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

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery Pub Date : 2025-05-18 DOI:10.1016/j.slasd.2025.100240

Somaya A. Abdel-Rahman , Moustafa T. Gabr

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引用次数: 0

Abstract

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.

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SLIT2/ROBO1相互作用高通量TR-FRET筛选方法的优化与开发

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，有助于肿瘤的进展、转移和治疗抵抗。SLIT2在各种恶性肿瘤中高表达，通过募集肿瘤相关巨噬细胞和破坏血管完整性来促进免疫逃避，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，使其成为一个潜在但尚未被充分开发的治疗靶点。然而，目前还没有SLIT2/ROBO1相互作用的小分子抑制剂存在。在此，我们描述了一种时间分辨荧光共振能量转移（TR-FRET）试验的开发和优化，用于高通量筛选靶向该途径的小分子抑制剂。利用重组SLIT2和ROBO1，我们建立了一种强大的检测方法，可以高通量筛选SLIT2/ROBO1抑制小分子化学文库。筛选蛋白质-蛋白质相互作用（PPI）抑制剂的重点化学文库，发现SMIFH2是SLIT2/ROBO1抑制剂，证明其能够以剂量依赖的方式破坏相互作用。我们的研究为SLIT2/ROBO1小分子抑制剂的鉴定提供了一个新的筛选平台，为未来针对该信号轴在癌症和其他疾病中的药物发现奠定了基础。

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来源期刊

SLAS Discovery Chemistry-Analytical Chemistry

CiteScore

7.00

自引率

3.20%

发文量

审稿时长

39 days

期刊介绍： Advancing Life Sciences R&D: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease. SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success. SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies. SLAS Discovery is of particular interest to those involved in analytical chemistry, applied microbiology, automation, biochemistry, bioengineering, biomedical optics, biotechnology, bioinformatics, cell biology, DNA science and technology, genetics, information technology, medicinal chemistry, molecular biology, natural products chemistry, organic chemistry, pharmacology, spectroscopy, and toxicology. SLAS Discovery is a member of the Committee on Publication Ethics (COPE) and was published previously (1996-2016) as the Journal of Biomolecular Screening (JBS).