Novel Approaches to Probe the Activity of Deubiquitinating Enzymes

Abstract

Ubiquitination is a highly conserved post-translational modification that regulates a multitude of critical cellular events. This process is orchestrated by a complex enzymatic network. Deubiquitinating enzymes (DUBs) are responsible for removal of ubiquitin from its conjugates. There are over 100 DUBs expressed in eukaryotes and several diseases are associated with their dysregulation, including cancer and neurodegeneration. Activity-based probes (ABPs) have been developed as an effective strategy to study DUBs. ABPs targeting these enzymes are typically based on a ubiquitin scaffold and incorporate an electrophilic group that reacts with an active site cysteine. These probes have greatly enhanced the mechanistic and structural understanding of DUBs but offer no external control over the timing of the reaction. In this work, ABPs consisting of a monoubiquitin recognition element and warheads with reactivity previously unexplored in the context of ABPs were designed and synthesised. The reactivity of these novel ABPs with DUBs was examined and new labelling strategies were developed to improve the study of these enzymes. New electrophilic probes with fluoride reactive groups were designed and synthesised. These probes were tested alongside previously reported electrophilic probes and demonstrated negligible reactivity with DUBs. Latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated thiol-ene coupling mechanism were developed. A novel labelling methodology was developed for these alkene-functionalised probes and reactivity was demonstrated against recombinant DUBs and endogenous DUBs within cell lysate. Specificity of this probe labelling was confirmed using inhibitor and proteomic studies. Novel assays to probe reversible and irreversible inhibitors for this enzyme were also demonstrated. In order to enhance the biocompatibility of this methodology, a milder source of UV light was used to initiate the reaction between DUBs and the alkene-functionalised probes. Alternative radical initiators were examined for a visible light-mediated thiol-ene reaction. Successful labelling of recombinant DUBs was achieved in both strategies, but visible light activation was limited by off-target reactivity in more complex systems. The visible light-mediated thiol-ene reaction and the thiol-yne reaction were also explored for non-templated protein conjugation, affording modest coupling in both cases. Overall, the novel electrophilic probes presented do not improve upon existing probes of similar reactivity. However, the work on alkene functionalised probes enables more finely resolved investigations of DUB activity in complex systems. In contrast to existing cysteine reactive probes, control over the timing of the enzyme-probe reaction is possible for the alkene functionalised probes as they are completely inert under ambient conditions, even upon probe binding. This is expected to help provide a better understanding of these enzymes and aid in the study and development of novel inhibitors. The visible light-mediated thiol-ene and thiol-yne reactions were found to have limited applications for bioconjugations.