Abdel-Rahman Somaya A, Gabr Moustafa T
Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, NY, NY 10065, USA; Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.
Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, NY, NY 10065, USA.
SLAS Discov. 2025 Jul;34:100240. doi: 10.1016/j.slasd.2025.100240. Epub 2025 May 18.
The SLIT2/ROBO1 signaling axis plays a critical role in cell migration, angiogenesis, and immune regulation, contributing to tumor progression, metastasis, and therapy resistance. SLIT2 is highly expressed in various malignancies, where it promotes immune evasion by recruiting tumor-associated macrophages and disrupting vascular integrity, ultimately diminishing therapeutic efficacy. Beyond cancer, SLIT2/ROBO1 is implicated in neural development, fibrosis, and vascular remodeling, making it a potential but underexplored therapeutic target. However, no small-molecule inhibitors of SLIT2/ROBO1 interaction currently exist. Herein, we describe the development and optimization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for high-throughput screening of small-molecule inhibitors targeting this pathway. Using recombinant SLIT2 and ROBO1, we established a robust assay that enables high-throughput screening (HTS) of chemical libraries of small molecules for SLIT2/ROBO1 inhibition. Screening a focused chemical library of protein-protein interaction (PPI) inhibitors identified SMIFH2 as a SLIT2/ROBO1 inhibitor, demonstrating its ability to disrupt the interaction in a dose-dependent manner. Our study introduces a novel screening platform for identifying small molecule inhibitors of SLIT2/ROBO1, laying the foundation for future drug discovery efforts aimed at targeting this signaling axis in cancer and other diseases.
SLIT2/ROBO1信号轴在细胞迁移、血管生成和免疫调节中起关键作用，促进肿瘤进展、转移和治疗抵抗。SLIT2在各种恶性肿瘤中高表达，通过招募肿瘤相关巨噬细胞和破坏血管完整性促进免疫逃逸，最终降低治疗效果。除癌症外，SLIT2/ROBO1还与神经发育、纤维化和血管重塑有关，使其成为一个有潜力但未被充分探索的治疗靶点。然而，目前尚无针对SLIT2/ROBO1相互作用的小分子抑制剂。在此，我们描述了一种用于高通量筛选靶向该通路的小分子抑制剂的时间分辨荧光共振能量转移（TR-FRET）分析方法的开发和优化。利用重组SLIT2和ROBO1，我们建立了一种强大的分析方法，能够对小分子化学文库进行高通量筛选以检测SLIT2/ROBO1抑制作用。筛选一个聚焦的蛋白质-蛋白质相互作用（PPI）抑制剂化学文库，确定SMIFH2为SLIT2/ROBO1抑制剂，证明其能够以剂量依赖方式破坏这种相互作用。我们的研究引入了一种用于鉴定SLIT2/ROBO1小分子抑制剂的新型筛选平台，为未来旨在靶向癌症和其他疾病中该信号轴的药物发现工作奠定了基础。
