Abstract
Small-molecule inducible degrons allow the conditional degradation of genetically modified proteins in diverse models from human cell lines to mice. However, these approaches vary in selectivity, potency, and suitability and have various limitations. In this thesis, we describe the development of BromoTag, a novel inducible degron system that consists of a BRD4 bromodomain variant that can be recruited to the VHL E3 ligase via new bumped proteolysis targeting chimaeras (PROTACs) to induce the degradation of the genetically engineered protein target. We developed an endogenous BromoTag platform that we used in the extensive optimisation of BromoTag bump-and-hole-PROTACs. Our cellular data collectively qualify PROTAC AGB1, a potent, rapid, and selective degrader of BromoTagged proteins. Furthermore, we established the mechanism of action underpinning AGB1’s exceptional activity and its aptness for application in human cell models due to its low cellular cytotoxicity and limited off-target activity.Further favourable pharmacokinetic profile in mice established AGB1’s suitability for in vivo application. We found an optimal CRISPR strategy for the homozygous integration of the BromoTag into HEK293, which we used to create and validate homozygous BromoTag knock-in HEK293 cell lines against targets MCM4, BRD4 and BRD2. We used these knock-in cell lines to demonstrate rapid, conditional degradation of BromoTagged proteins while targeting distinct and essential cellular processes. To probe the functional consequences of rapid, profound target depletion, we performed for the first time an unbiased proteomic analysis of a selectively degraded BRD4 to identify and characterise BRD4 regulated proteins. We found from this analysis potentially novel roles for previously identified BRD4 associating proteins ARC and CyclinT2.
This study demonstrates the development and application of the BromoTag, a small molecule induced degron that complements the arsenal of established degradation tools, providing scientists with a complementary and robust approach for interrogating the biological function of potentially therapeutic proteins in cells and in vivo.
| Date of Award | 2022 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Sponsors | Medical Research Council |
| Supervisor | Alessio Ciulli (Supervisor) & Julian Blow (Supervisor) |