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Trends in Pharmacological Sciences
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NLRP3 and pyroptosis blockers for treating inflammatory diseases

Open AccessPublished:May 03, 2022DOI:https://doi.org/10.1016/j.tips.2022.04.003

      Highlights

      • The nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome is a key mediator of pathological inflammation in many diseases.
      • There are several drug-like small molecules under clinical development as novel therapeutics targeting the NLRP3 inflammasome.
      • Pyroptosis is a unique approach to suppress chronic inflammation.
      • Understanding the mechanism of action by which small molecules block the NLRP3 inflammasome allows better molecular optimization.
      • Different clinical trials are running using agents that inhibit the NLRP3 inflammasome.
      The nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome has emerged as a key mediator of pathological inflammation in many diseases and is an exciting drug target. Here, we review the molecular basis of NLRP3 inhibition by drug-like small molecules under development as novel therapeutics. We also summarize recent strategies to block pyroptosis as a novel approach to suppress chronic inflammation. Major recent developments in this area include the elucidation of mechanisms of action (MoAs) by which small molecules block NLRP3 inflammasome assembly and gasdermin D (GSDMD)-induced pyroptosis. We also discuss the status of clinical trials using agents that block specific components of the NLRP3 pathway, including their potential clinical applications for the treatment of many diseases.

      Keywords

      Inflammasomes are novel targets for anti-inflammatory therapies

      NLRP3 (see Glossary) inflammasome is an intracellular protein complex that has emerged as a key mediator of inflammation in many pathologies. NLRP3 activation by microbes and danger signals (Figure 1) leads to the release of the proinflammatory cytokines IL-1β and IL-18, as well as to GSDMD-mediated pyroptosis, a proinflammatory type of cell death (Box 1).
      Figure 1
      Figure 1The nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome signaling pathway, which can result in cell pyroptosis.
      (A) Domain structure of NLRP3, with an N-terminal Pyrin domain (PYD), a central NACHT domain, and a C-terminal leucine-rich repeat (LRR) domain. (B) A range of stimuli, including microbes, particulate matter (misfolded proteins, crystals, and nanoparticles), and damage-associated molecules (e.g., extracellular ATP), induce the formation of NLRP3 oligomers and allow recruitment of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and the zymogen caspase-1, leading to activation of caspase-1 and the cleavage of gasdermin D (GSDMD). Caspase-1 also processes pro-IL-1β and pro-IL-18 to their mature forms. The N-terminal domain of GSDMD (p30) forms pores in the plasma membrane and facilitates the release of IL-1β and IL-18. The plasma membrane protein ninjurin-1 (NINJ1) mediates the extent of plasma membrane rupture, inducing pyroptotic cell death. Abbreviation: CARD, caspase activation and recruitment domain.
      Pyroptosis: a gasdermin-meditated type of inflammatory cell death
      Pyroptosis is a type of programmed necrotic cell death characterized by the permeabilization of the plasma membrane by gasdermin protein family members, leading to the subsequent release of intracellular contents (for recent review, see [
      • Broz P.
      • et al.
      The gasdermins, a protein family executing cell death and inflammation.
      ]). The presence of cytosolic constituents in the extracellular milieu is a signal that the innate immune system recognizes through specific receptors that elicit an inflammatory response aimed to repair tissue damage. For example, proinflammatory cytokines of the IL-1 family, such as the alarmin IL-1β, lack a signal peptide and, thus, their cellular release requires permeabilization of the plasma membrane [
      • Chan A.H.
      • Schroder K.
      Inflammasome signaling and regulation of interleukin-1 family cytokines.
      ]. Gasdermins are an evolutionary conserved family of proteins that share two well-defined structural domains: an N-terminal domain with intrinsic pore-forming properties and a C-terminal domain that represses the pore-forming activities of the N-terminal domain, connected by a linker sequence [
      • Broz P.
      • et al.
      The gasdermins, a protein family executing cell death and inflammation.
      ]. Linker cleavage of gasdermin proteins liberates the N-terminal fragment, which oligomerizes and forms pores in cellular membranes, including the plasma membrane. Several distinct proteases cleave the linker of specific gasdermin family members. GSDMD is processed by caspase-1/-4/-5/-8/-11 and neutrophil elastase, whereas GSDMB is processed by granzyme A, and GSDME is cleaved by caspase-3 [
      • Broz P.
      • et al.
      The gasdermins, a protein family executing cell death and inflammation.
      ,
      • Demarco B.
      • et al.
      Caspase-8-dependent gasdermin D cleavage promotes antimicrobial defense but confers susceptibility to TNF-induced lethality.
      ,
      • Zhou Z.
      • et al.
      Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells.
      ]. GSDMD plasma membrane pores are positively regulated by ROS [
      • Evavold C.L.
      • et al.
      Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.
      ] and negatively regulated by the scission of damaged membranes by the endosomal sorting complexes required for transport (ESCRT) machinery [
      • Broz P.
      • et al.
      The gasdermins, a protein family executing cell death and inflammation.
      ]. GSDMD pores are a negatively charged conduit for the direct passage of mature IL-1 cytokines [
      • Xia S.
      • et al.
      Gasdermin D pore structure reveals preferential release of mature interleukin-1.
      ], which have a neutral or positively charged surface [
      • Monteleone M.
      • et al.
      Interleukin-1β maturation triggers its relocation to the plasma membrane for gasdermin-D-dependent and -independent secretion.
      ]. In many settings, the extent of plasma membrane rupture induced by GSDMD pores in the dying cells is regulated by the protein ninjurin-1 [
      • Kayagaki N.
      • et al.
      NINJ1 mediates plasma membrane rupture during lytic cell death.
      ], leading to the release of intracellular contents, including HMGB1, inflammasome oligomers, and mitochondrial DNA, or even inducing the expulsion of neutrophil nuclear DNA leading to NETosis [
      • Broz P.
      • et al.
      The gasdermins, a protein family executing cell death and inflammation.
      ,
      • Torre-Minguela C.
      • et al.
      Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis.
      ,
      • Xia S.
      • et al.
      Gasdermin D pore structure reveals preferential release of mature interleukin-1.
      ,
      • Tan G.
      • et al.
      HMGB1 released from GSDME-mediated pyroptotic epithelial cells participates in the tumorigenesis of colitis-associated colorectal cancer through the ERK1/2 pathway.
      ].
      The past 5 years have seen rapid progress in the development of small-molecule inhibitors targeting the NLRP3 inflammasome pathway, with several NLRP3 and GSDMD inhibitors alleviating pathology in more than 100 preclinical disease models [
      • Corcoran S.E.
      • et al.
      Pharmacological inhibition of the nod-like receptor family pyrin domain containing 3 inflammasome with MCC950.
      ,
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ] (Tables 1 and 2 and Figure 2). These promising findings have fueled the rapid progression of NLRP3 inhibitors through human trials toward the clinic (Box 2), with GSDMD inhibitors closely following. Thus, it is both important and timely to review the landscape of emerging inflammasome drug candidates and their potential clinical applications. It is becoming particularly pressing to understand which inflammasome inhibitors to prioritize for trials in specific disease indications. Here, we discuss priority disease areas for NLRP3 inflammasome blockers, as well as the advantages and disadvantages of directly blocking the inflammasome (via NLRP3 inhibition) versus suppressing GSDMD-induced pyroptosis.
      Table 1NLRP3 inhibitors
      Abbreviations: AKT kinase, protein kinase B; DSS, dextran sulfate sodium; EAE, experimental autoimmune encephalomyelitis; FAK1, focal adhesion kinase 1; HSF1, heat shock factor 1; Hsp72, heat shock protein 72; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; N/A, not applicable; NAFLD, nonalcoholic fatty liver disease; ND, Not determined ; PGE2, prostaglandin E2; RPL10, ribosomal protein L10; STAT, signal transducer and activator of transcription; SYK, spleen tyrosine kinase; TBI, traumatic brain injury; TGF-β, transforming growth factor-β; TRPV2, transient receptor potential cation channel subfamily V member 2; USP, ubiquitin-specific protease.
      InhibitorMoAIC50
      IC50 values are included for IL-1β release from mouse macrophages stimulated with LPS and ATP or nigericin.
      Off-target effects (IC50)
      Known off-target inhibitory effects are summarized with IC50 values presented where possible.
      NLRP3-associated disease model
      Disease models in which inhibitors have been effective are summarized.
      MCC950

      Binds to NACHT domain, locking NLRP3 in an inactive conformation

      Inhibits ATPase activity
      7.5 nMCarbonic anhydrase 2 (11 μM)AD, PD, EAE, ischemic stroke, TBI, T2DM, age-related metabolic syndrome, diabetes-related co-morbidities: nephropathy, endothelial dysfunction, atherosclerosis, retinopathy, encephalopathy, cognitive impairment, ischemia–reperfusion injury, liver injury; NAFLD, NASH, liver fibrosis, IBD, colitis, and models reviewed in [
      • Corcoran S.E.
      • et al.
      Pharmacological inhibition of the nod-like receptor family pyrin domain containing 3 inflammasome with MCC950.
      ]
      CY-09

      Binds to Walker A site in NACHT domain

      Inhibits ATPase activity
      6 μMCytochrome P450 enzymes 1A2 (19 μM), 2C9 (8 μM), 2C19 (>50 μM), 2D6 (>50 μM), 3A4 (26 μM)Stroke, T2DM, diabetic retinopathy, diabetic liver injury, NAFLD, peritonitis, Muckle–Wells syndrome, pain, osteoarthritis, myocardial infarction, epilepsy, osteosarcoma, acute kidney injury, hemolytic uremic syndrome
      Bay-11-7082

