Activity‐Based Protein Profiling – Finding General Solutions to Specific Problems

Cravatt, Benjamin F.

doi:10.1002/ijch.202300029

Cited by 17 publications

(11 citation statements)

References 99 publications

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“…Identification of proteoform-selective molecules is an area of rapidly emerging interest. ,− As shown by type II inhibitors that target inactive forms of kinases, − we expect that targeting procaspases should emerge as a general strategy to selectively inactivate individual caspases in cells. An additional feature of procaspase inhibitors is that when compared with active caspase inhibitors, they should fully block downstream caspase activationactive caspase inhibitors are ill equipped to block the rapid caspase activation (<30 min)and subsequent cleavage of effector caspases that is observed during apoptosis.…”

Section: Discussionmentioning

confidence: 99%

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors

Castellón,

Ofori,

Burton

et al. 2024

J. Am. Chem. Soc.

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Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, smallmolecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometrybased chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

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Section: Discussionmentioning

confidence: 99%

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors

Castellón,

Ofori,

Burton

et al. 2024

J. Am. Chem. Soc.

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“…Yet, virtually nothing is known about the biosynthetic or catabolic pathways that regulate lyso-PS metabolism in these tissues. In humans, a majority of the phospholipases and lysophospholipases belong to the mSH family of enzymes, and a significant portion of this enzyme family still lacks the assignment of a physiologically relevant biochemical activity and an in vivo biological function. , Leveraging the repertoire of pharmacological and genetic tools available to interrogate the enzymes of the mSH family, ,− it is possible to determine if certain tissue-specific lipases might, in fact, be metabolic regulators of lyso-PS. For example, the major lyso-PS lipase ABHD12 is highly expressed in the brain and is known to regulate levels of lyso-PS only in the nervous system.…”

Section: Key Emerging Questionsmentioning

confidence: 99%

Lysophosphatidylserine: A Signaling Lipid with Implications in Human Diseases

Chakraborty,

Kamat

2024

Chem. Rev.

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Lysophosphatidylserine (lyso-PS) has emerged as yet another important signaling lysophospholipid in mammals, and deregulation in its metabolism has been directly linked to an array of human autoimmune and neurological disorders. It has an indispensable role in several biological processes in humans, and therefore, cellular concentrations of lyso-PS are tightly regulated to ensure optimal signaling and functioning in physiological settings. Given its biological importance, the past two decades have seen an explosion in the available literature toward our understanding of diverse aspects of lyso-PS metabolism and signaling and its association with human diseases. In this Review, we aim to comprehensively summarize different aspects of lyso-PS, such as its structure, biodistribution, chemical synthesis, and SAR studies with some synthetic analogs. From a biochemical perspective, we provide an exhaustive coverage of the diverse biological activities modulated by lyso-PSs, such as its metabolism and the receptors that respond to them in humans. We also briefly discuss the human diseases associated with aberrant lyso-PS metabolism and signaling and posit some future directions that may advance our understanding of lyso-PS-mediated mammalian physiology.

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“…A chemoproteomic method termed activity-based protein profiling (ABPP) that uses activity-based probes (ABPs) is a powerful platform to map proteome-wide reactive proteins and evaluate the functional states of enzymes in complex biological systems. 26, 27 Using ABPs that mimic native ubiquitin but are modified with an electrophilic warhead at the C-terminus to label the active site cysteines irreversibly, ABPP has led to the identification of new DUB families and characterization of DUB activity across conditions. 28 In a recent study, an ISG15-based ABP was used to detect deISGylating enzymes in human cell lysates and identified USP16 as an ISG15 cross-reactive DUB in addition to the previously-characterized DUBs such as USP5 and USP14.…”

Section: Introductionmentioning

confidence: 99%

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

Davis,

Omole,

Jung

et al. 2024

Preprint

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Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

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Activity‐Based Protein Profiling – Finding General Solutions to Specific Problems

Cited by 17 publications

References 99 publications

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors

Lysophosphatidylserine: A Signaling Lipid with Implications in Human Diseases

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

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