Development of Next-Generation Protein Degradation Systems

: BromoTag and Other PROTACs

Abstract

Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Presented in this thesis is the design and develop of the BromoTag, a new inducible degron system comprising of a Brd4 bromodomain L387A degron tag that enables direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. I describe a new stereoselective synthesis to achieve allele specific BET inhibitors in >99% ee, which enabled the synthesis of novel “bump-&-hole”-PROTACs as single stereoisomers. Extensive optimization and structure–activity relationships through validation of our “bump-&-hole”–PROTACs using a CRISPR knock-in cell line expressing a model target of BromoTag-Brd2 at endogenous levels, yielded AGB1, a potent, fast, and selective degrader of BromoTag’ed proteins.

Developing AGB1 led to the discovery of a simple bioisosteric, amide-toester substitution, between a PROTAC linker and a BET targeting ligand can increase degradation potency and bioactivity. Applying these findings to a series of amide/ester matched pairs of non-bumped BET PROTACs also showed that esters were more potent at degrading BET proteins than their amide counterparts, while retaining plasma stability. The enhancements in potency were later found to be driven by greater cell permeability rather than improvements in ternary complex formation.

Finally, I applied this amide-to-ester strategy to design and synthesise a series of novel heterotrivalent PROTACs, consisting of a trifunctional linker that conjugates a BET ligand, to both VHL and CRBN E3 ligase ligands. We identified AB3067, a compound which can potently degrade BET proteins through engagement of both VHL and CRBN E3 ligases simultaneously. AB3067 displays BET degradation even in the absence of either E3 ligase in VHL/CRBN knockout cell lines and provides a potential strategy to overcome PROTAC induced cell resistance.

Date of Award	2022
Original language	English
Awarding Institution	University of Dundee
Supervisor	Alessio Ciulli (Supervisor) & Gopal Sapkota (Supervisor)