      Inhibits ATPase activity

      Binding site unknown
      ~3 μMIKK (10 μM), USP7 (0.19 μM), USP21 (0.96 μM)TBI, spinal cord injury, T2DM, IBD, psoriasis, lupus nephritis, acute kidney injury, acute lung injury, wound healing
      Parthenolide

      Inhibits ATPase activity

      Binding site unknown
      1.4 μMIKK-β (7.5 μM), caspase-1 (10 μM), Hsp72 (>10 μM), FAK1 (>10 μM), RPL10 (>10 μM), JAK2 (4 μM)N/A
      BOT-4-one

      Inhibits ATPase activity

      Binding site unknown
      1.28 μMIKK-β (~10 μM)Peritonitis, dermatitis, psoriasis, arthritis
      MNS

      Inhibits ATPase activity

      Binding site unknown
      ~3 μMSyk (2.5 μM), Src kinase (29.3 μM), p97 (1.7 μM)Burn wound healing, airway inflammation
      OLT1177

      Inhibits ATPase activity

      Binding site unknown
      NDIn vivo NF-κB, IκB, IKK

      Increases oxidative metabolism in vivo
      AD, EAE, colitis, arthritis, allergic asthma, acute kidney injury
      INF39

      Inhibits ATPase activity

      Binding site unknown
      ~10 μMUnknownSpinal cord injury, IBD, colitis, drug-induced liver injury, pancreatitis, myocardial infarction
      HS-203873

      Inhibits ATPase activity by competing with ATP binding~50 μMUnknownN/A
      Tranilast

      Binds to NACHT disrupting NLRP3–NLRP3 interactions and blocking oligomerization~25 μMNumerous, e.g., TRPV2 (<75 μM), inhibition of histamine (100 μM), PGE2 (30 μM), and cytokine release (TGF-β, IL-6, IL-8, IL-5), collagen synthesisT2DM, NASH, colitis, peritonitis, Muckle–Wells syndrome, atherosclerosis, nanoparticle cerebral toxicity, myocardial infarction
      Oridonin

      Covalently modifies Cys279 in NACHT and attenuates NLRP3–NEK7 interaction0.75 μMNumerous, e.g., AKT1 (8.4 μM), AKT2 (8.9 μM), HSF1, cMyc, NF-κB, JAK/STAT, p38 MAPKTBI, T2DM, alcohol-induced hepatotoxicity, liver fibrosis, IBD, ischemia–reperfusion injury, peritonitis, gouty arthritis, myocardial infarction, hemolytic uremic syndrome, acute lung injury, acute liver injury, ventilator-induced lung injury, hearing loss, pleurisy
      RRx-001

      Binds to Cys409 in NACHT and attenuates NLRP3–NEK7 interaction117 nMNumerous, e.g., inhibition of glucose 6-phosphate dehydrogenase and NF-κB, hemoglobin binding, apoptosisEAE, DSS colitis, allergic asthma
      JC-171

      Attenuates NLRP3–ASC interaction. Binding site unknown8.45 μMUnknownEAE
      a Abbreviations: AKT kinase, protein kinase B; DSS, dextran sulfate sodium; EAE, experimental autoimmune encephalomyelitis; FAK1, focal adhesion kinase 1; HSF1, heat shock factor 1; Hsp72, heat shock protein 72; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; N/A, not applicable; NAFLD, nonalcoholic fatty liver disease; ND, Not determined ; PGE2, prostaglandin E2; RPL10, ribosomal protein L10; STAT, signal transducer and activator of transcription; SYK, spleen tyrosine kinase; TBI, traumatic brain injury; TGF-β, transforming growth factor-β; TRPV2, transient receptor potential cation channel subfamily V member 2; USP, ubiquitin-specific protease.
      b IC50 values are included for IL-1β release from mouse macrophages stimulated with LPS and ATP or nigericin.
      c Known off-target inhibitory effects are summarized with IC50 values presented where possible.
      d Disease models in which inhibitors have been effective are summarized.
      Table 2GSDMD inhibitors
      Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; N/A, not applicable; Nrf2, nuclear factor erythroid 2-related factor 2.
      InhibitorMoAIC50
      IC50 values are included for inflammasome-stimulated cell death.
      Off-target effects (IC50)
      Known off-target inhibitory effects are summarized, with IC50 values presented where possible.
      GSDMD-associated disease model
      Disease models in which inhibitors have been effective are summarized.
      Necrosulfonamide

      Binds directly to C191 blocking GSDMD oligomerization~10 μMMLKL (0.2 μM)LPS-induced sepsis, LPS-induced pulmonary fibrosis
      Dimethyl fumarate

      Succinates C191, blocking GSDMD oligomerization<10 μMNumerous, e.g., aldehyde dehydrogenase (0.15–1.45 μM), dopamine beta-hydroxylase (<10 μM), caspase-1 (~0.25 μM), caspase-3 (~2 μM), GSDME (<10 μM)LPS-induced sepsis, familial Mediterranean fever, autoimmune encephalitis
      Disulfiram

      Modifies C191, blocking GSDMD oligomerization~10 μMNumerous, e.g., activates Nrf2, inhibits NFkB signaling, GAPDH, cyclooxygenase 2, NLRP3 signalingLPS-induced sepsis, pneumonia, renal fibrosis, gouty arthritis
      Antioxidants and mTORC1 inhibitorsBlock ROS-induced GSDMD oligomerizationN/AN/AN/A
      a Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; N/A, not applicable; Nrf2, nuclear factor erythroid 2-related factor 2.
      b IC50 values are included for inflammasome-stimulated cell death.
      c Known off-target inhibitory effects are summarized, with IC50 values presented where possible.
      d Disease models in which inhibitors have been effective are summarized.
      Figure 2
      Figure 2Mechanism of action of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inhibitors.
      (A) Inhibitors that block the ATPase activity of NLRP3. MCC950 binds to the NACHT domain, preventing NLRP3 ATPase activity and keeping NLRP3 inactive [
      • Coll R.C.
      • et al.
      MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.
      ,
      • Tapia-Abellán A.
      • et al.
      MCC950 closes the active conformation of NLRP3 to an inactive state.
      ,
      • Vande Walle L.
      • et al.
      MCC950/CRID3 potently targets the NACHT domain of wildtype NLRP3 but not disease-associated mutants for inflammasome inhibition.
      ,
      • Hochheiser I.V.
      • et al.
      Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3.
      ,
      • Dekker C.
      • et al.
      Crystal structure of NLRP3 NACHT domain with an inhibitor defines mechanism of inflammasome inhibition.
      ]. CY-09 and HS-203873 compete with ATP binding, preventing ATPase activity [
      • Jiang H.
      • et al.
      Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders.
      ,
      • Liao K.-C.
      • et al.
      Application of immobilized ATP to the study of NLRP inflammasomes.
      ,
      • Gastaldi S.
      • et al.
      Chemical modulation of the 1-(piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one scaffold as a novel NLRP3 inhibitor.
      ]. BAY-11-7082, parthenolide, 3,4-methylenedioxy-β-nitrostyrene (MNS), OLT1177, BOT-4-one, and INF-39 all inhibit the ATPase activity of NLRP3, but their binding sites are not yet described [
      • Shim D.-W.
      • et al.
      BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination.
      ,
      • Juliana C.
      • et al.
      Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome.
      ,
      • He Y.
      • et al.
      3,4-Methylenedioxy-β-nitrostyrene inhibits NLRP3 inflammasome activation by blocking assembly of the inflammasome.
      ,
      • Marchetti C.
      • et al.
      OLT1177, a β-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation.
      ,
      • Cocco M.
      • et al.
      Development of an acrylate derivative targeting the NLRP3 inflammasome for the treatment of inflammatory bowel disease.
      ]. (B) Inhibitors that block NLRP3 protein–protein interactions. Tranilast binds to NACHT and inhibits NLRP3–NLRP3 interactions blocking NLRP3 oligomerization [
      • Huang Y.
      • et al.
      Tranilast directly targets NLRP3 to treat inflammasome-driven diseases.
      ]. Oridonin binds to C279 and RRx-001 binds to C409, both of which attenuate Never in mitosis gen A-related kinase 7 (NEK7)–NLRP3 interactions [
      • He H.
      • et al.
      Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.
      ,
      • Chen Y.
      • et al.
      RRx-001 ameliorates inflammatory diseases by acting as a potent covalent NLRP3 inhibitor.
      ]. JC-171 attenuates NLRP3–adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) interactions [
      • Guo C.
      • et al.
      Development and characterization of a hydroxyl-sulfonamide analogue, 5-chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-methoxy-benzamide, as a novel NLRP3 inflammasome inhibitor for potential treatment of multiple sclerosis.
      ]. Abbreviation: CARD, caspase activation and recruitment domain.
      Early clinical trials of NLRP3 blockers
      Five companies have initiated clinical trials with specific NLRP3 inhibitors, although the results from most of these are not yet published. IFM Tre was acquired by Novartis in 2019, and their molecule IFM 2427 (later named DFV890) has reportedly completed Phase I safety trials. DFV890 will be tested in Phase II trials for the CAPS subtype familial cold autoinflammatory syndrome (NCT04868968) and knee osteoarthritis (NCT04886258). DFV890 has completed a Phase II trial for coronavirus disease 2019 (COVID-19) pneumonia (NCT04382053). DFV890 administrated to patients with impaired respiratory function because of COVID-19 pneumonia had no effect on disease severity as measured by the Acute Physiology and Chronic Health Evaluation II score, serum C-reactive protein levels, clinical status, or requirement for mechanical ventilation. Inflazome tested two molecules in Phase I trials. Inzomelid (IZD174; NCT04086602) is a brain-penetrant molecule, whereas Somalix (IZD334) is peripherally restricted and was tested in a Phase I and a Phase IB trial in CAPS (NCT04015076). No results from these trials have been released, but Roche acquired Inflazome in 2020i and Phase II trials might be initiated in the near future. In June 2020, NodTheraii announced a Phase I trial with their NLRP3 inhibitor NT-0167, but no further information on this trial is available. Recently, NodThera announced that they initiated a Phase I safety trial with a lead NLRP3 inhibitor NT-0796 (ACTRN12621001082897)iii,iv. Zydus Cadila developed MCC950-derivative molecules [
      • Agarwal S.
      • et al.
      Identification of a novel orally bioavailable NLRP3 inflammasome inhibitor.
      ,
      • Kennedy C.R.
      • et al.
      A probe for NLRP3 inflammasome inhibitor MCC950 identifies carbonic anhydrase 2 as a novel target.
      ] and has initiated a Phase I safety trial for their molecule ZYIL1 (NCT04972188)v,vi. In addition, OLT1177 (aka dapansutrile) is being developed by Olatec Therapeutics and has concluded a Phase IB trial (NCT03534297) with no serious reported adverse events, in which it improved left ventricular ejection fraction in patients with heart failure and reduced ejection fraction. Dapansutrile was also tested in a Phase IIA trial in which it reduced joint pain during gout flares (EudraCT 2016-000943-14) [
      • Wohlford G.F.
      • et al.
      Phase 1B, randomized, double-blinded, dose escalation, single-center, repeat dose safety and pharmacodynamics study of the oral NLRP3 inhibitor dapansutrile in subjects with NYHA II-III systolic heart failure.
      ,
      • Klück V.
      • et al.
      Dapansutrile, an oral selective NLRP3 inflammasome inhibitor, for treatment of gout flares: an open-label, dose-adaptive, proof-of-concept, phase 2a trial.
      ].

