Development Of Synthetic Peptide Substrates To Study Tyrosine Kinase Activity

Abstract

Kinases are key targets for cancer therapeutics due to their role in development and progression of cancer. Despite this, the majority of new kinase inhibitors and studies remain focused on a small subset of target kinases, leaving much of the kinome understudied. To aid researchers in kinase studies and inhibitor design, we implemented a streamlined phosphoproteomics pipeline to determine substrate profiles of kinases and used this information to design synthetic peptide substrates to study kinase activity. Using Bruton’s tyrosine kinase (BTK) as a proof-of-concept, we used phosphoproteomics to identify in vitro substrates of BTK, and input these substrates into our KINATEST-ID V2.1 R-package to identify amino acid preferences. We designed and characterized novel BTK synthetic substrates, as well as established a correlation between the assigned KINATEST-ID score for each substrate and biochemical performance. To increase applicability of the BTK substrates, we integrated a known terbium-chelation motif into the substrate sequence for use in a time-resolved terbium luminescence assay to monitor BTK activity over time. These workflows were then applied to the understudied TAM family of receptor tyrosine kinases, Tyro3, Axl, and Mer. We designed a set of synthetic substrates for each TAM kinase, which can be used in activity assays. We also found that Tyro3 had differential phosphorylation patterns of the synthetic substrates, allowing for a Tyro3 synthetic substrate to be specific for Tyro3 over Axl or Mer. Overall, we demonstrate optimized workflows for studying substrate profiles of tyrosine kinases and design and characterization of synthetic peptide substrates. This work can be applied towards in vitro kinase activity studies and inhibitor screening and characterization.