Temperature-related intensity change (TRIC)-based high-throughput screening enables the discovery of small molecule CD28 binders.

CD28 is a pivotal costimulatory receptor involved in T cell activation and immune regulation, positioning it as a key therapeutic target for inflammatory diseases, including inflammatory bowel disease (IBD). Despite its potential, small molecules targeting CD28 are still limited. To fill this gap, we developed a high-throughput screening (HTS) platform based on Temperature-Related Intensity Change (TRIC) technology, enabling rapid, immobilization-free screening of chemical libraries of small molecules. Using the Dianthus instrument, we applied our optimized TRIC assay for CD28 (signal-to-noise ratio of 21.99) to screen two MedChemExpress libraries: Small Molecule Immuno-Oncology Compounds (SMIOC) and Protein-Protein Interaction Inhibitors (PPII), identifying 50 initial hits. Following exclusion of compounds with dye interference or aggregation artifacts, 12 candidates were prioritized for further validation. Microscale thermophoresis (MST) confirmed dose-dependent binding of seven compounds to CD28, with affinities in the micromolar range. Surface plasmon resonance (SPR) further validated two compounds, EABP 02,303 and CTEP, as CD28 binders. These results demonstrate that our TRIC-based HTS platform is robust, scalable, and effective for identifying small molecule CD28 binders. The incorporation of orthogonal validation supports the reliability of our findings and highlights the feasibility of small-molecule discovery targeting CD28.