Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

Chad S Hewitt; Chittaranjan Das; Daniel P Flaherty

doi:10.3390/biom12010062

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

Biomolecules. 2022 Jan 1;12(1):62. doi: 10.3390/biom12010062.

Authors

Chad S Hewitt¹, Chittaranjan Das², Daniel P Flaherty^{1

3

4}

Affiliations

¹ Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
² Department of Chemistry, College of Science, Purdue University, West Lafayette, IN 47907, USA.
³ Purdue Center for Cancer Research, Hanson Life Sciences Research Building, Purdue University, West Lafayette, IN 47907, USA.
⁴ Purdue Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA.

Abstract

There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbV^{Q40V/T66K/V70F}-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

Keywords: DUBs; UCHL3; activity-based probes; ubiquitin variants.

MeSH terms

Amino Acid Substitution*
Cell Line, Tumor
Humans
Mutation, Missense*
Ubiquitin Thiolesterase* / chemistry
Ubiquitin Thiolesterase* / genetics
Ubiquitin Thiolesterase* / metabolism
Ubiquitin* / chemistry
Ubiquitin* / genetics
Ubiquitin* / metabolism

Substances

UCHL1 protein, human
Ubiquitin
UCHL3 protein, human
Ubiquitin Thiolesterase