Cellular Assays for Dynamic Quantification of Deubiquitinase Activity and Inhibition
Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved..
Deubiquitinases (DUBs) are proteolytic enzymes that catalyze the removal of ubiquitin from protein substrates. The critical role of DUBs in regulating protein ubiquitination makes them attractive drug targets in oncology, neurodegenerative disease, and antiviral development. Biochemical assays for quantifying DUB activity have enabled characterization of substrate preferences and discovery of small molecule inhibitors. However, assessing the efficacy of these inhibitors in cellular contexts to support clinical drug development has been limited by a lack of tractable cell-based assays. To address this gap, we developed a two-color flow cytometry-based assay that allows for sensitive quantification of DUB activity and inhibition in living cells. The utility of this system was demonstrated by quantifying the potency of GRL0617 against the viral DUB SARS-CoV-2 PLpro, identifying potential GRL0617 resistance mutations, and performing structure-function analysis of the vOTU domain from the recently emerged Yezo virus. In addition, the system was optimized for cellular DUBs by modifying a GFP-targeting nanobody to recruit USP7 and USP28 to benchmark a panel of reported inhibitors and assess inhibition kinetics. Together, these results demonstrate the utility of these assays for studying DUB biology in a cellular context with potential to aid in inhibitor discovery and development.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:435 |
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| Enthalten in: |
Journal of molecular biology - 435(2023), 23 vom: 01. Dez., Seite 168316 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Moghadasi, Seyed Arad [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 29.11.2023 Date Revised 13.12.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jmb.2023.168316 |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved. | ||
| 520 | |a Deubiquitinases (DUBs) are proteolytic enzymes that catalyze the removal of ubiquitin from protein substrates. The critical role of DUBs in regulating protein ubiquitination makes them attractive drug targets in oncology, neurodegenerative disease, and antiviral development. Biochemical assays for quantifying DUB activity have enabled characterization of substrate preferences and discovery of small molecule inhibitors. However, assessing the efficacy of these inhibitors in cellular contexts to support clinical drug development has been limited by a lack of tractable cell-based assays. To address this gap, we developed a two-color flow cytometry-based assay that allows for sensitive quantification of DUB activity and inhibition in living cells. The utility of this system was demonstrated by quantifying the potency of GRL0617 against the viral DUB SARS-CoV-2 PLpro, identifying potential GRL0617 resistance mutations, and performing structure-function analysis of the vOTU domain from the recently emerged Yezo virus. In addition, the system was optimized for cellular DUBs by modifying a GFP-targeting nanobody to recruit USP7 and USP28 to benchmark a panel of reported inhibitors and assess inhibition kinetics. Together, these results demonstrate the utility of these assays for studying DUB biology in a cellular context with potential to aid in inhibitor discovery and development | ||
| 650 | 4 | |a Journal Article | |
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| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
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| 650 | 4 | |a deubiquitinase (DUB) activity | |
| 650 | 4 | |a deubiquitinase (DUB) inhibition | |
| 650 | 4 | |a ubiquitin (Ub) biology | |
| 650 | 4 | |a viral deubiquitinases (DUBs) PL(pro) and vOTU | |
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