Synthetic Substrate Discovery for Hck Kinase via Phage Display.

Protein tyrosine kinases govern essential cellular processes, including proliferation, survival, and migration. Dysregulation of this enzyme family has been implicated in multiple hallmarks of cancer. These kinases catalyze tyrosine phosphorylation in a tightly controlled manner, with substrate recognition dictated by the local chemical environment of surrounding residues. Although several strategies have been developed to define protein tyrosine kinase substrate specificity, these approaches have notable limitations in scale and resolution. To address these challenges, we present a modernized phage display platform integrated with next-generation sequencing, enabling comprehensive and high-throughput profiling of tyrosine kinase substrates. This method enabled the simultaneous assessment of the relative phosphorylation of billions of potential substrates for Hck kinase, followed by analysis of positional and motif enrichment to guide iterative substrate design. The novel substrates developed through this process exhibited robust kinetic behavior (Km < 30 μM) when evaluated by HPLC/MS assays, yielding insights into Hck substrate preferences within multipositional sequence contexts.