      The inflammasomes

      The inflammasome pathway is an important innate immune effector mechanism triggered in response to infection and tissue injury. Inflammasomes elicit an inflammatory response that aims to eliminate invading pathogens and repair damaged tissues, restoring homeostasis. Inflammasome assembly involves a series of protein oligomerization events induced after the recognition of specific molecular patterns from pathogens, cell damage, or altered homeostatic conditions, resulting in the formation of large multiprotein signaling platforms that activate the protease caspase-1 (Figure 1). The best-described inflammasomes are formed by the following sensor proteins: (i) nucleotide-binding oligomerization domain with leucine-rich repeat (NLR) and pyrin domain-containing 1 (NLRP1); (ii) NLRP3 (Figure 1); (iii) NLRP6; (iv) NLR family apoptosis inhibitory protein (NAIP), which functions in concert with the NLR family, caspase activation and recruitment domain (CARD)-containing 4 (NLRC4); (v) absent in melanoma 2 (AIM2); and (vi) Pyrin (PYD) [
      • Christgen S.
      • et al.
      Toward targeting inflammasomes: insights into their regulation and activation.
      ]. These inflammasome sensor proteins recognize distinct perturbations of homeostasis and form oligomers able to recruit the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) into the inflammasome through PYD–PYD or CARD–CARD homotypic domain interactions (Figure 1). ASC then oligomerizes to form helical filaments in which the zymogen pro-caspase-1 is recruited via CARD–CARD domain interactions to favor its activation [
      • Christgen S.
      • et al.
      Toward targeting inflammasomes: insights into their regulation and activation.
      ]. Caspase-1 subsequently induces the proteolytic maturation of proinflammatory cytokines of the IL-1 family, such as IL-1β and IL-18 (Figure 1) [
      • Christgen S.
      • et al.
      Toward targeting inflammasomes: insights into their regulation and activation.
      ]. Caspase-1 also processes the protein GSDMD, which releases its lytic N-terminal domain (N-GSDMD), which binds to the plasma membrane and oligomerizes to form pores, allowing the release of mature IL-1β and IL-18 (Figure 1). If the burden of plasma membrane pores is sufficiently high, this results in a proinflammatory necrotic type of cell death called pyroptosis (Box 1) [
      • Broz P.
      • et al.
      The gasdermins, a protein family executing cell death and inflammation.
      ]. During pyroptosis, the release of intracellular contents, including inflammasome oligomers, amplifies the inflammatory response and contributes to inflammatory pathology [
      • Baroja-Mazo A.
      • et al.
      The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response.
      ].
      Among inflammasomes, the NLRP3 inflammasome is the most promiscuous, because it can be activated in response to diverse triggers, including pathogen- and danger-associated molecular patterns. Activators of the NLRP3 inflammasome include an array of viral and bacterial proteins (e.g., bacterial toxins, such as nigericin or viroporins), intracellular molecules released into the extracellular fluid upon cellular damage (such as extracellular ATP), which engages the purinergic receptor type 2, family X, subunit 7 (P2X7), and insoluble particles [such as uric acid crystals, amyloid-β (Aβ) depositions, or silica particles] [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ,
      • Christgen S.
      • et al.
      Toward targeting inflammasomes: insights into their regulation and activation.
      ]. Most of these triggers activate NLRP3 by inducing a decrease in the cytosolic concentration of potassium ions (K+), which promotes a conformational change in the inactive NLRP3 oligomer, allowing it to interact with negatively charged lipids in the dispersed trans-Golgi network, thereby promoting the formation of active NLRP3 oligomers that initiate inflammasome signaling [
      • Tapia-Abellán A.
      • et al.
      Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation.
      ,
      • Andreeva L.
      • et al.
      NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation.
      ]. NLRP3 can be also activated through the non-canonical inflammasome pathway, in which intracellular bacterial lipopolysaccharide (LPS) and arachidonic acid-derived oxidized phospholipids activate caspase-4/5 (caspase-11 in mice). These caspases cleave GSDMD, promoting pore formation by inducing the insertion of the N-GSDMD into the plasma membrane. GSDMD pores allows K+ efflux and the subsequent activation of the NLRP3 inflammasome, activating caspase-1. In the non-canonical inflammasome pathway, the activation of IL-1β and IL-18 depends on caspase-1, whereas the activation of GSDMD and pyroptosis development depends on caspase-4/5/11 [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ].
      Pathological functions for NLRP3 signaling in humans were first discovered in studies that characterized a group of rare autoinflammatory diseases called cryopyrin-associated periodic syndromes (CAPS). These patients harbor gain-of-function mutations in NLRP3 that promote inflammasome activation and result in an inflammation-related phenotype characterized by periodic fevers, sterile urticaria, and joint inflammation [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ,
      • Christgen S.
      • et al.
      Toward targeting inflammasomes: insights into their regulation and activation.
      ]. The wild-type NLRP3 inflammasome is activated and contributes to the inflammatory response during infection with viruses [including influenza, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), or chikungunya), fungi (e.g., Candida albicans), and bacteria [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ,
      • Vora S.M.
      • et al.
      Inflammasome activation at the crux of severe COVID-19.
      ]; here, the NLRP3 inflammasome not only mediates an important innate immune defense mechanism, but can also be responsible for immunopathology in some infection settings. NLRP3 targeting would be expected to have fewer immunosuppressive effects compared with existing anti-IL-1 therapies, because different pathogen-recognizing inflammasomes, such as NLRP1 and NLRC4, could still produce IL-1β [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ]. One peculiarity of the NLRP3 inflammasome is its propensity for dysregulation during the initiation and course of many chronic inflammatory diseases, including gout, rheumatoid arthritis, and inflammatory bowel disease (IBD) [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ]. The pathophysiological functions of the NLRP3 inflammasome are also emerging in several metabolic and neuroinflammatory diseases, in which NLRP3 appears to drive a subclinical inflammatory state several years before clinical symptoms appear. Therefore, NLRP3 is involved in the pathogenesis of atherosclerosis, diabetes, metabolic syndrome, and nonalcoholic steatohepatitis (NASH), as well as in pathologies of the central nervous system, including Alzheimer’s (AD) and Parkinson’s disease (PD), multiple sclerosis (MS), and stroke [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ].

