Open AccessNovel Bruton’s Tyrosine Kinase (BTK) Substrates for Time-Resolved Luminescence Assays
Author(s) -
Naomi E. Widstrom,
Minervo Perez,
Erica D. Pratt,
Jason L. Heier,
John F. Blankenhorn,
Lindsay Breidenbach,
Hannah Peterson,
Laurie L. Parker
Publication year - 2022
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.2c00106
Subject(s) - bruton's tyrosine kinase , tyrosine kinase , in vitro , kinase , ibrutinib , chemistry , cancer research , biochemistry , computational biology , signal transduction , medicine , biology , immunology , leukemia , chronic lymphocytic leukemia
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.