Quantitative measurement of PROTAC intracellular accumulation

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that hijack the cell's ubiquitin-proteasome system for targeted protein degradation.
• Successful PROTAC efficacy relies on intracellular accumulation, target engagement, and formation of a ternary complex with the protein of interest (POI) and an E3 ligase.
• PROTACs can be non-covalent or covalent (irreversible/reversible), necessitating distinct evaluation methods.

Purpose of the Study:

• To develop and validate a high-throughput assay for prioritizing different types of Bruton's tyrosine kinase (BTK) PROTACs.
• To quantitatively assess intracellular accumulation and target engagement of PROTAC candidates.
• To enable efficient evaluation of PROTAC efficacy for drug discovery.

Main Methods:

• Utilized a high-throughput screening approach to analyze PROTAC candidates.
• Employed quantitative measurement of intracellular PROTAC accumulation.
• Incorporated kinase binding assays and the NanoBRET target engagement platform for comprehensive evaluation.

Main Results:

• Successfully established a high-throughput assay for prioritizing BTK PROTACs.
• Demonstrated quantitative measurement of intracellular PROTAC levels.
• Validated the assay's ability to assess target engagement using NanoBRET.

Conclusions:

• The developed assay provides a robust method for evaluating and prioritizing PROTAC candidates based on intracellular behavior and target engagement.
• This platform facilitates efficient drug discovery by enabling rapid assessment of PROTAC efficacy.
• The assay is applicable to various PROTAC types, including non-covalent and covalent inhibitors.