      Drug-like small molecules blocking NLRP3

      Numerous small molecules that directly or indirectly inhibit NLRP3 signaling have been reported. Here, we focus on direct inhibitors of NLRP3 the MoAs of which are identified (Table 1 and Figure 2). The NACHT domain mediates the oligomerization of active NLRP3 and contains a nucleotide-binding domain (NBD) that hydrolyzes ATP. ATP binding and hydrolysis are essential for NLRP3 function, because mutating the Walker A ATP-binding site blocks NLRP3 signaling [
      • Sandall C.F.
      • et al.
      ATP-binding and hydrolysis in inflammasome activation.
      ]. Disrupting the ability of NLRP3 to hydrolyze ATP is a common mode of action for several small molecules (Table 1 and Figure 2).
      Before the description of the inflammasome in 2002, research at Pfizer identified a group of sulfonylurea-containing compounds (e.g., glyburide) that blocked IL-1β processing in response to prototypical NLRP3 stimuli, such as LPS and ATP [
      • Perregaux D.G.
      • et al.
      Identification and characterization of a novel class of interleukin-1 post-translational processing inhibitors.
      ]. Based on these studies, several cytokine release inhibitory drugs (CRIDs) were developed that blocked IL-1β release from human monocytes with reported IC50s in the nanomolar range [
      • Laliberte R.E.
      • et al.
      Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing.
      ]. In 2009, Lamkanfi and colleagues linked sulfonylureas to NLRP3 when they demonstrated that glyburide specifically inhibited NLRP3 signaling [
      • Lamkanfi M.
      • et al.
      Glyburide inhibits the cryopyrin/Nalp3 inflammasome.
      ]. Building on these observations, Coll et al. [
      • Coll R.C.
      • et al.
      A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.
      ] investigated one of the compounds developed at Pfizer called CRID3 (aka CP-456,773), renaming this compound MCC950. The authors demonstrated that MCC950 (CRID3) was a nanomolar potent and specific inhibitor of NLRP3 signaling that was active in several models of inflammatory disease (Table 1 and Figure 2) [
      • Coll R.C.
      • et al.
      A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.
      ]. MCC950 (CRID3) has since become the most widely used tool compound for NLRP3 inhibition in research studies, with MCC950 being the more commonly used name for this compound. MoA studies demonstrated that MCC950 directly and noncovalently interacts with the NACHT domain (Table 1 and Figure 2) [
      • Coll R.C.
      • et al.
      MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.
      ,
      • Tapia-Abellán A.
      • et al.
      MCC950 closes the active conformation of NLRP3 to an inactive state.
      ,
      • Vande Walle L.
      • et al.
      MCC950/CRID3 potently targets the NACHT domain of wildtype NLRP3 but not disease-associated mutants for inflammasome inhibition.
      ]. MCC950 blocks the ATPase activity of NLRP3 [
      • Coll R.C.
      • et al.
      MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.
      ], and intramolecular bioluminescence resonance energy transfer (BRET) assays show that MCC950 can shift NLRP3 from an active to an inactive conformation [
      • Tapia-Abellán A.
      • et al.
      MCC950 closes the active conformation of NLRP3 to an inactive state.
      ].
      Recently, the biophysical basis of the MoA of MCC950 has become clearer, with reports of the cryo-electron (cryo-EM) microscopy structure of full-length human NLRP3 in complex with ADP and MCC950 [
      • Hochheiser I.V.
      • et al.
      Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3.
      ] and the crystal structure of the human NLRP3 NACHT domain in complex with the MCC950 derivative NP3-146 [
      • Dekker C.
      • et al.
      Crystal structure of NLRP3 NACHT domain with an inhibitor defines mechanism of inflammasome inhibition.
      ]. Both of these structures of inactive NLRP3 observe that, remarkably, MCC950/NP3-146 forms interactions with multiple subdomains in NACHT, including the NBD, helical domain 1 (HD1), winged-helix domain (WHD), and helical domain 2 (HD2). Within this binding pocket, the central sulfonylurea of MCC950/NP3-146 forms a hydrogen bond with the Walker A site and also interacts with two key arginine residues (R351 and R578). These structures reveal how MCC950/NP3-146 specifically locks NLRP3 in an inactive state, preventing ATP hydrolysis and the structural rearrangements necessary to form an active oligomer that nucleates inflammasome assembly. Importantly, MCC950 also interacts with active NLRP3 and inhibits hyperactive CAPS-mutant NLRP3 [
      • Coll R.C.
      • et al.
      MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.
      ,
      • Tapia-Abellán A.
      • et al.
      MCC950 closes the active conformation of NLRP3 to an inactive state.
      ]. The structural basis for the ability of MCC950 to inhibit the active NLRP3 conformer remains to be determined. However, MCC950 does not inhibit the mouse NLRP3 L351P hyperactive mutation (corresponding to human L353P) because this mutation affects MCC950 binding to NLRP3 [
      • Vande Walle L.
      • et al.
      MCC950/CRID3 potently targets the NACHT domain of wildtype NLRP3 but not disease-associated mutants for inflammasome inhibition.
      ].
      Several MCC950 derivative molecules that potently inhibit NLRP3 are reported, including N-cyano-sulfoximineurea derivatives [
      • Agarwal S.
      • et al.
      Discovery of N-cyano-sulfoximineurea derivatives as potent and orally bioavailable NLRP3 inflammasome inhibitors.
      ] and alkenyl sulfonylurea derivatives [
      • Agarwal S.
      • et al.
      Identification of a novel orally bioavailable NLRP3 inflammasome inhibitor.
      ]. These derivatives may have a similar MoA to MCC950. As mentioned above, the type 2 diabetes mellitus (T2DM) drug, glyburide, is also a sulfonylurea that inhibits NLRP3 [
      • Lamkanfi M.
      • et al.
      Glyburide inhibits the cryopyrin/Nalp3 inflammasome.
      ] and several other sulfonylureas, such as sulofenur and glimepiride, can also inhibit NLRP3 signaling [
      • Hill J.R.
      • et al.
      Sulfonylureas as concomitant insulin secretagogues and NLRP3 inflammasome inhibitors.
      ]. Interestingly, when the cyclohexylurea group of glyburide is removed, it can still inhibit NLRP3 signaling [
      • Lamkanfi M.
      • et al.
      Glyburide inhibits the cryopyrin/Nalp3 inflammasome.
      ]; thus, the MoA of these sulfonylureas might be distinct from that of MCC950, and further investigations are required to determine whether they directly interact with NLRP3.
      Several covalent modifiers of the NLRP3 NACHT are now described. A compound screen for NLRP3 inhibitors identified the cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor C172, which prompted the authors to test the capacity of related compounds to inhibit NLRP3 activity [
      • Jiang H.
      • et al.
      Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders.
      ]. They identified CY-09, an analog of C172 that inhibits NLRP3 in the low micromolar range without affecting CFTR function. CY-09 directly interacts with NLRP3, as shown by microscale thermophoresis assays and interaction studies with biotinylated-CY-09 (Table 1 and Figure 2). CY-09 appears to compete with ATP for binding at the Walker A site to inhibit the ATPase activity of recombinant NLRP3 [
      • Jiang H.
      • et al.
      Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders.
      ].
      Several other compounds block the ATPase activity of NLRP3, but their binding sites are not yet identified. The vinyl sulfone Bay 11-7082, the sesquiterpene lactone parthenolide, and the benzoxathiole derivative BOT-4-one all block the ATPase activity of NLRP3 (Table 1 and Figure 2) [
      • Shim D.-W.
      • et al.
      BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination.
      ,
      • Juliana C.
      • et al.
      Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome.
      ], but also have other effects. For example, all three compounds inhibit IκB kinases (IKKs), which are part of the signaling pathway that activates the transcription factor nuclear factor (NF)-κB. NF-κB induces the transcription of pro-IL-1β and NLRP3, a process known as inflammasome priming [
      • Bauernfeind F.G.
      • et al.
      Cutting edge: NF-κB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression.
      ]. Therefore, compounds that inhibit NF-κB block priming and suppress IL-1β and NLRP3 activity independently of their direct effects on NLRP3. Parthenolide also inhibits caspase-1 and, thus, inhibits signaling by all canonical inflammasomes. In a small screen for kinase inhibitors, 3,4-methylenedioxy-β-nitrostyrene (MNS) was identified as a low micromolar inhibitor of NLRP3 signaling (Table 1 and Figure 2). This compound is a Syk kinase inhibitor, but suppresses NLRP3 activity independently of Syk by directly interacting with NLRP3 to block ATPase activity [
      • He Y.
      • et al.
      3,4-Methylenedioxy-β-nitrostyrene inhibits NLRP3 inflammasome activation by blocking assembly of the inflammasome.
      ]. OLT1177 (aka dapansutrile) is a β-sulfonyl nitrile compound that partially suppresses NLRP3 signaling (Table 1 and Figure 2). OLT1177 was shown to not only attenuate NLRP3 ATPase activity, but also significantly inhibit the NF-κB pathway in vivo, suggesting that it also affects NLRP3 priming [
      • Marchetti C.
      • et al.
      OLT1177, a β-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation.
      ].
      Based on previous work on electrophilic acrylic acids, Cocco and colleagues developed INF39 [
      • Cocco M.
      • et al.
      Development of an acrylate derivative targeting the NLRP3 inflammasome for the treatment of inflammatory bowel disease.
      ]. This compound is active in high micromolar concentrations (10–100 μM) and does not inhibit AIM2 or NLRC4 [
      • Shi Y.
      • et al.
      NLRP3 inflammasome inhibitor INF39 attenuated NLRP3 assembly in macrophages.
      ]. INF39 is a covalent inhibitor that attenuates NLRP3 structural changes as detected by BRET and suppresses NLRP3 ATPase activity (Table 1 and Figure 2). The capacity of NLRP3 to bind ATP can be exploited by using immobilized ATP matrices to purify NLRP3, which was the approach allowing Liao and colleagues [
      • Liao K.-C.
      • et al.
      Application of immobilized ATP to the study of NLRP inflammasomes.
      ] to identify a small molecule, HS-203873, that disrupted NLRP3–ATP interactions. HS-203873 was then confirmed to attenuate NLRP3 ATPase activity and signaling (Table 1 and Figure 2). Building on this work, a pharmacophore hybridization strategy using INF39 and HS-203873 identified several new compounds that could reduce NLRP3 ATPase activity. Molecular modeling of compounds such as INF120 with inactive ADP-bound NLRP3 suggests that they sit in a binding pocket that closely interacts with the ADP pocket [
      • Gastaldi S.
      • et al.
      Chemical modulation of the 1-(piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one scaffold as a novel NLRP3 inhibitor.
      ].
      Molecules that directly bind to NLRP3 and inhibit signaling by affecting protein–protein interactions (PPIs) are also identified. Oridonin is derived from traditional Chinese medicine and has broad anti-inflammatory activities, including inhibiting NLRP3 signaling. Microscale thermophoresis and pull-down studies with biotinylated oridonin confirmed that this compound directly interacts with NLRP3 to covalently modify the NLRP3 residue C279 in the NACHT domain (Table 1 and Figure 2); this attenuates NLRP3 interactions with Never in mitosis gen A-related kinase 7 (NEK7), suppressing inflammasome formation [
      • He H.
      • et al.
      Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.
      ]. The same authors also identified two other existing compounds, tranilast and RRx-001, as NLRP3 inhibitors. Tranilast is an antiallergy drug that inhibits NLRP3 signaling at micromolar concentrations. Based on pull-down studies with biotinylated tranilast, this drug appears to interact with the NLRP3 NACHT domain to disrupt NLRP3 intermolecular interactions, blocking NLRP3 oligomerization (Table 1 and Figure 2) [
      • Huang Y.
      • et al.
      Tranilast directly targets NLRP3 to treat inflammasome-driven diseases.
      ]. RRx-001 was developed as an anticancer molecule, but also potently inhibits NLRP3. RRx-001 covalently interacts with NLRP3 and blocks NLRP3–NEK7 interactions (Table 1 and Figure 2). Mutation of NLRP3 NACHT residue C409 abrogates the RRx-001 interaction, suggesting this as its binding site [
      • Chen Y.
      • et al.
      RRx-001 ameliorates inflammatory diseases by acting as a potent covalent NLRP3 inhibitor.
      ]. The hydroxyl sulfonamide JC-171 blocks NLRP3-dependent IL-1β release from mouse macrophages at micromolar concentrations. In co-immunoprecipitation assays, JC-171 decreased the levels of ASC pulled down by NLRP3, suggesting that it disrupts NLRP3–ASC PPIs (Table 1 and Figure 2) [
      • Guo C.
      • et al.
      Development and characterization of a hydroxyl-sulfonamide analogue, 5-chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-methoxy-benzamide, as a novel NLRP3 inflammasome inhibitor for potential treatment of multiple sclerosis.
      ].
      Blocking NLRP3 ATPase activity and PPIs has emerged as effective modes of NLRP3 inhibition. For potency and specificity, MCC950 remains the tool compound of choice for targeting NLRP3. Although reported off-target effects of MCC950 are minimal, a recent report observed that, at high micromolar concentrations, MCC950 could inhibit carbonic anhydrase 2 (CA2) (Table 1) [
      • Kennedy C.R.
      • et al.
      A probe for NLRP3 inflammasome inhibitor MCC950 identifies carbonic anhydrase 2 as a novel target.
      ]. This contrasts with a previous study that identified CA2 as a potential binding partner of CRIDs/MCC950, but found that these compounds did not affect CA2 enzymatic activity [
      • Laliberte R.E.
      • et al.
      Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing.
      ]. It has also been reported that MCC950 is able to block Cl efflux from nigericin-activated macrophages [
      • Jiang H.
      • et al.
      Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders.
      ]. Based on the recent cryo-EM and crystal structures of NLRP3 [
      • Andreeva L.
      • et al.
      NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation.
      ,
      • Hochheiser I.V.
      • et al.
      Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3.
      ,
      • Dekker C.
      • et al.
      Crystal structure of NLRP3 NACHT domain with an inhibitor defines mechanism of inflammasome inhibition.
      ], we anticipate that new structure-guided drug design, coupled with improved NLRP3 protein expression techniques, will facilitate the development of next-generation, highly specific NLRP3 inhibitors with distinct MoAs.

