Small molecules as tools for functional assessment of deubiquitinating enzyme function

Magin, Robert S.; Liu, Xiaoxi; Felix, Alejandra; Bratt, Ariana S.; Chan, Wai Cheung; Buhrlage, Sara J.

doi:10.1016/j.chembiol.2021.04.021

Cited by 10 publications

(7 citation statements)

References 64 publications

(137 reference statements)

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“…In summary, this study identified a subset of UPP targeting small molecules that inhibits BoNT/A LC activity in cells; two of the most efficacious compounds are DUB inhibitors. This is important as DUBs are considered druggable targets with significant clinical potential for various conditions (Magin et al, 2021). Initial studies described herein provide proof-ofconcept data indicating that small molecules targeting UPP can be useful for attenuating BoNT/A intoxication.…”

Section: Discussionmentioning

confidence: 84%

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity

et al. 2021

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Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.

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Section: Discussionmentioning

confidence: 84%

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity

et al. 2021

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“…The inhibition of DUB activity is subject to a growing interest in drug development, and several successful examples of potent DUB inhibitors have been published in recent years. ,, In this study, we developed the improved covalent OTUB2 inhibitor LN5P45 through optimization of the original OTUB2-COV1 inhibitor by addressing absolute stereochemistry and aromatic ring substituents. We determined the preferred absolute stereochemistry as (1 S ,2 S ), and the LN5P45 -OTUB2 co-crystal structure explained the molecular details of the inhibitor binding mode.…”

Section: Discussionmentioning

confidence: 99%

“…15 To further optimize the inhibitory properties of this compound, we first turned our attention to elucidating the preferred stereochemistry of the substituents attached to the cyclopropane, as the exact nature of the stereocenters in COV-1 were unknown. COV-1 is synthesized in two or three steps from 2-phenylcyclopropane-1-carboxylate (11) or its methyl (12) or ethyl ester (13) derivatives, as shown in Scheme S1. The stereochemistry in the final product originates from the stereochemical nature of the starting compound and does not change during the course of the synthesis.…”

Section: ■ Introductionmentioning

confidence: 99%

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2

Gan,

de Vries,

Akkermans

et al. 2023

ACS Chem. Biol.

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Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.

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“…Indeed, one of the biggest challenges in the field is the identification of DUB substrates. With the growing availability of selective DUB inhibitors, the application of chemical genomic approaches will facilitate the identification of DUB substrates and novel DUB biology (Magin et al, 2021). Most in vitro screens commonly use activity-based probes or simplified substrates such as Ub-AMC or di-Ub chains (Pruneda and Komander, 2019).…”

Section: Future Perspectivesmentioning

confidence: 99%

Deubiquitinases: From mechanisms to their inhibition by small molecules

2022

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Small molecules as tools for functional assessment of deubiquitinating enzyme function

Cited by 10 publications

References 64 publications

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2

Deubiquitinases: From mechanisms to their inhibition by small molecules

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