High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds using HiBiT CRISPR Cell Lines

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Targeted protein degradation (TPD) compounds, such as molecular glues and proteolysis targeting chimeras (PROTACs), represent a novel therapeutic strategy.
• These compounds harness the cell's ubiquitin-proteasomal pathway (UPP) to degrade specific proteins.
• High-throughput profiling of TPD compounds is challenging due to the complexity of cellular degradation pathways.

Purpose of the Study:

• To develop a robust and scalable protocol for screening TPD compounds.
• To enable real-time and kinetic monitoring of target protein degradation.
• To facilitate the discovery and optimization of small molecule therapeutics.

Main Methods:

• Utilized CRISPR/Cas9 for endogenous tagging of target proteins with the HiBiT tag.
• Developed cell lines with luminescent protein reporters for monitoring degradation.
• Employed luminescent plate-based readers for quantitative signal detection.
• Established protocols for live-cell kinetic and endpoint lytic assays.
• Integrated calculation of key degradation parameters (rate, Dmax, DC50, Dmax50) and multiplexing with viability assays.

Main Results:

• Demonstrated a reliable method for measuring compound-induced protein degradation.
• Enabled real-time kinetic and endpoint measurements of degradation.
• Facilitated rapid discovery and triaging of early-stage TPD compounds.
• Maintained endogenous protein expression and regulation within relevant cellular contexts.

Conclusions:

• CRISPR/Cas9-based HiBiT tagging provides an efficient platform for TPD compound screening.
• This approach supports the optimization of lead therapeutic compounds by maintaining physiological relevance.
• The developed protocol accelerates the drug discovery pipeline for TPD therapeutics.