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Published on: December 1, 2020
First-in-Class Small Molecule ROBO2 Binders Identified through Integrated Virtual Screening and Biophysical
Abstract:
Roundabout homolog 2 (ROBO2) is a transmembrane receptor implicated in glioblastoma progression through its interaction with Slit2-mediated signaling pathways. Dysregulated Slit2-ROBO2 signaling enhances tumor cell migration, invasion, and tissue infiltration, while elevated ROBO2 levels contribute to an immunosuppressive tumor microenvironment supporting GBM aggressiveness and highlighting ROBO2 as a therapeutic target. Despite its therapeutic relevance, no ROBO2-targeted small molecules have been reported. To address this gap, we performed a structure-based virtual screening campaign targeting ROBO2, followed by experimental validation with Dianthus TRIC platform and microscale thermophoresis (MST). Fifteen compounds were screened for ROBO2 binding, from which four candidates exhibited robust TRIC signals. Subsequent affinity measurements revealed that two small molecules, Z1334432986 and Z1692774161, bind ROBO2 in a reproducible concentration-dependent manner, with dissociation constants (Kd) of 40.8±4.8 μM and 25.8±16.95 μM, respectively. Molecular docking with validated hits revealed a shared ROBO2 binding pocket defined by conserved anchor residues (ASN354, SER366, ASP385) and accommodation of distinct ligand conformations within the ROBO2 binding pocket. This work establishes a screening pipeline for identifying ROBO2-targeted small molecules and lays the foundation for developing therapeutics aimed at disrupting Slit-ROBO2 signaling in GBM.
Insights
Researchers identified novel small molecules targeting Roundabout homolog 2 (ROBO2) for glioblastoma treatment. This discovery paves the way for developing new therapies against aggressive brain tumors by disrupting Slit-ROBO2 signaling pathways.
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.
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