Biophysical Derisking of Non-ATP Ligand Engagement in FGR Tyrosine Kinase: A Computational Strategy for Cancer-Relevant Kinase Discovery

Abstract Non-ATP kinase ligand engagement is attractive for cancer-relevant kinase discovery, but computational evidence can be easily overinterpreted. Apparent surface binding in docking or molecular dynamics may reflect weak nonspecific contact, canonical-site misassignment, transient local retention, or trajectory-average artifacts rather than a reproducible non-ATP engagement mode. Here, we present a computational biophysical derisking strategy for evaluating a non-ATP ligand-engagement hypothesis in human FGR tyrosine kinase using a structurally undisclosed probe ligand, UNL/Analog 7. The workflow integrates construct and residue audit, ATP-site exclusion using an FGR-specific crystallographic reference, local contact persistence, apo-holo pocket comparison, hydration analysis, RMSF/SASA interpretation, HDX-mimic peptide-window prioritization, and exploratory state classification. The analysis was designed to classify whether the ligand-engagement hypothesis merited experimental follow-up, not to establish inhibition, functional allostery, or ligandability. In the analyzed FGR trajectory, UNL/Analog 7 showed persistent engagement of a predefined non-ATP LCLA region while remaining separated from the sequence-mapped ATP-site reference. Apo comparison indicated that the LCLA groove was not newly formed, but instead represented a pre-existing weak surface groove that became more organized in ligand-associated holo states. Holo ligand-associated frames showed lower local water occupancy and reduced regional C-alpha mobility relative to apo. Solvent exposure analysis was mixed, with the strongest HDX-mimic support localized to a peripheral LCLA window and central windows classified more cautiously. Exploratory state analysis separated recurrent holo groove substates with similar ligand proximity but different hydration levels; these states were interpreted as descriptive substates, not validated kinetic states. Overall, this study supports a bounded derisking conclusion: UNL/Analog 7 shows trajectory-supported, ATP-site-excluded engagement of a pre-existing non-ATP FGR groove. The result justifies experimental prioritization by binding assays, HDX-MS, mutagenesis, kinase assays, and structural follow-up, but does not establish biochemical inhibition, functional allostery, cryptic-pocket emergence, thermodynamic water displacement, or validated ligandability.