A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation

• Published in: Scientific reports Vol. 11; no. 1; p. 15319
• Main Authors: Nizami, Sohaib, Millar, Val, Arunasalam, Kanisa, Zarganes-Tzitzikas, Tryfon, Brough, David, Tresadern, Gary, Brennan, Paul E., Davis, John B., Ebner, Daniel, Di Daniel, Elena
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.07.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/154; 631/154/1435; 631/154/2426; 631/154/436; 631/154/556; 631/378/1689; 631/378/87; 692/699/375; Animals; Apoptosis; Bone marrow; CARD Signaling Adaptor Proteins - metabolism; Caspase 1 - biosynthesis; Caspase-1; Cells, Cultured; Dimethyl Sulfoxide - pharmacology; Drug Discovery; Drug Evaluation, Preclinical - methods; Furans - pharmacology; Genes, Reporter; High-Throughput Screening Assays - methods; Hsp90 protein; Humanities and Social Sciences; IL-1β; Indenes - pharmacology; Inflammasomes; Inflammasomes - drug effects; Inflammation; Inflammatory diseases; Inhibitors; Interleukin-1beta - biosynthesis; Lipopolysaccharides - pharmacology; Macrophages; Macrophages - drug effects; Mice; multidisciplinary; Nigericin - pharmacology; NLR Family, Pyrin Domain-Containing 3 Protein - antagonists & inhibitors; Phenotype; Potassium; Proteins; Pyroptosis; Pyroptosis - drug effects; R&D; Recombinant Proteins - metabolism; Research & development; Science; Science (multidisciplinary); Small Molecule Libraries; Sulfonamides - pharmacology; Therapeutic targets
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-021-94850-w

Inhibition of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome has recently emerged as a promising therapeutic target for several inflammatory diseases. After priming and activation by inflammation triggers, NLRP3 forms a complex with apoptosis-associated speck-like protein containing a CARD domain (ASC) followed by formation of the active inflammasome. Identification of inhibitors of NLRP3 activation requires a well-validated primary high-throughput assay followed by the deployment of a screening cascade of assays enabling studies of structure–activity relationship, compound selectivity and efficacy in disease models. We optimized a NLRP3-dependent fluorescent tagged ASC speck formation assay in murine immortalized bone marrow-derived macrophages and utilized it to screen a compound library of 81,000 small molecules. Our high-content screening assay yielded robust assay metrics and identified a number of inhibitors of NLRP3-dependent ASC speck formation, including compounds targeting HSP90, JAK and IKK-β. Additional assays to investigate inflammasome priming or activation, NLRP3 downstream effectors such as caspase-1, IL-1β and pyroptosis form the basis of a screening cascade to identify NLRP3 inflammasome inhibitors in drug discovery programs.