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A high-throughput screening approach identified a selective pro-caspase-10 probe that can inhibit its activity

ABSTRACT: Caspases are cysteine proteases that play a critical role in inflammation and apoptosis. Caspases have a functional role in either the intrinsic apoptotic pathway as initiators of apoptosis or in the extrinsic apoptotic pathway as executioners of apoptosis. Caspase-10 has been speculated to play a principal role as an initiator of apoptosis but the absence of adequate tools, i.e. selective probes, have hindered our understanding of this enzyme as well as the other caspases. Here, we engineered a tobacco etch virus (TEV)-cleavable construct that is compatible with high-throughput screening to identify selective caspase-10 inhibitors. We identified a set of promiscuous caspase inhibitors, including pifithrin-µ (PFT), an inhibitor of p53 transcriptional activity, as well as promising selective pro-caspase-10 and TEV protease inhibitors.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Jurkat Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Jose Castellon

LAB HEAD: Dr. Keriann M. Backus

PROVIDER: PXD051710 | Pride | 2025-02-04

REPOSITORIES: pride

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			Action	DRS
	2023-05-17-KB-FAIMS-PFT-chymo-JOC-1.pepXML	Pepxml
	2023-05-17-KB-FAIMS-PFT-chymo-JOC-1.raw	Raw
	2023-05-17-KB-FAIMS-PFT-chymo-JOC-2.pepXML	Pepxml
	2023-05-17-KB-FAIMS-PFT-chymo-JOC-2.raw	Raw
	2023-05-17-KB-FAIMS-PFT-doubledigest-JOC-5.pepXML	Pepxml

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A high-throughput screening approach identified a selective pro-caspase-10 probe that can inhibit its activity

Project description:Caspases are cysteine proteases that play a critical role in inflammation and apoptosis. Caspases have a functional role in either the intrinsic apoptotic pathway as initiators of apoptosis or in the extrinsic apoptotic pathway as executioners of apoptosis. Caspase-10 has been speculated to play a principal role as an initiator of apoptosis but the absence of adequate tools, i.e. selective probes, have hindered our understanding of this enzyme as well as the other caspases. Here, we engineered a tobacco etch virus (TEV)-cleavable construct that is compatible with high-throughput screening to identify selective caspase-10 inhibitors. We identified a set of promiscuous caspase inhibitors, including pifithrin-µ (PFT), an inhibitor of p53 transcriptional activity, as well as promising selective pro-caspase-10 and TEV protease inhibitors.

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An integrated chemoproteomic and screening platform identifies a functional and ligandable non-catalytic cysteine in caspase-2

Project description: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, small molecule inhibitors have emerged as useful tools for modulating caspase activity. Achieving high selectivity remains a central challenge for caspase-directed inhibitor development efforts, due to the high sequence and structure homology of all twelve human caspases. Here, using a chemoproteomic approach, we first identify a highly reactive non-catalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a TEV 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, with compound treatment blocking intramolecular caspase interactions. Focused medicinal chemistry combined with chemoproteomic target engagement analysis in lysates and in cells yielded both caspase-2 selective and promiscuous caspase inhibitors, together with structurally matched inactive control compounds. Application of this focused set of tool compounds to stratify caspase contributions to activation of intrinsic apoptosis indicate likely compensatory caspase-9 activity driving etoposide-mediated cell death in the context of caspase-2 inactivation. More broadly, our study highlights the utility of targeting non-conserved and non-catalytic cysteine residues for achieving improved selectivity profiles for highly homologous families of enzymes.

