The cellular thermal shift assay for evaluating drug target interactions in cells

Area of Science:

• Biochemistry
• Pharmacology
• Molecular Biology

Background:

• Thermal shift assays traditionally analyze purified proteins to detect ligand interactions.
• Drug discovery requires methods to assess target engagement within a cellular environment.

Purpose of the Study:

• To introduce and validate the cellular thermal shift assay (CETSA) for studying protein stabilization in cells.
• To demonstrate CETSA's application in assessing drug candidate target engagement using human kinases p38α and ERK1/2.

Main Methods:

• Cells are treated with compounds, heated to induce protein denaturation, lysed, and fractionated.
• Ligand-bound proteins remain soluble, while unbound proteins precipitate.
• Detection of soluble, stabilized proteins is achieved via quantitative western blotting or a high-throughput proximity-based assay.

Main Results:

• The cellular thermal shift assay (CETSA) successfully detects ligand-induced thermal stabilization of proteins in a cellular context.
• Two distinct detection protocols (western blotting and proximity-based assay) were established and validated.
• The proximity-based assay offers increased throughput for large-scale sample analysis.

Conclusions:

• CETSA provides a robust method for studying drug target engagement within intact cells.
• The assay is adaptable for both academic research and drug discovery applications.
• Both described CETSA detection methods can be completed within a single day.