High-Throughput Detection of Ligand-Protein Binding Using a SplitLuc Cellular Thermal Shift Assay

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Validating small molecule-protein binding in cellular models is challenging.
• Traditional cellular thermal shift assay (CETSA) methods are time-consuming and low-throughput.
• Need for efficient assays to assess target engagement in a cellular context.

Purpose of the Study:

• To develop and present a high-throughput method for target engagement assays.
• To adapt the cellular thermal shift assay (CETSA) for increased screening capacity.
• To enable efficient validation of small molecule-protein interactions in a cellular environment.

Main Methods:

• Implementation of split Nano Luciferase (SplitLuc) as a reporter system.
• Adaptation of CETSA principles for a reporter-based assay.
• Development of protocols for 384- and 1536-well plate formats.

Main Results:

• SplitLuc CETSA provides a quantitative measure of target engagement.
• The assay is amenable to high-throughput screening of compounds and concentrations.
• Demonstrates a shift in protein melting profiles upon small molecule binding.

Conclusions:

• SplitLuc CETSA is a powerful tool for drug discovery and target validation.
• This reporter-based assay significantly improves the efficiency of cellular target engagement screening.
• Facilitates the transition from biochemical assays to physiologically relevant cellular models.