Yekeen, A. A.; Meyer, C. J.; McCoy, M.; Posner, B.; Westover, K. D.

Kinases are pivotal cell signaling regulators and prominent drug targets. Short peptide substrates are widely used in kinase activity assays essential for investigating kinase biology and drug discovery. However, designing substrates with high activity and specificity remains challenging. Here, we present Subtimizer (substrate optimizer), a streamlined computational pipeline for structure-guided kinase peptide substrate design using AlphaFold-Multimer for structure modeling, ProteinMPNN for sequence design, and AlphaFold2-based interface evaluation. Applied to five kinases, four showed substantially improved activity (up to 350%) with designed peptides. Kinetic analyses revealed >2-fold reductions in Michaelis constant (Km), indicating improved enzyme-substrate affinity. Two designed peptides exhibited >5-fold improvement in selectivity. This study demonstrates AI-driven structure-guided protein design as an effective approach for developing potent and selective kinase substrates, facilitating assay development for drug discovery and functional investigation of the kinome.