Detecting ligand-protein interactions inside cells using reactive peptide tags and split luciferase

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Understanding ligand-protein interactions is crucial for drug discovery and cellular pathway analysis.
• Existing methods for in-cell interaction detection have limitations, especially for unstable ligands.

Purpose of the Study:

• To develop a novel method for detecting and quantifying ligand-protein interactions within living cells.
• To enable the estimation of binding affinities for these interactions.
• To facilitate the study of interactions involving unstable synthetic ligands.

Main Methods:

• Utilizing short peptide reactive tags appended to both ligands and proteins.
• Employing a split NanoLuc luciferase complementation assay.
• Implementing the method for in-cell detection and affinity estimation.

Main Results:

• Successfully detected ligand-protein interactions occurring inside cells.
• Quantified binding affinities of ligand-protein interactions.
• Demonstrated the ability to detect interactions with unstable synthetic ligands.

Conclusions:

• The developed method provides a robust platform for studying intracellular ligand-protein interactions.
• This technique enhances the ability to assess drug-target engagement and molecular interactions in a cellular context.
• It offers a valuable tool for drug discovery and chemical biology research.