      Blocking GSDMD: novel pyroptotic inhibitors

      GSDMD is a key effector of inflammasome signaling, because it controls pyroptosis and the resultant release of proinflammatory cellular contents (Box 1). Deficiency in GSDMD dramatically decreases acute IL-1β release upon inflammasome activation [
      • Broz P.
      • et al.
      The gasdermins, a protein family executing cell death and inflammation.
      ]. Given that GSDMD controls the release of IL-1β downstream of multiple inflammasomes, GSDMD is an attractive target to pharmacologically modulate signaling by many inflammasomes in distinct diseases. Although pyroptosis inhibitors will block cell lysis and passive release of proinflammatory cell contents, they will not block the inflammasome-driven maturation of IL-1β or IL-18 or other backup forms of cell death; thus, cytokine-mediated inflammatory pathology remains possible despite GSDMD blockade. Indeed, although GSDMD deficiency slows the kinetics of IL-1β release [
      • Monteleone M.
      • et al.
      Interleukin-1β maturation triggers its relocation to the plasma membrane for gasdermin-D-dependent and -independent secretion.
      ], substantial GSDMD-independent IL-1β release is observed at late time points in specific settings [
      • Monteleone M.
      • et al.
      Interleukin-1β maturation triggers its relocation to the plasma membrane for gasdermin-D-dependent and -independent secretion.
      ].
      The first pyroptosis blocker described was the complex natural polyphenol, punicalagin, which specifically impairs inflammasome-driven membrane permeabilization without affecting caspase-1 activation or processing of IL-1β or GSDMD [
      • Martin-Sanchez F.
      • et al.
      Inflammasome-dependent IL-1β release depends upon membrane permeabilisation.
      ,
      • Torre-Minguela C.
      • et al.
      Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis.
      ]. Although its MoA is incompletely understood, punicalagin affects plasma membrane fluidity and, thus, may interfere with insertion or oligomerization of N-GSDMD in the plasma membrane [
      • Martin-Sanchez F.
      • et al.
      Inflammasome-dependent IL-1β release depends upon membrane permeabilisation.
      ]. Punicalagin is also a potent antioxidant, and reactive oxygen species (ROS) facilitate pore assembly by N-GSDMD [
      • Evavold C.L.
      • et al.
      Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.
      ]; therefore, punicalagin could also affect pyroptosis by scavenging ROS. Given that GSDMD oligomerization is blocked by reducing agents and the replacement of cysteines at amino acid positions 39 and 192 (homologs of human C38 and C191) to Ala impairs N-GSDMD oligomerization [
      • Liu X.
      • et al.
      Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.
      ], the formation of disulfide bonds between the Cys residues could promote oligomerization and pore formation. In fact, pharmacological agents that directly bind or modify GSDMD C191 impair pore formation and pyroptosis (Table 2), and this is the mechanism by which necrosulfonamide, dimethyl fumarate, and disulfiram block GSDMD activity [
      • Humphries F.
      • et al.
      Succination inactivates gasdermin D and blocks pyroptosis.
      ,
      • Hu J.J.
      • et al.
      FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.
      ,
      • Rathkey J.K.
      • et al.
      Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis.
      ]. Given that these compounds also react with exposed Cys residues of other proteins, they have several off-target effects (Table 2). For example, necrosulfonamide blocks mixed lineage kinase domain-like pseudokinase (MLKL) oligomerization and necroptosis [
      • Sun L.
      • et al.
      Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.
      ], whereas disulfiram also suppresses caspase-1 activity [
      • Hu J.J.
      • et al.
      FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.
      ] (Table 2).

      Targeting NLRP3 in preclinical disease models

      Given that the NLRP3 inflammasome is implicated in driving the pathophysiology of many diseases, numerous preclinical studies using small-molecule blockers targeting NLRP3 in distinct animal models of disease have been conducted. Here, we discuss the impact of NLRP3 inhibitors in animal models of metabolic, inflammatory, and neurological disease. We focus on these pathologies because they are common human diseases that lack effective therapies and, thus, present opportunities for widespread and significant impacts on patient care, being potential priority diseases for the clinical application of inflammasome inhibitors.

      Neurological disorders

      Neuroinflammation driven by the NLRP3 inflammasome has been found to be important in the aging brain; in neurodegenerative diseases, such as MS, AD, and PD; and in the detrimental response to stroke [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ]. Here, we summarize preclinical studies performed with NLRP3 inhibitors in rodent models of neurological diseases.

      Alzheimer's disease

      Neuroinflammation is considered a risk factor for the development of AD, and several innate immune and inflammatory pathways are associated with this condition [
      • Heneka M.T.
      • et al.
      Innate immune activation in neurodegenerative disease.
      ]. Among them, the NLRP3 inflammasome is activated in microglia by Aβ aggregates, inducing the release of IL-1β driving AD symptomatology [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ,
      • Heneka M.T.
      • et al.
      Innate immune activation in neurodegenerative disease.
      ]. The nonsteroidal anti-inflammatory drug fenamate is an indirect blocker of NLRP3 activation via inhibition of the volume-regulated anion channel. Fenamate has therapeutic effects in a model of memory loss induced by Aβ administration and in the triple-transgenic mouse model of AD expressing transgenes for PS1 (methionine 146 to valine mutation), APPSwe, and Tau (proline 301 to leucine mutation), a mouse model that progressively develops both Aβ deposition and Tau pathology [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ]. This shows that AD symptoms can be ameliorated by blocking the inflammatory response. Indeed, other NLRP3 blockers, such as MCC950 and OLT1177 (Table 1 and Figure 2), restored cognitive function, including learning and memory ability, in the amyloid precursor protein (APP)/presenilin 1 mouse model of AD [
      • Dempsey C.
      • et al.
      Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-β and cognitive function in APP/PS1 mice.
      ,
      • Lonnemann N.
      • et al.
      The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer’s disease.
      ]. MCC950 also suppressed disease in other animal models of AD, including genetic models (e.g., senescence accelerated mouse-prone 8) and models of induced disease (in which disease is induced by the intracerebroventricular infusion of Aβ oligomers, or by streptozotocin), in which MCC950 improves spatial memory ability and abolishes memory impairments [
      • Li J.
      • et al.
      Protection of MCC950 against Alzheimer’s disease via inhibiting neuronal pyroptosis in SAMP8 mice.
      ,
      • Fekete C.
      • et al.
      Chronic amyloid β oligomer infusion evokes sustained inflammation and microglial changes in the rat hippocampus via NLRP3.
      ,
      • He X.-F.
      • et al.
      NLRP3-dependent microglial training impaired the clearance of amyloid-beta and aggravated the cognitive decline in Alzheimer’s disease.
      ]. In addition, MCC950 impaired exogenously seeded Tau pathology in Tau transgenic mice and blocked Tau-induced IL-1β release in microglia [
      • Stancu I.-C.
      • et al.
      Aggregated Tau activates NLRP3-ASC inflammasome exacerbating exogenously seeded and non-exogenously seeded Tau pathology in vivo.
      ,
      • Ising C.
      • et al.
      NLRP3 inflammasome activation drives tau pathology.
      ]. Thus, NLRP3 is a promising therapeutic target for preventing the neuroinflammation associated with AD, to improve cognitive function.

