Chemical genetic inhibition of E2 ubiquitin-conjugating enzymes

Spear, Luke 2023. Chemical genetic inhibition of E2 ubiquitin-conjugating enzymes. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The development of a general approach for the rapid and selective inhibition of enzymes in cells using a common tool holds great promise for research and therapeutic applications. In this study, we expanded on a previously reported chemogenetic strategy for kinase inhibition to address the challenge of inhibiting cysteine-dependent enzymes. We achieved selective inhibition of two E2 ubiquitinconjugating enzymes, UBE2L3 and UBE2D1, through bioorthogonal tethering of electrophilic warheads. The successful inhibition was demonstrated in biochemical assays, with consistent inhibition of polyubiquitin chain formation observed using SDS-PAGE analysis. Mass spectrometry data confirmed the inhibitor tethering to the protein and supported the reaction between a chloroacetamide warhead in one of the proposed inhibitor conjugates and the E2 catalytic cysteine. Further optimization of the strategy is necessary to achieve complete inhibition without residual polyubiquitination. The transferability of this chemogenetic approach was demonstrated by selective inhibition of a UBE2D1 variant using the same inhibitor complex that achieved success in UBE2L3. This indicates the potential application of the strategy to other enzymes within the same family and other cysteine-dependent enzyme classes. Additionally, efforts were made to investigate the inhibition of UBE2L3 in living mammalian cells using the NF-κB signaling pathway as a model for the E2 enzyme’s activity. Although successful UBE2L3 overexpression was achieved for wild-type protein, UBE2L3 variant expression requires further tuning.Cell viability assays revealed toxicity at higher concentrations of the successful inhibitor complex, highlighting the need for considering toxicity in future inhibition assays. While the results presented here offer promising insights into selective enzyme inhibition and the potential use of this strategy in various cysteine-dependent enzymes, further investigation and optimization are required. The study provides a foundation for future research on the development of the proposed strategy and the investigation of cellular functions and validation of therapeutic targets.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Chemistry
Date of First Compliant Deposit:	11 January 2024
Last Modified:	11 Jan 2024 11:31
URI:	https://orca.cardiff.ac.uk/id/eprint/165409

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