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

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.