2. Academic Validation
  3. A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation

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

  • Sci Rep. 2021 Jul 28;11(1):15319. doi: 10.1038/s41598-021-94850-w.
Sohaib Nizami  # 1 2,  Val Millar  # 1 2 3,  Kanisa Arunasalam 1 2,  Tryfon Zarganes-Tzitzikas 1 2,  David Brough 4 5,  Gary Tresadern 6,  Paul E Brennan 1 2,  John B Davis 1 2,  Daniel Ebner 7 8,  Elena Di Daniel 9 10
Affiliations

Affiliations

  • 1 Alzheimer's Research UK Oxford Drug Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK.
  • 2 Target Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK.
  • 3 National Phenotypic Screening Centre, Target Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK.
  • 4 Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, UK.
  • 5 Lydia Becker Institute of Immunology and Inflammation, University of Manchester, Manchester, M13 9PT, UK.
  • 6 Janssen Research and Development, Turnhoutseweg 30, 2340, Beerse, Belgium.
  • 7 Target Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK. daniel.ebner@ndm.ox.ac.uk.
  • 8 National Phenotypic Screening Centre, Target Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK. daniel.ebner@ndm.ox.ac.uk.
  • 9 Alzheimer's Research UK Oxford Drug Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK. elena.di-daniel@astx.com.
  • 10 Target Discovery Institute, NDM Research Building, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK. elena.di-daniel@astx.com.
  • # Contributed equally.
PMID: 34321581 DOI: 10.1038/s41598-021-94850-w
Abstract

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.

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