Identification of Selective Small Molecule Modulators of mRNA Processing Using a Multiplexed QuantiGene...

Area of Science:

• Molecular Biology
• Drug Discovery
• Assay Development

Background:

• High-throughput screening (HTS) with multiplexed, cell-based assays is crucial for identifying modulators of biological processes.
• Discovering small molecules that modulate mRNA processing requires sensitive and specific screening platforms.

Purpose of the Study:

• To develop a multiplexed, bead-based HTS assay for quantifying multiple target gene transcripts simultaneously.
• To identify selective small molecule modulators of mRNA processing using a novel QuantiGene assay on the Luminex platform.

Main Methods:

• Developed a multiplexed, bead-based QuantiGene assay on the Luminex platform for simultaneous gene transcript quantification.
• Implemented a dual-normalization hit-calling pipeline (plate- and well-based) to mitigate screening artifacts and plate variability.
• Validated hits using an orthogonal, four-point concentration-response real-time PCR (qPCR) assay in a disease-relevant cell line.

Main Results:

• The assay successfully identified unique compounds selectively modulating individual target genes involved in mRNA processing.
• A dual-normalization strategy reduced false negatives and improved hit confirmation rates.
• Chemical motif analysis revealed known and novel scaffolds enriched for specific targets, with only 5% of actives showing broad activity across three targets.

Conclusions:

• The developed multiplexed HTS platform is a scalable and reliable strategy for identifying selective small molecule modulators of mRNA processing.
• The assay demonstrates robustness and translational relevance, applicable to early drug discovery.
• The findings highlight the platform's capacity for detecting highly selective modulators of complex biological pathways.