Assay Development and Screening
Optimization of Assays for Challenging Targets for Lead Discovery and Screening
Protein phosphorylation by kinases is a major mechanism of cell signaling, and is involved in almost all aspects of cell biology. Kinase dysregulation is a key factor in diseases like cancer, and kinases are one of the major drug targets in oncology. However, despite decades of research and billions of dollars in drug discovery efforts on kinases, relatively few are well characterized. The majority of the ~90 tyrosine kinases are considered “orphans,” for which few to no substrates, and thus few details about biological pathways and roles, are known. Without substrates to use as activity probes, inhibitors for use as tool compounds or potential therapeutics cannot be discovered. We have developed a strategy to incorporate empirically-determined substrate profiling data into our KINATEST-ID bioinformatics pipeline to efficiently tackle the orphan kinase problem, determine substrate preferences and design novel substrate tools. Protease-digested peptides from cell lysates are stripped of pre-existing phosphates, then re-phosphorylated with a kinase of interest. The resulting phosphopeptides are enriched and analyzed using mass spectrometry. Phosphopeptide sequences are extracted from the peptide ID list and funneled through the KINATEST-ID pipeline using a set of scripts implemented in the open-source user interface GalaxyP, to define substrate sequence preferences and propose candidate optimal substrate peptides. Those are then synthesized and tested for phosphorylation efficiency by the target kinase. Using this approach, we have characterized substrate preferences for several understudied kinases for which few validated substrates were known, including FLT3 and two clinically relevant mutants, and BTK. Current and future efforts are to broaden the scope of kinases characterized using this streamlined phosphoproteomics/bioinformatics pipeline and proceed with systematically defining substrate information and developing novel tools for other orphaned kinases in the kinome.