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

Naomi E Widstrom, Minervo Perez, Erica D Pratt, Jason L. Heier, John F. Blankenhorn, Lindsay Breidenbach, Hannah Peterson, Laurie L Parker

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)1328-1333
Number of pages6
JournalACS Chemical Biology
Issue number6
StatePublished - Jun 17 2022

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health/National Cancer Institute through R01CA183571 and R33CA217780 (LLP), R01CA183571-S1 (MP), T32CA009138 (NEW and EDP), and T32AG029796 (JLH and JFB). The table of contents graphic was created with .

Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural


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