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Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

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 mole...

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Autores principales: Roth, Aileen, Gihring, Adrian, Göser, Florian, Peifer, Christian, Knippschild, Uwe, Bischof, Joachim
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401859/
https://www.ncbi.nlm.nih.gov/pubmed/34443486
http://dx.doi.org/10.3390/molecules26164898
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author Roth, Aileen
Gihring, Adrian
Göser, Florian
Peifer, Christian
Knippschild, Uwe
Bischof, Joachim
author_facet Roth, Aileen
Gihring, Adrian
Göser, Florian
Peifer, Christian
Knippschild, Uwe
Bischof, Joachim
author_sort Roth, Aileen
collection PubMed
description 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 IC(50) 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, radioactive-based 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.
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spelling pubmed-84018592021-08-29 Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example Roth, Aileen Gihring, Adrian Göser, Florian Peifer, Christian Knippschild, Uwe Bischof, Joachim Molecules Article 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 IC(50) 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, radioactive-based 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. MDPI 2021-08-12 /pmc/articles/PMC8401859/ /pubmed/34443486 http://dx.doi.org/10.3390/molecules26164898 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Roth, Aileen
Gihring, Adrian
Göser, Florian
Peifer, Christian
Knippschild, Uwe
Bischof, Joachim
Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example
title Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example
title_full Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example
title_fullStr Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example
title_full_unstemmed Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example
title_short Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example
title_sort assessing the inhibitory potential of kinase inhibitors in vitro: major pitfalls and suggestions for improving comparability of data using ck1 inhibitors as an example
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401859/
https://www.ncbi.nlm.nih.gov/pubmed/34443486
http://dx.doi.org/10.3390/molecules26164898
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