2025-01-14 | PXD046269 | Pride

An integrated chemoproteomic and screening platform identifies a functional and ligandable non-catalytic cysteine in caspase-2

2025-01-14 | PXD042362 | Pride

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Stucki2005 - caspase-3 metabolism

Project description:Jörg W. Stucki & Hans-Uwe Simon. Mathematical modeling of the regulation of caspase-3 activation and degradation. Journal of Theoretical Biology 234, 1 (2005). Caspases are thought to be important players in the execution process of apoptosis. Inhibitors of apoptosis (IAPs) are able to block caspases and therefore apoptosis. The fact that a subgroup of the IAP family inhibits active caspases implies that not each caspase activation necessarily leads to apoptosis. In such a scenario, however, processed and enzymically active caspases should somehow be removed. Indeed, IAP–caspase complexes covalently bind ubiquitin, resulting in degradation by the 26S proteasome. Following release from mitochondria, IAP antagonists (e.g. second mitochondrial activator of caspases (Smac)) inactivate IAPs. Moreover, although pro-apoptotic factors such as irradiation or anti-cancer drugs may release Smac from mitochondria in tumor cells, high cytoplasmic survivin and ML-IAP levels might be able to neutralize it and, consequently, IAPs would further be able to bind activated caspases. Here, we propose a simple mathematical model, describing the molecular interactions between Smac deactivators, Smac, IAPs, and caspase-3, including the requirements for both induction and prevention of apoptosis, respectively. In addition, we predict a novel mechanism of caspase-3 degradation that might be particularly relevant in long-living cells.

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Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain

Project description:Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Additional cellular activities, unrelated to cell death seem to be influenced by these enzymes. Identification of genes co-regulated with caspases could help to ascertain new biological roles for these proteases.To identify genes and pathways under the influence of caspase-2 we silenced its expression in U87MG glioblastoma cell line. Transcriptional expression profiles of cells transfected with caspase-2 siRNA or control siRNA were compared. Gene expression profiling was evaluated from 2 replicates of U87MG cells transfected with CASP2-siRNA, or with Control-siRNA.

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2023-03-08 | PXD039954 | Pride

RNAseq of untreated and CSF1-treated, CSF1+QVD-treated monocytes

Project description:Caspases, which are key effectors of apoptosis, have demonstrated non-apoptotic functions. One of these functions is the differentiation into macrophages of peripheral blood monocytes exposed to Colony-Stimulating Factor-1 (CSF1). Macrophage polarization plays an important role in the pathogenesis of diverse human diseases as cancer, leading us to explore if caspase inhibition would affect macrophage polarization. To explore the role of caspases in CSF1 differentiation, we used human monocytes sorted from buffy coats treated by cytokines. We reported that caspase inhibition delays the ex vivo differentiation of peripheral blood monocytes exposed to CSF1 and modifies the phenotype of generated macrophages, e.g. cell shape, surface markers. Moreover, by RNAseq, we observed that the macrophages generated in presence of CSF1 and QVD are different from CSF1-treated monocytes. This study confirms the importance of caspase activation in CSF1 differentiation.

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Non-lethal stress-induced discrete proteolysis is caspase-3/7 dependent

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Project description:Caspases, which are key effectors of apoptosis, have demonstrated non-apoptotic functions. One of these functions is the differentiation into macrophages of peripheral blood monocytes exposed to Colony-Stimulating Factor-1 (CSF1). Conversely, GM-CSF induces the differentiation of monocytes into macrophages in a caspase-independent manner. Macrophages generated by CSF1 and GM-CSF have distinct polarity. Macrophage polarization plays an important role in the pathogenesis of diverse human diseases as cancer, leading us to explore if caspase inhibition would affect macrophage polarization. To explore the role of caspases in CSF1 differentiation, we used human monocytes sorted from buffy coats treated by cytokines. We reported that caspase inhibition delays the ex vivo differentiation of peripheral blood monocytes exposed to CSF1 and modifies the phenotype of generated macrophages, e.g. cell shape, surface markers. Moreover, by RNAseq, we observed that the macrophages generated in presence of CSF1 and QVD are different from CSF1-treated monocytes and from GM-CSF-treated monocytes. Cell cycle and focal adhesion-related pathway genes were selectively down-regulated. This study confirms the importance of caspase activation in CSF1 differentiation.

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