High-Throughput Quantification of Surface Protein Internalization and Degradation

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Cell surface proteins regulate cellular processes and are implicated in diseases.
• Targeted drugs often act on cell surface proteins, with internalization and degradation properties gaining focus.
• High-throughput methods are needed to assess drug effects on cell surface proteins.

Purpose of the Study:

• To develop a generic, high-throughput assay for quantifying cell surface protein internalization and degradation.
• To enable simultaneous and independent measurement on a single-cell level.
• To facilitate drug discovery and development targeting cell surface proteins.

Main Methods:

• A generic high-throughput assay using HaloTag fusion proteins and differential fluorescent ligand permeability.
• Homogeneous assay performed with adherent live cells in a 96-well format.
• Channel rescaling for accurate quantification of surface and internal protein levels.

Main Results:

• Simultaneous and independent quantification of internalization and degradation of surface proteins.
• Applicability demonstrated on EGFR, HER2, and EpCAM using various small molecule ligands and protein affinity reagents.
• No labeling of screened molecules required, simplifying the process.

Conclusions:

• The developed assay provides a robust and versatile tool for high-throughput screening of drug candidates targeting cell surface proteins.
• This method aids in understanding drug pharmacokinetics, pharmacodynamics, and potential resistance mechanisms.
• The assay is applicable to various drug modalities and major cancer targets.