Fluorescence-Coupled Ubiquitination Assay as a High-Throughput Screening Strategy for Novel Cereblon Degraders

Cereblon
(CRBN)-based protein degradation, via molecular glue degraders
(MGDs) and proteolysis-targeting chimeras (PROTACs), is a promising
cancer treatment strategy in targeted protein degradation (TPD). However,
novel degraders discovery remains limited due to the lack of robust,
high-throughput screening (HTS) methods for processing pools of purified
compounds or complex chemical synthesis mixtures. Here, we introduce
an innovative HTS strategy that employs a highly sensitive, fluorescence-coupled
ubiquitination assay to identify CRBN-based degraders. This approach
tracks ubiquitinated target proteins via gel-based analyses, and thereby
progressively narrows down the list of potential degrader molecules
from large-scale compound libraries or chemical reaction mixtures.
Using this strategy, we identified LL-BPTF-8, a promising lead compound
of PROTAC degrader with high potency and selectivity that targets
the bromodomain PHD finger transcription factor (BPTF). Overall, our
method offers a low-cost, rapid, and versatile platform for the HTS
of protein degrader candidates, significantly streamlining the discovery
of novel degraders.