The Development of Cereblon Recruiting BromoTag Degraders for Targeted Protein Degradation

Abstract

Inducible degron systems are powerful technologies that induce the degradation of genetically tagged target proteins to allow the study of fundamental biology. Recently, a ‘bump-&-hole’ strategy, where a site-specific mutation induces a ‘hole’ in which an allele-specific probe possessing a ‘bump’ moiety can be selectively incorporated, has been employed to develop the novel degron system BromoTag. BromoTag utilises the heterobifunctional ‘bumped’ PROTAC AGB1 to hijack the von Hippel-Lindau E3 ligase and trigger the selective degradation of target proteins fused with mutant BRD4BD2(L387A) degron tags. As part of this research, we aimed to further expand the BromoTag toolkit via the incorporation of a corresponding Cereblon (CRBN)-recruiting BromoTag degrader.

We sought to design and synthesise a small series of CRBN-recruiting BromoTag degraders and assess their on-target potency and selectivity against the wildtype Bromodomain and Extra-Terminal (BET) proteins. The modification of two existing CRBN-recruiting PROTAC scaffolds, capable of potently degrading BET proteins, dBET6 and AB3067, led to the design and synthesis of a small library of five CRBN-recruiting BromoTag degraders. The potency and selectivity of these compounds was evaluated using Promega’s HiBiT lytic bioluminescence assay in tandem with HiBiT-tagged BromoTag A549 cell lines and HiBiT-tagged BET protein HEK293 cell lines. All compounds were found to induce on-target degradation of BromoTagged target proteins, however, selectivity against the endogenous BET proteins was variable, with prominent degradation of protein BRD3 being consistently observed. Compound 35 (ALH55) and 36 (ALH54) were identified as the most promising degraders, capable of potently degrading BromoTagged-BRD4 as a proof-of-concept target, and achieving high levels of degradation, despite also engaging in isoform-specific degradation of BRD3.

We observed that the amide to ester substitution and pomalidomide conjugated to a linker in the 4’ position with a 5’ fluorine atom was favourable for increased cellular potency and degradation of the BromoTagged target protein.

Date of Award	2023
Original language	English
Awarding Institution	University of Dundee
Supervisor	Alessio Ciulli (Supervisor) & William Farnaby (Supervisor)