Subtimizer: Computational Workflow for Structure-Guided Design of Potent and Selective Kinase Peptide Substrates
Year: 2026
Type: article
Abstract: 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 the Michaelis constant (Km), indicating improved enzyme–substrate affinity. Designed peptides for MET and ROS1 exhibited reciprocal selectivity, with 4-fold and 11-fold preferences for their intended targets, respectively. 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.
Institution: The University of Texas Southwestern Medical Center
Language: English
Subfield: Drug Discovery
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7128204171