First-in-Class Small Molecule ROBO2 Binders Identified through Integrated Virtual Screening and Biophysical...

Area of Science:

• Oncology
• Molecular Biology
• Drug Discovery

Background:

• Roundabout homolog 2 (ROBO2) is a transmembrane receptor involved in glioblastoma (GBM) progression.
• Dysregulated Slit2-ROBO2 signaling promotes tumor cell migration, invasion, and immunosuppression in GBM.
• ROBO2 represents a potential therapeutic target, but no specific small molecule inhibitors have been identified.

Purpose of the Study:

• To identify and validate small molecules that bind to ROBO2 for potential glioblastoma therapy.
• To establish a screening pipeline for discovering ROBO2-targeted therapeutics.

Main Methods:

• Structure-based virtual screening of compounds targeting ROBO2.
• Experimental validation using Dianthus TRIC platform and microscale thermophoresis (MST).
• Molecular docking to analyze binding interactions within the ROBO2 pocket.

Main Results:

• Fifteen compounds were screened, with four showing ROBO2 binding.
• Two small molecules, Z1334432986 and Z1692774161, demonstrated reproducible binding to ROBO2.
• Dissociation constants (Kd) were determined, with Z1692774161 showing higher affinity (25.8±16.95 μM).
• Molecular docking revealed a conserved binding pocket with specific anchor residues.

Conclusions:

• This study successfully identified two novel small molecules targeting ROBO2.
• A robust screening pipeline for ROBO2-targeted inhibitors was established.
• These findings provide a foundation for developing new GBM therapeutics that disrupt Slit-ROBO2 signaling.