Developing a Chemical Approach to Study E3 Ligase Biology

Khan, Anam (2025) Developing a Chemical Approach to Study E3 Ligase Biology. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The ubiquitin-proteasome system (UPS) is a highly regulated biological system in eukaryotic cells responsible for the correct disposal of proteins. E3 ubiquitin ligases are a family of proteins that catalyse the transfer of ubiquitin from an E2 ligase to the protein substrate marking it for disposal by the proteasome. With over 600 E3 ligases expressed by the human genome, each with its own substrate specificity profile, the need to understand E3 biology and to design drugs to target this system has become an important goal of current drug discovery efforts. However, due to the weak and transient nature of these interactions the ability to study the substrates of E3 ligases has been limited. The aim of this project is to develop a novel chemical methodology to monitor and identify E3 ligase substrates endogenously in cells. Using a unique bio-orthogonal ligand directed chemistry approach we successfully synthesised a library of compounds using N-acyl-N-alkyl sulfonamide (NASA) as a reactive group to target lysine residues of the binding pocket of the clinically relevant E3 Ligase Cereblon (CRBN). In vitro Surface Plasmon Resonance (SPR) were used to assay the binding of the novel compounds to the CRBN protein surface followed by cellular assays to test target engagement and endogenous protein labelling. Six novel Lystargeting probes were synthesised which demonstrated effective binding and labelling of CRBN, highlighting their promise as potential candidates for the identification of novel E3 ligase neo-substrates. To investigate the further application of this technology we synthesised components to build a probe to target tyrosine residues by using the concept of sulfur-triazole exchange (SuTEx) chemistry. Similarly, a modular component was designed and synthesised to incorporate a photo-cross linking element as an addition to the existing library of lysine targeting probes. The work in this thesis details the design and synthetic optimisation of these chemical probes and their initial biological investigation and applications.