Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe

Panyain, Nattawadee; Godinat, Aurélien; Thawani, Aditya R.; Lachiondo‐Ortega, Sofía; Mason, Katie; Elkhalifa, Sarah; Smith, Lisa M.; Harrigan, Jeanine A.; Tate, Edward W.

doi:10.1039/d1md00218j

Cited by 16 publications

(18 citation statements)

References 26 publications

(43 reference statements)

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“…Counterintuitively, CG173 shows weak and decreasing inhibition of cellular UCHL1 with longer incubation times. Whether this behavior and the toxicity are a general feature of the substituted 2-carboxy- N -cyanopyrrolidine scaffold 24 , 27 , 28 remains to be investigated from larger compound libraries.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, rational endeavors to improve the potency and specificity of DUB inhibitors are often hampered by the lack of structural information from specific inhibitor:DUB complexes 22 , 23 as is also the case for UCHL1. Following the publication of 2- and 3-carboxy- N -cyanopyrrolidines as UCHL1 inhibitors in the patent literature 24 , 25 , the Flaherty 26 , Tate 27 , 28 , and Ovaa/Geurink 29 labs published small molecule activity-based probes for UCHL1. These showed highly potent target engagement in cell lines, covalent inhibition in vitro, and enabled the visualization of UCHL1 activity in live zebrafish.…”

Section: Introductionmentioning

confidence: 99%

“…However, some reported compounds showed pronounced cytotoxicity, which is at odds with the viability of many UCHL1 knockout cell lines 30 . Moreover, various other covalently bound targets were identified which could not be disentangled with the used set of control probes 28 . In addition, no phenotype in protein abundance following UCHL1 inhibition was detected 28 .…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, various other covalently bound targets were identified which could not be disentangled with the used set of control probes 28 . In addition, no phenotype in protein abundance following UCHL1 inhibition was detected 28 .…”

Section: Introductionmentioning

confidence: 99%

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Structural basis for specific inhibition of the deubiquitinase UCHL1

et al. 2022

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Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structural basis for specific inhibition of the deubiquitinase UCHL1

et al. 2022

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“…MT16-001, a covalent inhibitor of UCH-L1 based on the thiazolyl cyanopyrrolidine backbone, binds to C90 in the active site of UCH-L1 (108). This inhibitor induces proliferation inhibition at sub-molar concentrations in B cell and lung cancer cell lines (which are known to be sensitive to UCH-L1 knockdown) and is more selective for UCH-L1 than other DUBs (108,109). However, based on the evidence reported to date (108), its selectivity profile has not been determined and further experiments are required to explore this.…”

Section: Uch-l1 Inhibitorsmentioning

confidence: 99%

Ubiquitin C‑terminal hydrolase‑L1: A new cancer marker and therapeutic target with dual effects (Review)

Wang

Zhang

et al. 2023

Oncol Lett

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Ubiquitin C-terminal hydrolase-L1 (UCH-L1), a member of the lesser-known deubiquitinating enzyme family, has deubiquitinase and ubiquitin (Ub) ligase activity and the role of stabilizing Ub. UCH-L1 was first discovered in the brain and is associated with regulating cell differentiation, proliferation, transcriptional regulation and numerous other biological processes. UCH-L1 is predominantly expressed in the brain and serves a role in tumor promotion or inhibition. There is still controversy about the effect of UCH-L1 dysregulation in cancer and its mechanisms are unknown. Extensive research to investigate the mechanism of UCH-L1 in different types of cancer is key for the future treatment of UCH-L1-associated cancer. The present review details the molecular structure and function of UCH-L1. The role of UCH-L1 in different types of cancer is also summarized and how novel treatment targets provide a theoretical foundation in cancer research is discussed.

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Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin‐K Activity Detection, Revealing Nuclear Activity

Dey,

Sinai‐Turyansky,

Yakobovich

et al. 2024

Advanced Science

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Cathepsin‐K (CTSK) is an osteoclast‐secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity‐based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro‐form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

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Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe

Abstract: Ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme (DUB), is a potential drug target in various cancers, and liver and lung fibrosis. However, bona fide functions and substrates of UCHL1...

Cited by 16 publications

References 26 publications

Structural basis for specific inhibition of the deubiquitinase UCHL1

Structural basis for specific inhibition of the deubiquitinase UCHL1

Ubiquitin C‑terminal hydrolase‑L1: A new cancer marker and therapeutic target with dual effects (Review)

Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin‐K Activity Detection, Revealing Nuclear Activity

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