Synthesis of activity-based probes for the metalloprotease CSN5

Amin, Siddique (2021) Synthesis of activity-based probes for the metalloprotease CSN5. MSc(R) thesis, University of Glasgow.

Abstract

The Cullin-RING E3 ligase (CRL) family consists of over 200 enzymes that are responsible for the ubiquitylation and regulation of a huge variety of proteins involved in the immune response, cell cycle control and cell proliferation. CRLs are activated by covalent attachment to the C-terminus of the ubiquitin-like modifier NEDD8 by forming an isopeptide bond in a process called NEDDylation. Inhibition of CRLs is mediated by the COP9 signalosome complex (CSN) which cleaves the isopeptide bond binding to NEDD8 and removes it. CRL homeostasis is disrupted by CSN overactivity and is associated with the pathogenesis of ~50% of all cancers in humans. The means by which the activity of the catalytic subunit of CSN (CSN5) is regulated is not fully understood and thus new tools are required to study its activity. CSN5 contains a JAMM domain with a Zn2+ atom facilitating its catalytic activity. The synthesis of several activity-based probes for CSN5 was attempted, each containing a zinc-binding group (ZBG) for attachment onto the catalytic site, a NEDD8 peptide to enable selectivity to CSN5 and a biotin tag for subsequent biochemical assays. Three zinc-binding groups were considered including hydroxamic acid, imidazole and a precursor of a potent small molecule CSN5 inhibitor, CSN5i-3. The multi-step synthesis of the CSN5i-3 precursor was attempted but not completed due to time constraints. In an endeavour to mimic the entire substrate of CSN5, the synthesis of NEDD8 was attempted but not successful. In a separate approach, a peptide of the NEDD8 C-terminal tail was synthesised and successfully biotinylated, however conjugation to the zinc-binding groups hydroxamic acid and imidazole were unsuccessful due to the low solubility of the peptide.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: NEDD8, Activity-Based Probe, COP9 signalosome complex, peptidomimetic, deubiquitinase, solid-phase peptide synthesis.
• Subjects: Q Science > QD Chemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Supervisor's Name: Jamieson, Dr. Andrew
• Date of Award: 2021
• Depositing User: Mr Siddique Amin
• Unique ID: glathesis:2021-82092
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 30 Mar 2021 08:09
• Last Modified: 30 Mar 2021 08:31
• Thesis DOI: 10.5525/gla.thesis.82092
• URI: https://theses.gla.ac.uk/id/eprint/82092