Optimization and development of a high-throughput TR-FRET screening assay for SLIT2/ROBO1 interaction

The SLIT2/ROBO1 signaling axis is a key regulator of cell migration, angiogenesis, and immune modulation, and is increasingly recognized for its role in tumor progression, metastasis, and resistance to therapy. Elevated SLIT2 expression in various cancers facilitates immune evasion by recruiting tumor-associated macrophages and compromising vascular integrity, thereby reducing the effectiveness of treatments. Outside of oncology, SLIT2/ROBO1 is also involved in neural development, fibrosis, and vascular remodeling, highlighting its potential as a broad therapeutic target. Despite its significance, no small-molecule inhibitors targeting the SLIT2/ROBO1 interaction have been developed to date. In this study, we report the development and optimization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay designed for high-throughput screening (HTS) of small-molecule inhibitors against this pathway. Using recombinant SLIT2 and ROBO1 proteins, we established a robust, scalable platform suitable for screening chemical libraries. Screening a focused library of protein–protein interaction inhibitors identified SMIFH2 as a dose-dependent inhibitor of SLIT2/ROBO1 binding. This work presents a novel assay system for identifying small-molecule modulators of SLIT2/ROBO1 and provides a foundation for therapeutic discovery targeting this signaling axis in cancer and other pathological conditions.