In the rapidly evolving field of drug discovery, one of the most significant challenges is targeting proteins that have long been deemed "undruggable." To overcome this, researchers have been focusing on alternative strategies, including the modulation of RNA splicing. At Evotec, we are leveraging cutting-edge high-throughput RT-qPCR technology to accelerate the identification and development of small molecule RNA splicing modulators.

RNA splicing is a crucial step in gene expression where introns are removed and exons are stitched together to form mature messenger RNA (mRNA). By manipulating this process, researchers can generate alternatively spliced RNA isoforms that either produce truncated, non-functional proteins or induce RNA degradation via the nonsense-mediated mRNA decay (NMD) pathway. This approach offers new opportunities to target previously inaccessible disease-related proteins.

To facilitate the discovery of RNA splicing modulators, Evotec has developed a fully automated, multiplex RT-qPCR screening platform with the capability to:

• Process up to 15,000 compounds per day using a miniaturized 384-well plate format.
• Analyze multiple gene targets within a single well, significantly increasing efficiency.
• Utilize advanced cell models, including patient-derived cells, primary cells, induced pluripotent stem cells (iPSCs), and organoids.
• Provide a cost-effective alternative to traditional reporter-gene assays.

The RT-qPCR platform is integrated with Genedata Screener software, which enables:

• Rapid characterization of extensive compound libraries.
• Identification of splicing modulators while filtering out cytotoxic compounds.
• Precise estimation of compound activity through changes in concentration-response curves.

This advanced computational approach ensures that the most promising compounds are quickly identified and prioritized for further development.

RNA-based screening is only the first step. Once gene expression hits are identified, their biological relevance is confirmed through secondary protein-based assays, including:

• Homogeneous Time-Resolved Fluorescence (HTRF) assays.
• HiBit tagging systems.
• Flow cytometry-based approaches.

These additional validation steps help confirm that small molecule modulators effectively influence protein levels, paving the way for lead optimization.

Evotec’s high-throughput RT-qPCR platform represents a powerful tool for drug discovery, offering a robust and scalable approach to modulating RNA splicing. By combining automation, advanced data analysis, and secondary protein assays, we are accelerating the development of novel therapeutics aimed at previously ‘undruggable’ disease mechanisms.

Stay tuned for further breakthroughs in RNA-targeted therapeutics!