Discovery of a first-in-class SLIT2 binder disrupting the SLIT2/ROBO1 axis

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 DEL-S1, 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, DEL-S1 was identified and confirmed to bind SLIT2 via temperature-related intensity change (TRIC) assay. Functional inhibition of the SLIT2/ROBO1 complex by DEL-S1 was demonstrated using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, yielding an IC50 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 DEL-S1 impairs SLIT2/ROBO1 complex formation by inducing conformational rearrangements. DEL-S1 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 DEL-S1. 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.