Calvo-Barreiro Laura, Upadhyay Saurabh, Gabr Moustafa T
Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, NY, NY 10065, USA.
Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, NY, NY 10065, USA.
SLAS Discov. 2025 Sep;35:100256. doi: 10.1016/j.slasd.2025.100256. Epub 2025 Aug 5.
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.
CD28是一种关键的共刺激受体，参与T细胞活化和免疫调节，使其成为包括炎症性肠病（IBD）在内的炎症性疾病的关键治疗靶点。尽管其具有潜力，但靶向CD28的小分子仍然有限。为了填补这一空白，我们基于温度相关强度变化（TRIC）技术开发了一个高通量筛选（HTS）平台，能够对小分子化学文库进行快速、无需固定化的筛选。使用石竹仪器，我们将优化后的用于CD28的TRIC检测方法（信噪比为21.99）应用于筛选两个MedChemExpress文库：小分子免疫肿瘤化合物（SMIOC）和蛋白质-蛋白质相互作用抑制剂（PPII），确定了50个初始命中化合物。在排除具有染料干扰或聚集假象的化合物后，优先选择12个候选化合物进行进一步验证。微量热泳动（MST）证实了7种化合物与CD28的剂量依赖性结合，亲和力在微摩尔范围内。表面等离子体共振（SPR）进一步验证了两种化合物EABP 02,303和CTEP为CD28结合剂。这些结果表明，我们基于TRIC的HTS平台对于识别小分子CD28结合剂是强大、可扩展且有效的。正交验证的纳入支持了我们研究结果的可靠性，并突出了靶向CD28的小分子发现的可行性。