      Parkinson's disease

      The development of PD is accompanied by the loss of dopaminergic neurons, the appearance of α-synuclein aggregates in the form of Lewy bodies, the activation of microglia, and the increase of proinflammatory cytokines, such as IL-1β. Aggregates of α-synuclein activate NLRP3 in microglia and induce the release of IL-1β [
      • Heneka M.T.
      • et al.
      Innate immune activation in neurodegenerative disease.
      ]. In several mouse models of PD, MCC950 (Table 1 and Figure 2) administration improved behavioral dysfunctions by mitigating motor deficits, reducing nigrostriatal dopaminergic degeneration, and decreasing neuroinflammation, alongside inhibiting proinflammatory microglia activation [
      • Cheng J.
      • et al.
      Microglial autophagy defect causes Parkinson disease-like symptoms by accelerating inflammasome activation in mice.
      ,
      • Gordon R.
      • et al.
      Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice.
      ,
      • Huang S.
      • et al.
      A selective NLRP3 inflammasome inhibitor attenuates behavioral deficits and neuroinflammation in a mouse model of Parkinson’s disease.
      ]. Therefore, NLRP3 blockers are potential disease-modifying treatments for PD.

      Multiple sclerosis

      Chronic inflammatory demyelination of central nervous system neurons characterizes MS, which gives rise to diffuse neurodegeneration in the entire brain [
      • Lünemann J.D.
      • et al.
      Targeting inflammasomes to treat neurological diseases.
      ]. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model of MS. Although MS is a complex condition, EAE reproduces the key pathological features of MS, such as axonal demyelination and inflammation. In EAE, several NLRP3 blockers, namely, MCC950, OLT1177, and JC-171 (Table 1 and Figure 2), can attenuate disease severity and prevent cognitive and functional deficits and can also improve hippocampal pathology and reduce neuronal demyelination [
      • Coll R.C.
      • et al.
      A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.
      ,
      • Guo C.
      • et al.
      Development and characterization of a hydroxyl-sulfonamide analogue, 5-chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-methoxy-benzamide, as a novel NLRP3 inflammasome inhibitor for potential treatment of multiple sclerosis.
      ,
      • Sánchez-Fernández A.
      • et al.
      OLT1177 (dapansutrile), a selective NLRP3 inflammasome inhibitor, ameliorates experimental autoimmune encephalomyelitis pathogenesis.
      ,
      • Hou B.
      • et al.
      Inhibition of the NLRP3-inflammasome prevents cognitive deficits in experimental autoimmune encephalomyelitis mice via the alteration of astrocyte phenotype.
      ,
      • Malhotra S.
      • et al.
      NLRP3 inflammasome as prognostic factor and therapeutic target in primary progressive multiple sclerosis patients.
      ]. In particular, JC-171 was effective in both prophylactic and therapeutic settings, suggesting that NLRP3 blockers have potential as treatment for established disease in MS [
      • Guo C.
      • et al.
      Development and characterization of a hydroxyl-sulfonamide analogue, 5-chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-methoxy-benzamide, as a novel NLRP3 inflammasome inhibitor for potential treatment of multiple sclerosis.
      ]. Indeed, patients with MS with increased inflammasome function and IL-1β production have significantly faster disease progression [
      • Malhotra S.
      • et al.
      NLRP3 inflammasome as prognostic factor and therapeutic target in primary progressive multiple sclerosis patients.
      ].

      Stroke

      The NLRP3 inflammasome drives inflammation in ischemia–reperfusion injury after transient middle cerebral artery occlusion. Treatment with CY-09 or MCC950 (Table 1 and Figure 2) in this model attenuates neurological deficits and long-term cognitive dysfunction [
      • Sun R.
      • et al.
      Low-density lipoprotein receptor (LDLR) regulates NLRP3-mediated neuronal pyroptosis following cerebral ischemia/reperfusion injury.
      ] as well as reduces infarct volume and protects blood–brain barrier integrity [
      • Franke M.
      • et al.
      The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice.
      ,
      • Palomino-Antolin A.
      • et al.
      Time-dependent dual effect of NLRP3 inflammasome in brain ischaemia.
      ,
      • Bellut M.
      • et al.
      NLPR3 inflammasome inhibition alleviates hypoxic endothelial cell death in vitro and protects blood-brain barrier integrity in murine stroke.
      ,
      • Ismael S.
      • et al.
      Inhibition of the NLRP3-inflammasome as a potential approach for neuroprotection after stroke.
      ,
      • Wang H.
      • et al.
      NLRP3 inflammasome activates interleukin-23/interleukin-17 axis during ischaemia-reperfusion injury in cerebral ischaemia in mice.
      ]. MCC950 also reduces brain injury and inflammation in a model of intracerebral hemorrhage and in the control cortical impact model of experimental traumatic brain injury [
      • Ren H.
      • et al.
      Selective NLRP3 (pyrin domain-containing protein 3) inflammasome inhibitor reduces brain injury after intracerebral hemorrhage.
      ,
      • Ismael S.
      • et al.
      MCC950, the selective inhibitor of nucleotide oligomerization domain-like receptor protein-3 inflammasome, protects mice against traumatic brain injury.
      ]. However, MCC950 treatment did not affect injury caused by ferric chloride (FeCl3) in a model of thrombotic stroke [
      • Lemarchand E.
      • et al.
      Extent of ischemic brain injury after thrombotic stroke is independent of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome.
      ]. The disease-driving role of the NLRP3 inflammasome following stroke suggests that NLRP3 blockers have therapeutic potential in stroke.

      Metabolic disorders

      Type 2 diabetes mellitus

      A low-grade chronic inflammatory response is one mechanism leading to the development of insulin resistance, T2DM, and associated comorbidities, including liver and kidney damage. In particular, the NLRP3 inflammasome is activated in response to several altered metabolic signals in T2DM, leading not only to the activation of caspase-1 and production of IL-1β, but also to pancreatic β cell failure [
      • He H.
      • et al.
      Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.
      ,
      • Huang Y.
      • et al.
      Tranilast directly targets NLRP3 to treat inflammasome-driven diseases.
      ]. The NLRP3 blockers CY-09, tranilast, and oridonin (Table 1 and Figure 2) have proven successful for reducing body weight and blood glucose, as well as promoting insulin sensitivity in a high-fat diet-induced animal model of T2DM [
      • Jiang H.
      • et al.
      Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders.
      ,
      • He H.
      • et al.
      Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.
      ,
      • Huang Y.
      • et al.
      Tranilast directly targets NLRP3 to treat inflammasome-driven diseases.
      ]. In keeping with this, the NLRP3 inhibitor MCC950 (Table 1 and Figure 2) was able to reduce body weight and blood glucose and increase insulin sensitivity in aged mice, suggesting that NLRP3 inhibitors provide therapy in age-related metabolic syndrome [
      • Marín-Aguilar F.
      • et al.
      NLRP3 inflammasome inhibition by MCC950 in aged mice improves health via enhanced autophagy and PPARα activity.
      ]. However, in the diabetic db/db mice, a genetic murine model of diabetes, MCC950 did not affect blood glucose or insulin resistance [
      • Kammoun H.L.
      • et al.
      Evidence against a role for NLRP3-driven islet inflammation in db/db mice.
      ,
      • Zhang C.
      • et al.
      A small molecule inhibitor MCC950 ameliorates kidney injury in diabetic nephropathy by inhibiting NLRP3 inflammasome activation.
      ]. However, MCC950 did improve several diabetes-associated comorbidities, including renal failure in diabetic db/db mice, which develop kidney injury [
      • Zhang C.
      • et al.
      A small molecule inhibitor MCC950 ameliorates kidney injury in diabetic nephropathy by inhibiting NLRP3 inflammasome activation.
      ,
      • Wu M.
      • et al.
      Inhibition of NLRP3 inflammasome ameliorates podocyte damage by suppressing lipid accumulation in diabetic nephropathy.
      ], and vascular endothelial dysfunction and atherosclerosis in adiponectin- or apolipoprotein-knockout mice, respectively [
      • Zhang J.
      • et al.
      A novel mechanism of diabetic vascular endothelial dysfunction: hypoadiponectinemia-induced NLRP3 inflammasome activation.
      ,
      • Sharma A.
      • et al.
      Specific NLRP3 inhibition protects against diabetes-associated atherosclerosis.
      ]. However, MCC950 increased renal inflammation in streptozotocin-induced diabetic mice [
      • Østergaard J.A.
      • et al.
      Adverse renal effects of NLRP3 inflammasome inhibition by MCC950 in an interventional model of diabetic kidney disease.
      ]. MCC950 ameliorated the neurological deficit and encephalopathy observed in the diabetic db/db mice and in the high-fat diet-induced diabetes model, improving anxiety- and depression-like behaviors [
      • Zhai Y.
      • et al.
      Inhibiting the NLRP3 inflammasome activation with MCC950 ameliorates diabetic encephalopathy in db/db mice.
      ,
      • Ward R.
      • et al.
      NLRP3 inflammasome inhibition with MCC950 improves diabetes-mediated cognitive impairment and vasoneuronal remodeling after ischemia.
      ,
      • Hong P.
      • et al.
      Inhibition of NLRP3 inflammasome ameliorates cerebral ischemia-reperfusion injury in diabetic mice.
      ]. Glyburide treatment in patients with diabetes reduced mortality in sepsis compared with patients not taking glyburide or in patients without diabetes, suggesting that glyburide exerts anti-inflammatory effects on the immune system by blocking the NLRP3 inflammasome [
      • Koh G.C.K.W.
      • et al.
      Glyburide is anti-inflammatory and associated with reduced mortality in melioidosis.
      ]. The NLRP3 inhibitor CY-09 (Table 1 and Figure 2) also protected against diabetic retinopathy and acute liver injury in a streptozotocin-induced hyperglycemic mouse model [
      • Zou W.
      • et al.
      ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributes to aberrant retinal angiogenesis in diabetic retinopathy.
      ,
      • Wang Q.
      • et al.
      Hyperglycemia aggravates acute liver injury by promoting liver-resident macrophage NLRP3 inflammasome activation via the inhibition of AMPK/mTOR-mediated autophagy induction.
      ].

