Orthogonal Temperature-Related Intensity Change and Time-Resolved Förster Resonance Energy Transfer High-Throughput Screening Platform for the Discovery of SLIT2 Binders.

SLIT2 is a secreted glycoprotein implicated in axon guidance, immune modulation, and tumor biology, whose extracellular and glycosylated nature can complicate conventional biophysical screening workflows. Here, we provide a complete, step-by-step protocol for an orthogonal high-throughput discovery pipeline that integrates temperature-related intensity change (TRIC) as a solution-based primary binding screen with time-resolved Förster resonance energy transfer (TR-FRET, homogeneous time-resolved fluorescence format) as a functional assay for inhibition of the SLIT2-ROBO1 interaction. The workflow is designed to be fast and convenient, uses low reaction volumes and low nanomolar protein concentrations to minimize material use, and includes built-in quality control steps to support reproducible hit triage. In TRIC (NanoTemper Dianthus), binding is detected as temperature-dependent fluorescence intensity changes of a labeled target protein under an infrared (IR)-mediated thermal gradient, enabling immobilization-free detection of small-molecule interactions and instrument-assisted filtering of autofluorescent, quenching, or aggregating compounds. Candidate binders are advanced to multi-point TRIC/microscale thermophoresis (MST) measurements on Monolith X to determine binding affinity (Kd). In TR-FRET, disruption of SLIT2-ROBO1 association is quantified by changes in the ratiometric 665/620 nm emission readout, measured with a time delay to suppress short-lived background fluorescence, enabling concentration-response analysis and reporting of relative IC(50) values (including partial inhibition behavior where applicable). Although presented using the SLIT2-ROBO1 extracellular interaction as a representative model system, this orthogonal screening strategy is designed to be adaptable to other extracellular protein-protein interactions where minimizing immobilization artifacts and fluorescence interference is critical. Key features • End-to-end pipeline: TRIC primary screen → artifact filtering → MST/Monolith X affinity → TR-FRET inhibition validation. • Homogeneous, immobilization-free assays compatible with 384-well HTS formats. • Reproducible analysis: standardized thresholds for TRIC hits and relative IC(50) reporting for partial inhibitors. • Generalizable to other extracellular glycoproteins and ligandable protein-protein interactions (PPIs).