Techniques to Study Inflammasome Activation and Inhibition by Small Molecules

Angosto-Bazarra, Diego; Molina-López, Cristina; Peñín‐Franch, Alejandro; Hurtado-Navarro, Laura; Pelegrı́n, Pablo

doi:10.3390/molecules26061704

Cited by 16 publications

(12 citation statements)

References 119 publications

(150 reference statements)

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“…Subsequently, inflammasome activation induces the production of pro-IL-1 and pro-IL-18 in an NF-β-dependent way [29] . In fact, inflammasomes are categorized and named based on the sensor protein that initiates their activation [30] . NLRs such as NLRP1b, NLRC4, and NLRP3 have well-established roles during inflammasome assembly and pyroptosis initiation [31] .…”

Section: Resultsmentioning

confidence: 99%

Association of pyroptosis and severeness of COVID-19 as revealed by integrated single-cell transcriptome data analysis

Yang

Zhang

et al. 2022

ImmunoInformatics

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Section: Resultsmentioning

confidence: 99%

Association of pyroptosis and severeness of COVID-19 as revealed by integrated single-cell transcriptome data analysis

Yang

Zhang

et al. 2022

ImmunoInformatics

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“…Different techniques could be used to study NLRP3 inhibitors [ 47 ]; the use of differentiated THP-1 cells treated with NLRP3 activating stimuli is suitable for the screening of large series of compounds [ 48 ]. All the synthesised compounds ( Figure 2 ) were evaluated for their NLRP3 inhibitory activity by measuring their ability to prevent NLRP3-dependent pyroptosis in differentiated THP-1 cells.…”

Section: Resultsmentioning

confidence: 99%

Chemical Modulation of the 1-(Piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one Scaffold as a Novel NLRP3 Inhibitor

et al. 2021

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In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1β release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1β release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein–ligand binding that might explain the activity of the compounds.

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“…To date, several approaches have been used for the identification of small molecule CASP1/NLRP3 inhibitors. 28–30 Conventional Western blot detection of cleaved caspase 1 and NLRP3 protein and by ELISA of released caspase 1 and the downstream production of pro-inflammatory factors (eg, IL-1β, IL-18) in cell lysates and supernatants (cell culture media, serum, etc.) are the most commonly used methods, but are not ideal for HTP screening of small molecule compound libraries.…”

Section: Discussionmentioning

confidence: 99%

CASPorter: A Novel Inducible Human CASP1/NALP3/ASC Inflammasome Biosensor

C¹,

Beard²,

Yang³

et al. 2022

JIR

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Background Following our 2015 elucidation of the CASP1/NALP3 inflammasome mechanism of glucocorticoid (GC)-resistance in pediatric acute lymphoblastic leukemia (ALL) patients, we engineered a cell-based CASP1/NALP3 reporter system suitable for high-throughput screening (HTS) of small molecule libraries, with the purpose of identifying compounds capable of inhibiting the CASP1/NALP3 inflammasome and synergizing with GC drugs for the treatment of GC-resistant ALL patients and various autoinflammatory diseases. Methods A Dox-controlled system was utilized to induce the expression of the ASC transgene in HEK293 cells while simultaneously overexpressing NLRP3 and CASP1 . ASC/CASP1/NALP3 inflammasome complex formation was confirmed by co-immunoprecipitation (co-IP) experiments. Next, a LV fluorescence-based biosensor ( CASP orter) was transduced in the HEK293-iASC-NLRP3/CASP1 cell line to monitor the real-time activation of CASP1/NALP3 inflammasome in live cells. The applicability and effectiveness of the CASP orter cell line were tested by co-treatment with Dox and four known CASP1/NLRP3 inhibitors (MCC950, Glyburide, VX-765 and VRT-043198). Inflammasome activation and inhibitions were assessed by Western blotting, fluorescence microscopy and flow cytometry (FC) methods. Results Dox treatment significantly induced ASC expression and increased levels of cleaved and catalytically active CASP1, co-IPs further demonstrated that CASP1 was pulled-down with NLRP3 in HEK293-iASC-NLRP3/CASP1 cells after induction of ASC by Dox treatment. In HEK293-iASC-NLRP3/CASP1- CASP orter cell system, cleavage of the CASP1 consensus site (YVAD) in the CASP orter protein after Dox treatment causing excitation/emission of green fluorescence and the 71% GFP+ cell population increase quantified by FC (78.1% vs 6.90%). Dox-induced activation of the NLRP3 inflammasome was dose-dependently inhibited by Dox co-treatment with four known CASP1/NLRP3 inhibitors. Conclusion We have established a cell-based CASP1/NLRP3 inflammasome model, utilizing a fluorescence biosensor as readout for qualitatively observing and quantitatively determining the activation of caspase 1 and NLRP3 inflammasomes in living cells and easily define the inhibitory effect of inhibitors with high efficacy.

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Techniques to Study Inflammasome Activation and Inhibition by Small Molecules

Cited by 16 publications

References 119 publications

Association of pyroptosis and severeness of COVID-19 as revealed by integrated single-cell transcriptome data analysis

Association of pyroptosis and severeness of COVID-19 as revealed by integrated single-cell transcriptome data analysis

Chemical Modulation of the 1-(Piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one Scaffold as a Novel NLRP3 Inhibitor

CASPorter: A Novel Inducible Human CASP1/NALP3/ASC Inflammasome Biosensor

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