      NASH, liver damage, and fibrosis

      Liver steatosis or nonalcoholic fatty liver disease (NAFLD) is the result of the accumulation of fat in the liver and is a common complication in diabetes. Liver steatosis could evolve to NASH when inflammatory cytokines are produced in the liver, promoting hepatic inflammation, fibrosis, and loss of function. Liver steatosis, NASH, and fibrosis are all associated with NLRP3 inflammasome signaling [
      • Mridha A.R.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      ]. A high-fat diet in mice is commonly used to model NAFLD, and the use of the NLRP3 blockers CY-09, tranilast, or oridonin (Table 1 and Figure 2) decreased the accumulation of intracellular lipid droplets in the liver and improved liver steatosis [
      • Jiang H.
      • et al.
      Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders.
      ,
      • He H.
      • et al.
      Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.
      ,
      • Wang X.
      • et al.
      NLRP3 inflammasome inhibitor CY-09 reduces hepatic steatosis in experimental NAFLD mice.
      ,
      • Sun K.
      • et al.
      Sleeve gastroplasty combined with the NLRP3 inflammasome inhibitor CY-09 reduces body weight, improves insulin resistance and alleviates hepatic steatosis in mouse model.
      ]. In particular, CY-09 also improved hepatic steatosis after bariatric surgery [
      • Sun K.
      • et al.
      Sleeve gastroplasty combined with the NLRP3 inflammasome inhibitor CY-09 reduces body weight, improves insulin resistance and alleviates hepatic steatosis in mouse model.
      ] and was able to suppress inflammatory cytokine expression in the liver [
      • Jiang H.
      • et al.
      Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders.
      ]. Liver steatosis is also an age-related symptom and, in old mice, treatment with MCC950 reduced liver fat accumulation and improved hepatic function [
      • Marín-Aguilar F.
      • et al.
      NLRP3 inflammasome inhibition by MCC950 in aged mice improves health via enhanced autophagy and PPARα activity.
      ]. A diet deficient in methionine and choline results in liver inflammation, NASH, and fibrosis, and co-administration of NLRP3 inhibitors (MCC950, tranilast, or IFM-514; Table 1 and Figure 2) with this diet suppressed the severity of hepatic inflammation and fibrosis [
      • Mridha A.R.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      ,
      • Uno M.
      • et al.
      Tranilast, an antifibrogenic agent, ameliorates a dietary rat model of nonalcoholic steatohepatitis.
      ,
      • Torres S.
      • et al.
      The specific NLRP3 antagonist IFM-514 decreases fibrosis and inflammation in experimental murine non-alcoholic steatohepatitis.
      ]. NLRP3 blockade also decreased macrophage and neutrophil infiltration to the liver [
      • Mridha A.R.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      ] and caspase-1 activation [
      • Torres S.
      • et al.
      The specific NLRP3 antagonist IFM-514 decreases fibrosis and inflammation in experimental murine non-alcoholic steatohepatitis.
      ] and downregulated expression of TGF-β and its target genes, which drive collagen production [
      • Uno M.
      • et al.
      Tranilast, an antifibrogenic agent, ameliorates a dietary rat model of nonalcoholic steatohepatitis.
      ]. Liver fibrosis also occurs in the helminthic infectious disease schistosomiasis, in which MCC950 treatment reduced infection-induced hepatic fibrosis markers [
      • Zhang W.-J.
      • et al.
      NLRP3 inflammasome activation from Kupffer cells is involved in liver fibrosis of Schistosoma japonicum-infected mice via NF-κB.
      ]. Liver injury and fibrosis also develop during bile duct ligation, in which MCC950 treatment to inhibit NLRP3 activation and IL-1β and IL-18 production alleviated neutrophil infiltration and hepatic cell death [
      • Qu J.
      • et al.
      The selective NLRP3 inflammasome inhibitor MCC950 alleviates cholestatic liver injury and fibrosis in mice.
      ]. Alcohol addiction and severe alcohol consumption is a frequent cause of liver damage and fibrosis. The NLRP3 blocker oridonin (Table 1 and Figure 2) attenuated liver inflammation and injury in a model of chronic alcohol consumption [
      • Yan S.-L.
      • et al.
      Oridonin attenuates the effects of chronic alcohol consumption inducing oxidative, glycative and inflammatory injury in the mouse liver.
      ]. Intriguingly, MCC950 also reduced alcohol consumption and preference [
      • Lowe P.P.
      • et al.
      Inhibition of the inflammasome signaling cascade reduces alcohol consumption in female but not male mice.
      ], and the alcoholism drug disulfiram (Table 2) suppressed NLRP3 signaling by inhibiting GSDMD [
      • Hu J.J.
      • et al.
      FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.
      ]. NLRP3 blockers were also successfully used in other types of hepatic damage, in particular in models of acute liver injury induced by thioacetamide treatment during severe hyperglycemia. In this model, CY-09 (Table 1 and Figure 2) alleviated acute liver injury by reducing levels of transaminases in blood, improving histological liver damage, and decreasing hepatic cell death [
      • Wang Q.
      • et al.
      Hyperglycemia aggravates acute liver injury by promoting liver-resident macrophage NLRP3 inflammasome activation via the inhibition of AMPK/mTOR-mediated autophagy induction.
      ]. Similarly, oridonin treatment attenuated carbon tetrachloride-induced chronic liver injury and fibrosis [
      • Liu D.
      • et al.
      Oridonin ameliorates carbon tetrachloride-induced liver fibrosis in mice through inhibition of the NLRP3 inflammasome.
      ].

