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Title: Bactacs : a chemical tool for targeted protein degradation within bacteria
Author: Brodie, Glen
ISNI:       0000 0005 0286 6948
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2020
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Targeted protein degradation is a promising new technology that allows for the modulation of cellular protein concentrations. The technology can be used to target previously undruggable proteins for degradation, thus has tremendous potential as both a therapeutic strategy and as a new tool to study protein function. Protacs (Proteolysis Targeting Chimeras) are small bi-functional molecules capable of recruiting the eukaryotic ubiquitin–proteasome system (UPS) and targeting it to a protein of interest, ultimately resulting in the protein of interest’s degradation. Protacs, however, are exclusive to eukaryotes due to bacteria lacking the ubiquitin–proteasome system. Taking inspiration from Protacs, this thesis details the development of a new tool that allows for the targeted degradation of proteins in bacteria, which will herein be referred to as Bactacs (Bacterial Proteolysis Targeting Chimeras). Chapter 1 serves as an introduction into antibiotic resistance and details how targeted protein degradation could overcome some of the limitations posed by traditional therapeutic strategies when treating bacterial infection, highlighting the real-world benefits of such a technology in bacteria. The chapter then goes on to discuss the benefits of targeted protein degradation as a tool for studying proteins in bacteria, before introducing and describing Protac technology itself. The chapter then goes on to discuss various bacterial protease systems, and the “tags” used by these systems to signal a protein for degradation in bacteria. The chapter then concludes with an introduction to the Bactac and how Bactacs could be used to potentially hijack bacterial proteolysis machineries. Chapter 2 details the design, synthesis, and biological testing of the first generation of Bactac molecules. The chapter highlights the challenges involved in creating a suitable assay to determine protein degradation, and highlights issues with the expression of the required recombinant fusion protein — mSA-EGFP. The chapter also details the synthesis of useful “linker” molecules. Chapter 3 begins by introducing the HaloTag system to be used as an alternative to the recombinant protein system used in chapter 2. The chapter then details the design, synthesis, and biological testing of the second generation of Bactac molecules to be used in conjunction with the HaloTag system. The chapter discusses the use of the CAPA assay, used to determine Bactac accumulation within bacterial cells, as well as other physical techniques used to determine if the Bactacs were binding their targets. The chapter then highlights difficulties in using fluorescence-based assays to determine protein degradation. The chapter then concludes with the successful degradation of a HaloTag fusion protein by two different Bactac compounds — namely LLL-HTL-I and ADM-HTL-II — which was determined by quantitative western blotting. Chapter 4 summarises the findings of the PhD project, whilst chapter 5 discusses future work to be carried out. Experimental protocols, as well as experimental data, are described for both the biological and chemical components of this project in chapters 6 and 7 respectively. An electronic appendix is also attached separately containing NMRs and fluorescence data.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QR Microbiology