HiBiT-RR and nLuc^K0 Tagging Systems for Monitoring Targeted Protein Degradation

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Targeted protein degradation (TPD) is a key strategy in modern drug discovery, particularly for developing Proteolysis-Targeting Chimeras (PROTACs).
• Accurate measurement of target protein degradation is crucial for TPD drug development.
• NanoLuciferase (nLuc) and split-nLuc systems are sensitive reporters but can be subject to artifactual degradation due to lysine residues.

Purpose of the Study:

• To develop and validate novel reporter tags that mitigate artifactual degradation in TPD assays.
• To introduce lysine-free variants of nLuc tags, HiBiT-RR and nLuc^K0, for more reliable TPD screening.

Main Methods:

• Generation of lysine-free reporter tags (HiBiT-RR and nLuc^K0) by replacing all lysine residues with arginine.
• Implementation of these tags in cellular degradation assays using both transiently and endogenously tagged proteins.
• Validation of degradation events using immunoblotting techniques.

Main Results:

• Lysine-free HiBiT-RR and nLuc^K0 tags effectively prevent tag-driven degradation artifacts.
• These modified tags enable more accurate and sensitive measurement of target protein degradation in cellular assays.
• The protocols are applicable to various expression systems, including endogenously tagged proteins.

Conclusions:

• Lysine-free reporter tags significantly improve the reliability of TPD screening assays.
• HiBiT-RR and nLuc^K0 are valuable tools for advancing drug discovery efforts utilizing targeted protein degradation.
• These optimized reporters reduce the risk of false positives, leading to more robust TPD candidate identification.