A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators.

Area of Science:

• Molecular Biology
• Genetics
• Pharmacology

Background:

• Alternative splicing is a key regulator of gene function and a therapeutic target for human diseases.
• Current methods for identifying splicing-modulating compounds are limited, and the splicing effects of existing drugs are largely unknown.

Purpose of the Study:

• To develop a high-throughput screening assay for cellular exon inclusion.
• To identify known bioactive compounds that modulate alternative splicing, specifically targeting MAPT exon 10.

Main Methods:

• Development of a two-color fluorescent reporter assay for exon inclusion.
• Screening of known bioactive compound libraries using the reporter assay.
• Validation of hits in endogenous MAPT mRNA splicing.
• Microarray analysis to assess global splicing changes.

Main Results:

• A novel reporter assay was established, accommodating various cassette exons and minimizing transcriptional/translational artifacts.
• Screening identified compounds that alter MAPT exon 10 splicing in both reporter and endogenous contexts.
• Digoxin, a cardiac drug, was identified as a novel splicing modulator.
• Positive compounds affected distinct signal transduction pathways and modulated diverse sets of exons.

Conclusions:

• Currently prescribed cardiotonic steroids modulate alternative splicing.
• The developed reporter assay is effective for screening drugs that alter exon inclusion.
• This work opens new possibilities for drug repurposing and the discovery of novel splicing-targeting therapeutics.