A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Regulators of G-protein signaling (RGS) proteins are crucial in cellular signaling pathways.
• Identifying small molecule modulators of RGS proteins is vital for understanding their biology and for drug discovery.
• Existing methods for screening protein-protein interactions can be limited in speed and efficiency.

Purpose of the Study:

• To develop an advanced yeast 2-hybrid (YTH) assay for studying RGS proteins, specifically GalphaZ and RGS-Z1.
• To create a multiplexed luciferase-based assay for high-throughput screening of small molecule modulators of protein-protein interactions.

Main Methods:

• Developed and evaluated two distinct luciferase reporter gene systems (firefly and Renilla) for YTH interactions.
• Adapted a Gal4-responsive luciferase reporter system for YTH assays.
• Implemented a multiplexed assay by leveraging the different substrate specificities of firefly and Renilla luciferase.

Main Results:

• Both firefly and Renilla luciferase reporter genes showed a significant 40- to 50-fold increase in luminescence for interacting YTH fusion proteins compared to controls.
• The multiplexed assay demonstrated enhanced speed, sensitivity, simplicity, quantification, and efficiency for YTH applications.

Conclusions:

• The multiplexed luciferase-based YTH platform significantly enhances the efficiency and throughput of identifying small molecule modulators of protein-protein interactions.
• This advanced YTH assay serves as a valuable tool for drug discovery efforts, facilitating the identification of potential therapeutic leads.