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

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, UbVQ40V/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.

Ubiquitin Engineering for Interrogating the Ubiquitin–Proteasome System and Novel Therapeutic Strategies

“…The resulting UbV (UbV T9F/T66K ) served as a UCHL1-selective UbV-ABP, but it unexpectedly displayed preferential binding for UCHL3 over UCHL1 when used in small cell lung cancer cell lysates [ 79 ]. In a subsequent study, the same group focused on designing UCHL3-selective UbV-ABPs and developed UbV Q40V/T66K/V70F -ABP, which showed outstanding UCHL3 inhibition in vitro and reactivity for UCHL3 in MDA-MB-231 breast cancer cells without cross-reactivity with other DUBs [ 81 ].…”

Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1

“…For the past 20 years, LDN-57444 (Figure ) has been the gold standard small molecule probe to study UCHL1 but has since been invalidated as a UCHL1 inhibitor due to its inability to engage the target in cells . Among DUBs, UCHL1 has one of the strongest binding affinities for Ub ( K d = 100 nM), − rendering traditional reversible small molecule inhibition of the Ub-UCHL1 protein–protein interaction difficult to achieve. Recent research has shifted toward covalent inhibition strategies to overcome the high thermodynamic barrier.…”

The Multifaceted Role of the Ubiquitin Proteasome System in Pathogenesis and Diseases

“…UCHL3 is another UCH-family DUB that regulates DNA repair and is overexpressed in many cancers [ 17 , 18 ]. Building on their previous UCHL1 work, Hewitt and colleagues generated a selective triple-mutant UbV-ABP for UCHL3 and validated its function in multiple human cell lines [ 19 ].…”