Novel Bruton’s Tyrosine Kinase (BTK) Substrates for Time-Resolved Luminescence Assays

Bruton’s tyrosine kinase (BTK) is a well-documented target for cancer therapeutics due to its role in B-cell signaling pathways. However, inhibitor design is hindered by lack of tools to assess kinase activity. We used in vitro phosphoproteomics to determine BTK’s substrate preferences and applied t...

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Bibliographic Details
Published inACS chemical biology Vol. 17; no. 6; pp. 1328 - 1333
Main Authors Widstrom, Naomi E., Perez, Minervo, Pratt, Erica D., Heier, Jason L., Blankenhorn, John F., Breidenbach, Lindsay, Peterson, Hannah, Parker, Laurie L.
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 17.06.2022
Subjects
Agammaglobulinaemia Tyrosine Kinase
Luminescence
Luminescent Measurements
Phosphorylation
Protein Kinase Inhibitors - chemistry
Protein Kinase Inhibitors - pharmacology
Terbium
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Summary:Bruton’s tyrosine kinase (BTK) is a well-documented target for cancer therapeutics due to its role in B-cell signaling pathways. However, inhibitor design is hindered by lack of tools to assess kinase activity. We used in vitro phosphoproteomics to determine BTK’s substrate preferences and applied this information to our updated data processing pipeline, KINATEST-ID 2.1.0. This pipeline generates a position-specific scoring matrix for BTK and a list of candidate synthetic substrates, each given a score. Characterization of selected synthetic substrates demonstrated a correlation between KINATEST-ID 2.1.0 score and biochemical performance in in vitro kinase assays. Additionally, by incorporating a known terbium-chelation motif, we adapted synthetic substrates for use in an antibody-free time-resolved terbium luminescence assay. This assay has applications in high-throughput inhibitor screening.
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Author Contributions
NEW, MP, and LLP conceived the project. NEW, MP, JLH, HP, and LB performed experiments. NEW, MP, JFB, and EDP analyzed data. JFB designed and wrote initial R scripts; EDP wrote the updated and consolidated KINATEST-ID v 2.1.0 R package. NEW, EDP, and LLP wrote the manuscript. NEW and MP contributed equally.
ISSN:1554-8929
1554-8937
DOI:10.1021/acschembio.2c00106