A bump and hole approach to the development of selective degrons

Brennan, PJ

Oxford Thesis Collection

Thesis

A bump and hole approach to the development of selective degrons

Abstract:: 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.

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.

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%.

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APA Style

Brennan, P. J. (2023). A bump and hole approach to the development of selective degrons [PhD thesis]. University of Oxford.

MLA Style

Brennan, PJ. A Bump and Hole Approach to the Development of Selective Degrons. 2023. University of Oxford, PhD thesis.

Chicago Style

Brennan, PJ. 2023. “A Bump and Hole Approach to the Development of Selective Degrons.” PhD thesis, University of Oxford.
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