| Summary: | <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>
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