Abstract

Proteolysis targeting chimera
(PROTAC) have emerged as an attractive new modality in drug discovery with the
potential to deliver the next generation of medicines. Novel, reliable and
quantitative high-throughput protein degradation assays are required for PROTAC
discovery. BRD4 is a chromatin reader protein in the nucleus that recognizes
and binds acetylated histones and plays a key role in epigenetic memory and
transcription regulation. BRD4 plays a critical role in cancer and inflammation
and is the major target of BET inhibitors currently in clinical development.
Here, we demonstrated that HiBiT-technology-based assays could be developed to
study PROTAC-driven BRD4 degradation. The assay was developed in 384 well
format for PROTAC compound screening to deliver DC₅₀, D_max
and hook effect data. Furthermore, we developed a recruitment assay to monitor
formation of the ternary complex between BRD4:PROTAC:E3 ligase, and could
demonstrate a bell-shaped response with formation of this complex at nanomolar
concentration of PROTAC for either Cereblon or VHL and dissociation at higher
concentrations. The assay technology has been extended to multiple targets and
is now routinely deployed in discovery projects.