Development of a Cell-Based Assay Using a Split-Luciferase Reporter for Compound Screening

Area of Science:

• Molecular Biology
• Cancer Therapeutics
• Drug Discovery

Background:

• Recurrent mutations in spliceosome components highlight the spliceosome as a cancer therapy target.
• Limited small molecules targeting the spliceosome are available due to the lack of robust screening methods.
• Previous work established a split luciferase reporter for detecting spliceosome subunit levels (small nuclear ribonucleoproteins, snRNPs).

Purpose of the Study:

• To develop a robust and sensitive cell-based assay for screening small molecules that target the spliceosome.
• To improve upon a previously developed genetic reporter system for enhanced compound screening capabilities.

Main Methods:

• Utilized a split luciferase genetic reporter system to monitor cellular levels of small nuclear ribonucleoproteins (snRNPs).
• Optimized the assay by incorporating cell lysis buffer from blue native polyacrylamide gel electrophoresis (BN-PAGE) to enhance sensitivity and robustness.
• Applied the improved assay conditions for high-throughput screening to identify bioactive small molecules.

Main Results:

• The use of BN-PAGE cell lysis buffer significantly improved the sensitivity and robustness of the snRNP detection assay.
• The optimized assay successfully identified a small molecule capable of altering the reporter activity, indicating spliceosome modulation.
• The developed method demonstrates potential for screening against other cellular macromolecular complexes.

Conclusions:

• The enhanced cell-based assay provides a powerful tool for discovering small molecules that target the spliceosome.
• This improved screening approach facilitates the identification of novel cancer therapeutics by targeting spliceosome machinery.
• The methodology is adaptable for discovering bioactive molecules targeting various cellular complexes.