Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example
Autor: | Roth, Aileen, Gihring, Adrian, Göser, Florian, Peifer, Christian, Knippschild, Uwe, Bischof, Joachim |
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Jazyk: | angličtina |
Rok vydání: | 2021 |
Předmět: |
Ki
CK1 workflow inhibitor constant Organic chemistry IC50 Proteinkinase CK1 In Vitro Techniques Article Protein kinase Workflow Inhibitory Concentration 50 QD241-441 Protein kinases ddc:610 Phosphorylation Protein Kinase Inhibitors Enzyme Assays standardization Casein Kinase I small molecule inhibitor protein kinase Inhibitor constant Standardization Kinetics Small molecule inhibitor DDC 610 / Medicine & health |
Zdroj: | Molecules, Vol 26, Iss 4898, p 4898 (2021) Molecules Volume 26 Issue 16 |
ISSN: | 1420-3049 |
Popis: | Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactivebased in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors. publishedVersion |
Databáze: | OpenAIRE |
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