Optimization and Evaluation of Complementary Degrader Discovery Assays for Application in Screening

Area of Science:

• Biochemistry
• Drug Discovery
• Molecular Biology

Background:

• Targeted protein degradation (TPD) using molecular glues is a promising pharmaceutical strategy.
• Molecular glues modulate E3 ligase interactions to induce degradation of specific target proteins.
• Developing rational discovery strategies for molecular glues presents challenges, requiring robust validation to exclude nonspecific effects.

Purpose of the Study:

• To optimize and compare two cell-based assays for molecular glue degrader discovery.
• To evaluate the strengths and limitations of signal inhibition and signal rescue assay formats.
• To guide screening efforts by identifying reliable methods for hit validation.

Main Methods:

• Development of two orthogonal cell-based, target-centric assays: time-resolved FRET (signal inhibition) and TPD-coupled cell growth (signal rescue).
• Screening of approximately 1000 compounds, including reference compounds and known frequent hitters (FHs).
• Comparative analysis of statistical performance and hit populations between the two assay formats.

Main Results:

• The signal rescue assay format demonstrated reliable and specific capture of active target degraders.
• The signal rescue assay efficiently filtered out interfering compounds and FHs.
• This format successfully identified lower potency hits, expanding chemical starting points for drug discovery.

Conclusions:

• The signal rescue assay is a robust and specific method for identifying molecular glue degraders.
• This assay format offers advantages in hit validation and broadening chemical diversity in early drug discovery.
• Optimized assays are crucial for guiding screening efforts in targeted protein degradation drug discovery.