Effective cell-free drug screening protocol for protein-protein interaction

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions.
• Targeting PPIs with small molecules is difficult due to complex interfaces, necessitating large-scale screening.
• Dysregulation of key PPIs, like NF-κB, is implicated in numerous diseases.

Purpose of the Study:

• To develop a simplified, cost-effective, and robust protocol for screening direct PPI inhibitors.
• To enable high-throughput screening of large compound libraries for PPI modulators.
• To validate the protocol's efficacy using the NF-κB signaling pathway.

Main Methods:

• Engineered a bacterial expression system for producing split-Renilla luciferase (RL) fusion proteins.
• Utilized a split-RL complementation assay to monitor PPI in vitro.
• Validated compound efficacy in mammalian cells using the same complementation assay to ensure cell permeability.

Main Results:

• Demonstrated efficient and cost-effective production of RL fusion proteins for large-scale screening.
• Successfully applied the assay to identify direct inhibitors of NF-κB, a critical transcription factor.
• The protocol proved straightforward and robust for identifying PPI inhibitors.

Conclusions:

• The developed split-RL complementation assay provides a powerful tool for discovering direct PPI inhibitors.
• This method is suitable for screening numerous protein pairs and can be adapted for various research and therapeutic applications.
• The protocol facilitates the identification of small molecules targeting challenging protein-protein interactions.