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 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.

Assays for tyrosine phosphorylation in human cells.

Tyrosine kinases are important for many cellular processes and disruption of their regulation is a factor in diseases like cancer, therefore they are a major target of anticancer drugs. There are many ways to measure tyrosine kinase activity in cells by monitoring endogenous substrate phosphorylation, or by using peptide substrates and incubating them with cell lysates containing active kinases. However, most of these strategies rely on antibodies and/or are limited in how accurately they model the intracellular environment. In cases in which activity needs to be measured in cells, but endogenous substrates are not known and/or suitable phosphospecific antibodies are not available, cell-deliverable peptide substrates can be an alternative and can provide information on activation and inhibition of kinases in intact, live cells. In this chapter, we review this methodology and provide a protocol for measuring Abl kinase activity in human cells using enzyme-linked immunosorbent assay (ELISA) with a generic antiphosphotyrosine antibody for detection.