A bump and hole approach to the development of selective degrons
<p>The aim of this study was to develop novel immunomodulatory imide drug (IMiD) analogues which would not induce degradation of endogenous proteins but would induce degradation of a suitably modified zinc finger (ZF) degron motif, therefore providing an IMiD/ZF degron system that could be rel...
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| Format: | Thesis |
| Language: | English |
| Published: |
2023
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| _version_ | 1817930639428550656 |
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| author | Brennan, PJ |
| author2 | Conway, S |
| author_facet | Conway, S Brennan, PJ |
| author_sort | Brennan, PJ |
| collection | OXFORD |
| description | <p>The aim of this study was to develop novel immunomodulatory imide drug (IMiD) analogues which would not induce degradation of endogenous proteins but would induce degradation of a suitably modified zinc finger (ZF) degron motif, therefore providing an IMiD/ZF degron system that could be reliably used for target validation. To achieve this, we employed ‘bump and hole’ methodology to identify a ligand that selectively induces degradation of a mutant degron but not the wild type (WT) ZF. Traditional IMiDs, such as thalidomide, lenalidomide and pomalidomide, are unsuitable for target validation due to off-target degradation of transcription factors IKZF1 and IKZF3, as well as other endogenous proteins.</p>
<p>A library of ‘bumped’ IMiD analogues were screened by molecular docking, after which a refined library of ‘bumped’ IMiD analogues was chemically synthesised and tested for degradation ability against the WT ZF degron sequence, and a ‘hole’-containing glutamine-to-alanine (Q147A) mutant, using a flow cytometry-based fluorescence assay. The four most promising candidate molecules were then screened against a library of 8380 ZF mutant sequences using a fluorescence activated cell sorting (FACS) degradation assay, yielding IMiD/degron pairs with high selectivity.</p>
<p>Further flow cytometry and western blot assays confirmed the high selectivity of these IMID/degron pairs. No degradation of endogenous IKZF1 or IKZF3 was observed in Jurkat cells incubated with either compound 88 or 26 for 18 h at 10 μM concentration. The most selective IMID/degron pair – compound 26 and a Q147W/N149E/Q150I mutant ZF – was then used to degrade TRIM28, a disease-relevant protein with no known small molecule ligands, with DMAX > 80%.</p> |
| first_indexed | 2024-09-25T04:01:30Z |
| format | Thesis |
| id | oxford-uuid:3ef0ed59-98eb-4bb5-9eee-7d4877f5671b |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:09:19Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:3ef0ed59-98eb-4bb5-9eee-7d4877f5671b2024-10-04T07:46:00ZA bump and hole approach to the development of selective degronsThesishttp://purl.org/coar/resource_type/c_db06uuid:3ef0ed59-98eb-4bb5-9eee-7d4877f5671bBiologySynthetic BiologyChemistryChemical BiologyEnglishHyrax Deposit2023Brennan, PJConway, SDeane, C<p>The aim of this study was to develop novel immunomodulatory imide drug (IMiD) analogues which would not induce degradation of endogenous proteins but would induce degradation of a suitably modified zinc finger (ZF) degron motif, therefore providing an IMiD/ZF degron system that could be reliably used for target validation. To achieve this, we employed ‘bump and hole’ methodology to identify a ligand that selectively induces degradation of a mutant degron but not the wild type (WT) ZF. Traditional IMiDs, such as thalidomide, lenalidomide and pomalidomide, are unsuitable for target validation due to off-target degradation of transcription factors IKZF1 and IKZF3, as well as other endogenous proteins.</p> <p>A library of ‘bumped’ IMiD analogues were screened by molecular docking, after which a refined library of ‘bumped’ IMiD analogues was chemically synthesised and tested for degradation ability against the WT ZF degron sequence, and a ‘hole’-containing glutamine-to-alanine (Q147A) mutant, using a flow cytometry-based fluorescence assay. The four most promising candidate molecules were then screened against a library of 8380 ZF mutant sequences using a fluorescence activated cell sorting (FACS) degradation assay, yielding IMiD/degron pairs with high selectivity.</p> <p>Further flow cytometry and western blot assays confirmed the high selectivity of these IMID/degron pairs. No degradation of endogenous IKZF1 or IKZF3 was observed in Jurkat cells incubated with either compound 88 or 26 for 18 h at 10 μM concentration. The most selective IMID/degron pair – compound 26 and a Q147W/N149E/Q150I mutant ZF – was then used to degrade TRIM28, a disease-relevant protein with no known small molecule ligands, with DMAX > 80%.</p> |
| spellingShingle | Biology Synthetic Biology Chemistry Chemical Biology Brennan, PJ A bump and hole approach to the development of selective degrons |
| title | A bump and hole approach to the development of selective degrons |
| title_full | A bump and hole approach to the development of selective degrons |
| title_fullStr | A bump and hole approach to the development of selective degrons |
| title_full_unstemmed | A bump and hole approach to the development of selective degrons |
| title_short | A bump and hole approach to the development of selective degrons |
| title_sort | bump and hole approach to the development of selective degrons |
| topic | Biology Synthetic Biology Chemistry Chemical Biology |
| work_keys_str_mv | AT brennanpj abumpandholeapproachtothedevelopmentofselectivedegrons AT brennanpj bumpandholeapproachtothedevelopmentofselectivedegrons |