Harnessing bioreactivity for protein covalent probes against endogenous targets
<p>Various small-molecule drugs that covalently modify their targets have been discovered and designed to date. Covalent reaction has also been identified in the interaction between the cells of the human body and microorganisms, both from the immune system to pathogens and from pathogens to h...
| Autore principale: | |
|---|---|
| Altri autori: | |
| Natura: | Tesi |
| Lingua: | English |
| Pubblicazione: |
2021
|
| _version_ | 1826299090857623552 |
|---|---|
| author | Scheu, AHA |
| author2 | Howarth, M |
| author_facet | Howarth, M Scheu, AHA |
| author_sort | Scheu, AHA |
| collection | OXFORD |
| description | <p>Various small-molecule drugs that covalently modify their targets have been discovered and designed to date. Covalent reaction has also been identified in the interaction between the cells of the human body and microorganisms, both from the immune system to pathogens and from pathogens to host cells. Harnessing bioreactivity found in covalent pathogen adhesion, in this work I have demonstrated the use of a self activating protein domain from Neisseria meningitidis for covalent reaction against endogenous targets.</p>
<p>N. meningitidis iron-regulated protein C (FrpC) contains a ‘self-processing module’ (SPM); subject to calcium-activation, SPM cleaves the protein backbone at an Asp-Pro bond, forming a reactive anhydride from the Asp-Pro cleavage site. This protein anhydride can facilitate protein protein crosslinking via reaction with lysine residues. SPM can also be introduced recombinantly to introduce a reactive anhydride to non-FrpC proteins. In this thesis I established ‘NeissLock’, a method for targeted protein protein conjugation after SPM-mediated biochemical activation. First, I designed and implemented ‘NeissDist’, a tool to identify protein complexes in which a binder protein activated by SPM is suitably positioned for covalent attachment to a target protein of interest. I identified the complexes of Ornithine Decarboxylase Antizyme (OAZ) binding to Ornithine Decarboxylase (ODC) and Transforming Growth Factor alpha (TGFα) binding to Epidermal Growth Factor Receptor (EGFR) as model systems to study NeissLock conjugation.</p>
<p>NeissLock-mediated conjugation of OAZ to ODC shows effective (>50% yield) and specific coupling to the target protein, over a broad pH range. Using the OAZ/ODC system, I characterised conjugation to multiple target residues, showing flexibility in the site of reaction, and gained insight into linker design for efficient anhydride formation. Having shown specific conjugation of TGFα to EGFR on cells, NeissLock technology may allow broad compatibility with further protein-protein complexes as well as a range of therapeutic and diagnostic applications. </p> |
| first_indexed | 2024-03-07T04:56:37Z |
| format | Thesis |
| id | oxford-uuid:d6d3c428-cee6-4461-84cb-4347b73e5d1e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:56:37Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | oxford-uuid:d6d3c428-cee6-4461-84cb-4347b73e5d1e2022-03-27T08:36:30ZHarnessing bioreactivity for protein covalent probes against endogenous targetsThesishttp://purl.org/coar/resource_type/c_db06uuid:d6d3c428-cee6-4461-84cb-4347b73e5d1eEnglishHyrax Deposit2021Scheu, AHAHowarth, MSeymour, L<p>Various small-molecule drugs that covalently modify their targets have been discovered and designed to date. Covalent reaction has also been identified in the interaction between the cells of the human body and microorganisms, both from the immune system to pathogens and from pathogens to host cells. Harnessing bioreactivity found in covalent pathogen adhesion, in this work I have demonstrated the use of a self activating protein domain from Neisseria meningitidis for covalent reaction against endogenous targets.</p> <p>N. meningitidis iron-regulated protein C (FrpC) contains a ‘self-processing module’ (SPM); subject to calcium-activation, SPM cleaves the protein backbone at an Asp-Pro bond, forming a reactive anhydride from the Asp-Pro cleavage site. This protein anhydride can facilitate protein protein crosslinking via reaction with lysine residues. SPM can also be introduced recombinantly to introduce a reactive anhydride to non-FrpC proteins. In this thesis I established ‘NeissLock’, a method for targeted protein protein conjugation after SPM-mediated biochemical activation. First, I designed and implemented ‘NeissDist’, a tool to identify protein complexes in which a binder protein activated by SPM is suitably positioned for covalent attachment to a target protein of interest. I identified the complexes of Ornithine Decarboxylase Antizyme (OAZ) binding to Ornithine Decarboxylase (ODC) and Transforming Growth Factor alpha (TGFα) binding to Epidermal Growth Factor Receptor (EGFR) as model systems to study NeissLock conjugation.</p> <p>NeissLock-mediated conjugation of OAZ to ODC shows effective (>50% yield) and specific coupling to the target protein, over a broad pH range. Using the OAZ/ODC system, I characterised conjugation to multiple target residues, showing flexibility in the site of reaction, and gained insight into linker design for efficient anhydride formation. Having shown specific conjugation of TGFα to EGFR on cells, NeissLock technology may allow broad compatibility with further protein-protein complexes as well as a range of therapeutic and diagnostic applications. </p> |
| spellingShingle | Scheu, AHA Harnessing bioreactivity for protein covalent probes against endogenous targets |
| title | Harnessing bioreactivity for protein covalent probes against endogenous targets |
| title_full | Harnessing bioreactivity for protein covalent probes against endogenous targets |
| title_fullStr | Harnessing bioreactivity for protein covalent probes against endogenous targets |
| title_full_unstemmed | Harnessing bioreactivity for protein covalent probes against endogenous targets |
| title_short | Harnessing bioreactivity for protein covalent probes against endogenous targets |
| title_sort | harnessing bioreactivity for protein covalent probes against endogenous targets |
| work_keys_str_mv | AT scheuaha harnessingbioreactivityforproteincovalentprobesagainstendogenoustargets |