Yuan Shaoren, Abdel-Rahman Somaya A, Vázquez Nelson García, Nada Hossam, Calvo-Barreiro Laura, Kuncewicz Katarzyna, Gabr Moustafa T
bioRxiv. 2025 Jun 22:2025.06.22.660907. doi: 10.1101/2025.06.22.660907.
The SLIT2/ROBO1 signaling axis plays a critical role in neural development, immune regulation, and tumor progression, including glioblastoma. However, small molecule inhibitors targeting this protein-protein interaction remain unexplored. Herein, we report the discovery and validation of , a first-in-class small molecule that binds to SLIT2 and disrupts its interaction with ROBO1. Using a DNA-encoded library (DEL) screen of 4.2 billion compounds, was identified and confirmed to bind SLIT2 via temperature-related intensity change (TRIC) assay. Functional inhibition of the SLIT2/ROBO1 complex by was demonstrated using a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, yielding an IC of 68.8 ± 12.5 µM. Molecular docking and molecular dynamics (MD) simulations revealed key interaction hotspots at the SLIT2 binding interface and confirmed that impairs SLIT2/ROBO1 complex formation by inducing conformational rearrangements. exhibited favorable ADME properties, including satisfactory plasma and microsomal stability, low cytotoxicity, and minimal hERG liability. To facilitate structure-activity relationship (SAR) exploration, we designed and implemented a modular, one-pot synthetic route leveraging cyanuric chloride reactivity, enabling rapid derivatization of the triazine scaffold of . This strategy yielded structurally diverse analogs, including water-soluble carboxylate derivatives with preserved SLIT2/ROBO1 inhibitory activity. Together, this work establishes a novel chemical scaffold targeting SLIT2 and introduces a flexible synthetic platform to support further optimization toward therapeutic development.
SLIT2/ROBO1信号轴在神经发育、免疫调节以及包括胶质母细胞瘤在内的肿瘤进展中起着关键作用。然而，针对这种蛋白质-蛋白质相互作用的小分子抑制剂仍未得到探索。在此，我们报告了一种一流的小分子的发现与验证，该小分子与SLIT2结合并破坏其与ROBO1的相互作用。通过对42亿种化合物进行DNA编码文库（DEL）筛选，鉴定出该小分子，并通过温度相关强度变化（TRIC）分析证实其与SLIT2结合。使用时间分辨荧光共振能量转移（TR-FRET）分析证明该小分子对SLIT2/ROBO1复合物具有功能抑制作用，其IC50为68.8±12.5μM。分子对接和分子动力学（MD）模拟揭示了SLIT2结合界面的关键相互作用热点，并证实该小分子通过诱导构象重排损害SLIT2/ROBO1复合物的形成。该小分子具有良好的吸收、分布、代谢和排泄（ADME）特性，包括令人满意的血浆和微粒体稳定性、低细胞毒性以及最小的人醚-去极化激活的钾离子通道（hERG）风险。为了便于探索构效关系（SAR），我们设计并实施了一种利用三聚氯氰反应性的模块化一锅合成路线，能够对该小分子的三嗪支架进行快速衍生化。该策略产生了结构多样的类似物，包括具有保留的SLIT2/ROBO1抑制活性的水溶性羧酸盐衍生物。总之，这项工作建立了一种靶向SLIT2的新型化学支架，并引入了一个灵活的合成平台，以支持进一步优化用于治疗开发。
