Abstract 648: Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays
Authors: Mueller Daniel G. , Gericke Andreas , Totzke Frank , Heidemann-Dinger Carolin , Ketterer Constance , Kraemer Diane , Weber Thomas , Kubbutat Michael H. 
Published: March 2024

Essential biological signaling pathways affecting e.g., proliferation, differentiation, migration, and apoptosis are regulated by protein kinases. Deregulation of protein kinases is observed in many tumor cells and frequently the development and progression of human cancers and other diseases is found to be causally connected to altered activity of specific protein kinases.

Therefore, protein kinases have become a prime molecular target for therapeutic intervention. Multiple small molecule inhibitors targeting different kinases are currently in clinical use for treatment of various types of cancer and other diseases.

A first step in preclinical development of new compounds is testing candidate substances in biochemical in-vitro assays, either based on binding of the compounds to their target or based on alterations of the in-vitro activity of the target kinase monitored in biochemical activity assays.

Depending on the actual mode of action of a compound, the relevance of the results of such in-vitro assays may depend on the actual setup of the assay.

Mainly for reasons of cost and technical feasibility, the use of generic substrates for in-vitro kinase activity assays is well established. While many of those substrates are highly artificial and can only be considered as generic phosphate-group acceptors, they have been successfully used in the past in the development of many approved small molecule kinase inhibitors currently in clinical use.

However, even while many kinases may show activity with generic substrates, for some physiological substrates are required. Members of e.g. the RAF family and other members of the MAPK pathway are highly substrate specific and will not show kinase activity with generic substrates.

Such kinases which have been established with generic substrates are rarely switched to more physiological substrates as most often this will result in increased complexity and associated cost of preclinical compound development.

We have compared the in-vitro activity of WEE1 using different in-vitro activity assay readouts like autophosphorylation, phosphorylation of generic substrates and phosphorylation of its physiological substrate CDK1. In addition, we examined the substrate specificity of WEE1 towards CDK1 alone and in complex with Cyclin B1.

We compared the potency of a selection of WEE1 inhibitors when different substrates were used.

While in early preclinical development the use of rather artificial biochemical assays has been successfully been applied in the past, it should be taken into consideration that at least for the kinase target in focus, the identification, establishment and use of a more physiologically relevant substrate may increase the value of early-stage compound screening results significantly.

Citation Format: Daniel G. Mueller, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Diane Kraemer, Thomas Weber, Michael H. Kubbutat. Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 648.