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

Abstract

Targeted protein degradation (TPD) mediated by molecular glues is an innovative pharmaceutical paradigm. By binding to and modulating the surface of an E3-ligase component, molecular glue degraders can facilitate the recruitment of a specific target protein (or vice versa) and ultimately invoke target degradation. This mode of action results in specific challenges for the development of rational discovery strategies, and complex hit validation workflows may be required to reliably eliminate compounds that elicit nonspecific effects. With the aim to guide screening efforts, we optimized two orthogonal cell-based, target-centric assays for degrader discovery: (1) a time-resolved FRET assay directly quantifying the levels of a target protein and its degradation (signal inhibition) and (2) an assay coupling TPD to cell growth (signal rescue). To enable a deeper understanding of the individual assays’ strengths and limitations, we compared their statistical performance as well as respective hit populations by screening a specifically designed collection of about 1000 compounds containing well-annotated reference compounds and known frequent hitters (FHs). We found that the signal rescue format reliably and specifically captured active target degraders while efficiently filtering out interfering or FH compounds. Importantly, this format achieved to retrieve lower potency hits, which might be desirable in order to confidently include as many diverse chemical starting points as possible at the start of a drug discovery project.