Discovery of a First-in-Class SLIT2 Binder Disrupting the SLIT2/ROBO1 Axis via DNA-Encoded Library (DEL) Screening

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 IC ₅₀ 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.