Hulme, Alison

Richardson, Julia

Śmieja, Connor Bruce

Engineering and Physical Sciences Research Council (EPSRC)

2025-07-31T14:17:09Z

2025-07-31T14:17:09Z

2025-07-31

Ubiquitin (Ub) is conjugated to proteins through the Ubiquitin-proteasome system (UPS), acting as a post-translational modification that tunes signalling pathways and controls protein longevity. While the UPS is thought to be dysregulated in many types of disease, there is a lack of existing small molecule tools with which it can be studied. Developing new tool molecules would provide alternative approaches to examine the mechanisms of the UPS and unlock opportunities to discover future drug candidates. A structure-based approach was employed to identify a ligand that would act as a molecular glue, binding between UBE2D1 and Ubiquitin, an interaction which enhances substrate ubiquitination. The Sidhu group produced a ubiquitin variant, UbV.D1.1, which bound tightly and selectively to UBE2D1, even over its close homologues in the UBE2D family.† In particular, the mutation of a -Leu-Thr- sequence in ubiquitin to a -Phe-Trp- sequence strongly contributed to the enhanced binding, with the residues engaging additional key interactions. Therefore, a library of cyclic dipeptides based on the chemistry of this -Phe-Trp- sequence of UbV.D1.1 were synthesised as a starting point for the identification of molecular glues stabilising the Ub/UBE2D1 interaction. The compounds were tested for their ability to perturb ubiquitination reactions consisting of purified E1, E2, E3 and substrate proteins. Visualisation using SDS-PAGE revealed that cyclo[ᴅ-Phe-ʟ-Trp] (CBS006) inhibited ubiquitination at high μM concentrations as typically used in primary screening. Further examination revealed that, rather than acting as a molecular glue to the Ub/UBE2D1 interaction, CBS006 inhibited activation of Ub by the E1 enzyme. However, when CBS006 was resynthesized via a different synthetic route, the new sample did not reproduce the initially observed inhibition. This disparity is under further investigation in the Hulme group. Next, fluorine-labelled Ub-mimicking peptides (UMPs) based on those studied by Zhao and co-workers‡ were synthesised. Fluorine NMR experiments including a CPMG pulse sequence were performed on the peptides to study their sensitivity to the presence of the E1 enzyme. Preliminary results highlighted one of these peptides as being particularly sensitive towards binding to the E1, having a C2 value of 56%. This peptide has potential for future use as a spy molecule in screening small molecule libraries to discover E1 binders in a high-throughput manner. Finally, a novel cleavable linker for peptide synthesis, termed COLAZ, was designed and synthesised. The COLAZ linker bestows peptides with a fluorescent label that it stores in a photostable state throughout peptide synthesis. The suitability of the COLAZ linker for peptide synthesis was demonstrated by tracking the synthesis of a model dipeptide using LCMS, TLC and IR. Finally, the COLAZ linker was used to synthesise novel side-chain derivatised UMPs.

https://hdl.handle.net/1842/43752

http://dx.doi.org/10.7488/era/6285

en

The University of Edinburgh

2026-07-31

Ubiquitin-proteasome system (UPS)

some system (UPS)

Ubiquitination

E1 enzyme

Fluorine-labelled peptides

Discovery of tool compounds for study of the ubiquitin-proteasome system

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy

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