Abstract 6966: Apparent potency of small molecule inhibitors is influenced by the choice of substrate in LIMK biochemical in-vitro kinase activity assays

Abstract Protein kinases are essential for the regulation of many biological processes, e.g. proliferation, differentiation, migration, and apoptosis. Deregulated kinase activity is observed in tumor cells and altered activity of specific kinases is essential for development and progression of cancer and other diseases. Consequently, regulation of protein kinase activity became a target for therapeutic intervention. Intensive research resulted in the development of many small molecule kinase inhibitors (SMI) being clinically approved as targeted therapies. Functional biochemical in-vitro assays are essential requirements to assess SMIs effects on target kinase activity after validation of specific kinases being molecular drivers of a pathological condition. Only limited information is gained by monitoring the interaction of kinase and compound in pure binding assays, but more conclusive data can be generated by in-vitro kinase activity assays. However, the relevance of results from both approaches depends on how well the assay setup responds to the mode-of-action of the compounds. Recombinant proteins and generic substrates are well established in development of protein kinase assays due to cost and technical feasibility considerations. Especially some generic substrates are highly artificial e.g. the commonly used Tyrosine-kinase substrate Poly(Glu/Tyr)4:1 and may be considered as phosphate group acceptors only. However, such generic substrates have successfully been used for development of numerous clinically approved SMIs. Other kinases however are not compatible with generic substrates and can only be established using less artificial substrates or even require their respective physiological in-vivo substrate for in-vitro activity. These kinases include several members of the RAF and MAPK family. Functional in-vitro assay using a generic substrate are rarely replaced by physiological substrate due to more complex assay conditions and increased cost for drug screening. We compared biochemical in-vitro kinase activity assays for the LIM kinases, LIMK1 and LIMK2, using either generic substrates or the published physiological substrates, CFL1 and CFL2. We further evaluated these results by comparing them in different assays, using radiometric and luminescent read-out technologies. Several published LIMK inhibitors displayed significant differences in the relative potency when tested with generic or physiological substrates. Generic substrates for biochemical in-vitro kinase activity assays in preclinical drug development have been well established and successfully applied in the past. However, our data indicate, that the choice of a more physiological substrate should be considered carefully, as it might significantly increase the relevance and therefore value of the results obtained from early-stage compound screening. Citation Format: Robert Torka, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Rademann, Thomas Weber, Daniel Mueller. Apparent potency of small molecule inhibitors is influenced by the choice of substrate in LIMK biochemical in-vitro kinase activity assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6966.