smFASTIA: A high-sensitivity platform for purification-free kinetic screening of protein–small molecule interactions

Elucidating the binding kinetics of protein-small molecule interactions is critical for optimizing drug efficacy and understanding mechanism of action. However, establishing structure-kinetic relationships is often hindered by the throughput of conditional assays that require protein purification. Herein, we present smFASTIA, a rapid, purification-free screening platform that integrates cell-free protein synthesis with high-sensitivity bio-layer interferometry. Using the SpyTag003-SpyCatcher003 system, we immobilized target proteins at high density directly from crude reaction mixtures, enabling the detection of weak small-molecule signals. Using human carbonic anhydrase II and its inhibitor as a model, we screened 32 variants. Instead of relying on equilibrium analysis, we utilized the initial slope of the association phase as a kinetic indicator proportional to the association rate constant. This approach successfully discriminated variants based on their kinetic profiles. Validation by surface plasmon resonance confirmed that variants classified as non-binders in our screening included those with significantly reduced on-rates, demonstrating the platform's ability to filter based on association kinetics. This workflow enables the kinetic assessment of dozens of variants within two days, providing a powerful tool for accelerating SKR studies and protein engineering.