into the target kinase, creating an ATP analog-sensitive kinase allele (ASKA). The mutation in the kinase active site, typically at the “gatekeeper” residue, will cause a slight conformational change of the ATP binding pocket. This unique, active site structure has been used by Prof. Shokat to develop a novel class of “orthogonal” ATP competitive inhibitors. These inhibitors will not recognize or bind to WT kinases, but specifically bind and inhibit the genetically mutated target kinase. Insertion of ASKA mutations will dramatically increase the sensitivity of the kinase to the orthogonal inhibitor, lowering the IC50 value of the ASKA up to several thousand folds compared to the WT kinase. This leads to the inducible and specific pharmacological inhibition of the mutant kinase. Importantly, any kinase that will be modified with KinaseSwitchTM technology will be sensitive to one of three orthogonal inhibitors (typically Na-PP1, NM-PP1 or MB-PP1). This eliminates the need for specific drugs in order to study the pharmacological inhibition of the target kinase. KinaseSwitchTM alleles have been published for many kinases and the technology is broadly applicable for serine/threonine and tyrosine kinases at various locations within the kinome tree.

KinaseSwitchTM target validation and compound profiling technology is regulating kinase function by inhibiting the enzymatic activity of the drug target, while leaving the kinase protein itself intact. Therefore, the kinase may still act in its spatial-temporal context and serve its function in protein complexes. It is therefore not surprising that a comparison of kinase knockout and KinaseSwitchTM   phenotypes revealed interesting differences, alluding to the multiple and independent roles of the kinase protein itself and its associated enzymatic activity (reviewed in Knight and Shokat; Cell. 2007 Feb 9;128(3):425-30)

At TaconicArtemis, KinaseSwitchTM technology is now being used to generate innovative mouse models in which the WT function of the kinase is eliminated and replaced by ASKA mutations. Historically, for target validation approaches using mouse models, knockout (KO) technology has proven to be a useful tool in understanding the in vivo role of multiple kinases. However, knockout technology is not without limitations, as it irreversibly alters the genomic structure of the gene and loss of the kinase protein may lead to embryonic lethality. Use of specific recombinases (tissue specific Cre and inducible CreERT2 strains) can circumvent some of these issues, but also include additional breeding efforts. In contrast, KinaseSwitchTM chemical genetics uses the orthogonal compounds to inhibit kinase activity at any selected time during animal development. Just like any ATP competitive inhibitor, this inhibition is inducible and reversible. Thereby, KinaseSwitchTM technology mimics most truthfully the effect a compound might have on the kinase in a clinical setting.

More complex mouse models can also be generated by breeding KinaseSwitchTM mice to relevant disease models. The inhibition of a kinase during an established disease state, e.g. in a breast cancer model, will provide valuable insights into the therapeutic potential of the kinase target. During the drug discovery process KinaseSwitchTM can provide useful reference points to better understand the inhibition profile of small molecule drugs. Taconic believes that this technology will not only be used for the generation and analysis of mouse models but should also find its way for in vitro applications to profile drugs. One example could be the generation of KinaseSwitchTM mutant ES cells that could be used for differentiation studies. By using KinaseSwitchTM, target kinases could be inhibited in differentiated cardiomyocytes or neurons to provide insights into toxic side effects associated with kinase inhibition in these cell types. Such in vitro applications would allow ES cell lines to be integrated in the drug discovery process and become a tool during compound screening activities.

At Taconic, KinaseSwitchTM represents the latest technology to modify gene function not only at the DNA and RNA level by using knockouts and transgenic RNAi models, but also providing access to the latest tools to modify the protein itself.  We look forward to further developing and applying this technology with our customers.