Development of macrocyclic peptides as multifunctional probes in chemical biology

Thesis

Multifunctional probes combine a target engagement motif with at least one additional application, and have been used in a myriad of ways in chemical biology to identify the roles of their target in normal and disease settings. In this thesis, multifunctionality is installed to selective cyclic peptide inhibitors of epigenetic proteins: CP2 which inhibits histone demethylases 4A-C (KDM4A-C), and TiP1 which inhibits ten eleven translocation enzymes (TETs).

Multifunctional peptide probes were synthesised for epigenetic targets by addition of unnatural amino acids as click chemistry handles, or through installing photoreactive groups. Cu(I)-catalysed azide-alkyne cycloaddition click chemistry was used to synthesise peptide conjugates. Both CP2- and TiP1-based affinity peptide probes were produced by conjugating biotin to the peptides, and target engagement of biotinylated peptides was tested in cell lysates. Photoreactive multifunctional peptides were also assessed, for both KDM4A and TET activity, however these did not specifically crosslink their targets under the conditions tested.

E3 ligase binders were also installed to produce cyclic peptide proteolysis recruiting chimeras (PROTACs), to promote target protein degradation through the ubiquitin-proteasome system. A panel of ten CP2-based PROTACs was synthesised, using different linkers, recruiting either von Hippel Lindau (VHL) or cereblon (CRBN) E3 ligases. KDM4A protein degradation was established in cells transiently overexpressing HaloTagged and Flag tagged KDM4A (HaloFlagKDM4A) using HaloTag targeting PROTACs. Conditions established using HaloPROTACs were used to test the panel of CP2-PROTACs.

Authors: Roper, G

• DOI: 10.5287/ora-e2gdnxgdo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English