Development of prime and capture methods for proximity labelling of E3 ligases

Muganyi-Ingleton, Jenine (2022) Development of prime and capture methods for proximity labelling of E3 ligases. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Understanding the function of E3 ligases without genetic manipulation has proven to be a challenge. E3 ligases are responsible for numerous biological processes although very little is known about them. The unique approach of synthesising activity or chemical probes in the biochemical industry has emerged over the years. in this study we will explore the foundations of synthesising an activity probe that focuses primarily on the E3 ligase. Using x-ray crystallography, we were able to identify potential binding sites in which the activity probe could anchor onto before labelling the protein by nucleophilic attack. Prior knowledge of the relationship of thalidomide and E3 ligase Cereblon , meant that thalidomide was a suitable candidate for binding , whilst Biotin could be used to label the E3 ligase Cereblon. This work provides the foundation of synthesising an activity probe with dual functionality held in place by a central core to understand the E3 ligase and its substrates. This approach can also be utilized in other protein discoveries e.g., cause and effect of mutated proteins. In this study ,we synthesised the building blocks of a ‘prime’ and ‘capture’ probe optimising the conditions by incorporating a tosylate ligand directed modality .Various synthetic methods have been trialled to analyse the stability of the prime and capture probe chemically as a potential biological tool for protein labelling without genetic manipulation.