      Intestinal inflammation

      Although the NLRP3 inflammasome is implicated in the development of IBD, including Crohn’s disease and ulcerative colitis [
      • Mangan M.S.J.
      • et al.
      Targeting the NLRP3 inflammasome in inflammatory diseases.
      ], the etiology of these diseases in humans is complex and associated with chronic dysfunction of the immune response in the intestine. Animal models of intestinal inflammation often include toxic compounds in the drinking water to induce intestinal epithelial damage and subsequent inflammation; here, the most widely used chemical is synthetic sulfated polysaccharide dextran sulfate sodium (DSS) [
      • Eichele D.D.
      • Kharbanda K.K.
      Dextran sodium sulfate colitis murine model: an indispensable tool for advancing our understanding of inflammatory bowel diseases pathogenesis.
      ]. DSS activates pathological NLRP3 inflammasome signaling, because DSS-induced IBD is a model in which inflammatory symptoms strongly depend on this inflammasome [
      • Bauer C.
      • et al.
      Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome.
      ]. The NLRP3 blockers MCC950, OLT1177, INF39, tranilast, or the benzo[d]imidazole derivate Fc11a-2 (Table 1 and Figure 2) successfully treated DSS-induced colitis [
      • Wu D.
      • et al.
      Formononetin administration ameliorates dextran sulfate sodium-induced acute colitis by inhibiting NLRP3 inflammasome signaling pathway.
      ,
      • Wohlford G.F.
      • et al.
      Phase 1B, randomized, double-blinded, dose escalation, single-center, repeat dose safety and pharmacodynamics study of the oral NLRP3 inhibitor dapansutrile in subjects with NYHA II-III systolic heart failure.
      ,
      • Umiker B.
      • et al.
      The NLRP3 inflammasome mediates DSS-induced intestinal inflammation in Nod2 knockout mice.
      ,
      • Neudecker V.
      • et al.
      Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome.
      ,
      • Oizumi T.
      • et al.
      NLRP3 inflammasome inhibitor OLT1177 suppresses onset of inflammation in mice with dextran sulfate sodium-induced colitis.
      ,
      • Pu Z.
      • et al.
      Systematic understanding of the mechanism and effects of arctigenin attenuates inflammation in dextran sulfate sodium-induced acute colitis through suppression of NLRP3 inflammasome by SIRT1.
      ,
      • Sun X.
      • et al.
      Rectal administration of tranilast ameliorated acute colitis in mice through increased expression of heme oxygenase-1.
      ]. In particular, these compounds improved body-weight gain, decreased disease activity index, attenuated colon shortening, and reduced histopathological scores of intestinal damage and intestinal macroscopic damage index [
      • Wu D.
      • et al.
      Formononetin administration ameliorates dextran sulfate sodium-induced acute colitis by inhibiting NLRP3 inflammasome signaling pathway.
      ,
      • Wohlford G.F.
      • et al.
      Phase 1B, randomized, double-blinded, dose escalation, single-center, repeat dose safety and pharmacodynamics study of the oral NLRP3 inhibitor dapansutrile in subjects with NYHA II-III systolic heart failure.
      ,
      • Umiker B.
      • et al.
      The NLRP3 inflammasome mediates DSS-induced intestinal inflammation in Nod2 knockout mice.
      ,
      • Neudecker V.
      • et al.
      Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome.
      ,
      • Oizumi T.
      • et al.
      NLRP3 inflammasome inhibitor OLT1177 suppresses onset of inflammation in mice with dextran sulfate sodium-induced colitis.
      ,
      • Pu Z.
      • et al.
      Systematic understanding of the mechanism and effects of arctigenin attenuates inflammation in dextran sulfate sodium-induced acute colitis through suppression of NLRP3 inflammasome by SIRT1.
      ,
      • Sun X.
      • et al.
      Rectal administration of tranilast ameliorated acute colitis in mice through increased expression of heme oxygenase-1.
      ]. When combined with other drugs, NLRP3 inhibitors potentiated their protective effect in DSS-induced colitis, suggesting their use in preventing the onset of colitis. MCC950 synergized with the NF-κB inhibitor celastrol [
      • Saber S.
      • et al.
      Celastrol augments sensitivity of NLRP3 to CP-456773 by modulating HSP-90 and inducing autophagy in dextran sodium sulphate-induced colitis in rats.
      ] and metformin [
      • Saber S.
      • El-Kader E.M.A.
      Novel complementary coloprotective effects of metformin and MCC950 by modulating HSP90/NLRP3 interaction and inducing autophagy in rats.
      ]. OLT1177 (Table 1 and Figure 2) similarly boosted the effect of the purinergic inhibitor brilliant blue G [
      • Saber S.
      • et al.
      BBG enhances OLT1177-induced NLRP3 inflammasome inactivation by targeting P2X7R/NLRP3 and MyD88/NF-κB signaling in DSS-induced colitis in rats.
      ], and INF39 potentiated the effects of arctigenin, a phenylpropanoid dibenzylbutyrolactone ligand [
      • Pu Z.
      • et al.
      Systematic understanding of the mechanism and effects of arctigenin attenuates inflammation in dextran sulfate sodium-induced acute colitis through suppression of NLRP3 inflammasome by SIRT1.
      ]. However, therapeutic treatment with OLT1177 after murine DSS treatment and the establishment of disease did not affect colitis disease indices [
      • Oizumi T.
      • et al.
      NLRP3 inflammasome inhibitor OLT1177 suppresses onset of inflammation in mice with dextran sulfate sodium-induced colitis.
      ]. In other models of colitis, NLRP3 blockers improved intestinal disease. In the Winnie mouse model of spontaneous chronic colitis, MCC950 treatment improved body-weight gain, colon length, disease activity index, and histopathological scores and reduced proinflammatory cytokine production [
      • Perera A.P.
      • et al.
      MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice.
      ]. Colitis caused by di- or trinitrobenzene sulfonic acid or by oxazolone was ameliorated by treatment with the NLRP3 blockers INF39, oridonin, and tranilast (Table 1 and Figure 2) [
      • Cocco M.
      • et al.
      Development of an acrylate derivative targeting the NLRP3 inflammasome for the treatment of inflammatory bowel disease.
      ,
      • Pellegrini C.
      • et al.
      A comparative study on the efficacy of NLRP3 inflammasome signaling inhibitors in a pre-clinical model of bowel inflammation.
      ,
      • Chu H.Q.
      • et al.
      Protective effects of tranilast on oxazolone-induced rat colitis through a mast cell-dependent pathway.
      ,
      • Wang S.
      • et al.
      Oridonin’s therapeutic effect: suppressing Th1/Th17 simultaneously in a mouse model of Crohn’s disease.
      ]. In these models, NLRP3 inhibitors suppressed Th1/Th17 cells and reduced mast cell, neutrophil, and macrophage infiltration in the intestine [
      • Cocco M.
      • et al.
      Development of an acrylate derivative targeting the NLRP3 inflammasome for the treatment of inflammatory bowel disease.
      ,
      • Pellegrini C.
      • et al.
      A comparative study on the efficacy of NLRP3 inflammasome signaling inhibitors in a pre-clinical model of bowel inflammation.
      ,
      • Chu H.Q.
      • et al.
      Protective effects of tranilast on oxazolone-induced rat colitis through a mast cell-dependent pathway.
      ,
      • Wang S.
      • et al.
      Oridonin’s therapeutic effect: suppressing Th1/Th17 simultaneously in a mouse model of Crohn’s disease.
      ]. Therefore, NLRP3 blockade is an interesting and promising strategy for the management of bowel inflammation.

      Concluding remarks and future perspectives

      Although some NLRP3 inhibitors are entering early clinical trials (Box 2), the best clinical indications for NLRP3 inflammasome blockade remain to be determined. Choosing an indication for NLRP3 inhibitors is challenging given the breadth of conditions that are associated with NLRP3 (see Outstanding questions). Early proof-of-concept trials will likely focus on CAPS, a set of rare NLRP3 hereditary diseases, but given limited patient populations, other conditions must be chosen to robustly test NLRP3 inhibitor efficacy. Despite the success of NLRP3 blockers in preventing or treating over a hundred preclinical animal models of disease, we need to determine when, where, and how NLRP3 is activated in human disease to be able to translate preclinical studies into effective clinical therapies. In any case, preclinical models suggest that chronic inflammation related to NLRP3-activating mutations, digestive, metabolic, or neurodegenerative diseases would be promising indications for Phase II clinical trials. However, such trials require patient stratification to enable selection of individuals with strong NLRP3 activation. There is currently a lack of biomarkers to indicate NLRP3 inflammasome activity and for assessing target engagement. Approaches such as measuring extracellular vesicle RNA signatures specifically associated with NLRP3 or determining NLRP3 activity in blood cells could be fruitful [
      • Budden C.F.
      • et al.
      Inflammasome-induced extracellular vesicles harbour distinct RNA signatures and alter bystander macrophage responses.
      ,
      • Martínez-García J.J.
      • et al.
      P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis.
      ]. The involvement of other inflammasomes in diseases is still emerging, with several nonmicrobial activators of NLRC4, AIM2, PYD, and NLRP1 inflammasomes now identified. Therapies that block the action of multiple inflammasomes, such as molecules that block ASC oligomerization [
      • Soriano-Teruel P.M.
      • et al.
      Identification of an ASC oligomerization inhibitor for the treatment of inflammatory diseases.
      ], could be promising approaches as pan-inflammasome therapeutics. Similarly, compounds that block GSDMD and pyroptosis to suppress the execution phase of inflammasome signaling are emerging as novel approaches to ameliorate inflammation, but could be bypassed by the execution of alternative forms of cell death. Thus, inflammasome blockers show enormous promise as a new generation of anti-inflammatory drugs for a wide range of conditions currently lacking curative treatments.
      What clinical applications will show the best efficacy for NLRP3-targeting molecules?
      When, where, and how is NLRP3 activated in human disease?
      Would pan-inflammasome inhibitors offer additional therapeutic benefit to NLRP3-specific blockers?
      Will GSDMD and pyroptosis inhibitors be effective anti-inflammatory therapies?

      Acknowledgments

      K.S. is supported by the National Health and Medical Research Council of Australia (Fellowships 1141131 and 2009075 and grant 2009677 ). R.C.C. is supported by an Academy of Medical Sciences Springboard Award ( SBF005\1104 ), a Royal Society Research Grant ( RGS\R1\201127 ), and a Biotechnology and Biological Sciences Research Council New Investigator Research Grant ( BB/V016741/1 ). P.P. is supported by the MCIN/AEI/10.13039/501100011033 (grant PID2020-116709RB-I00), the Fundación Séneca (grants 20859/PI/18 , 21081/PDC/19, and 0003/COVI/20 ), the Instituto Salud Carlos III (grant DTS21/00080 ), the EU Horizon 2020 project PlasticHeal (grant 965196 ), and the European Research Council (grant ERC-2019-PoC 899636 ).

      Declaration of interests

      R.C.C. and K.S. are co-inventors on patent applications for NLRP3 inhibitors, which have been licensed to Inflazome Ltd. K.S. served on the Scientific Advisory Board of Inflazome in 2016–2017 and serves as a consultant to Quench Bio and Novartis. R.C.C. is a consultant for BioAge Labs. P.P. is co-inventor on a patent application to use the NLRP3 inflammasome as biomarker of disease, which has been licensed to Viva In Vitro Diagnostics SL, a company co-funded by P.P. P.P. is also scientific consultant of Glenmark Ltd.

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      Glossary

      Cryopyrin-associated periodic syndromes (CAPS)
      rare autoinflammatory disorders resulting from gain-of-function mutations in NLRP3 and characterized by periodic fevers, sterile urticaria, and joint inflammation. CAPS incorporate three overlapping disease entities: familial cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome (MWS), and neonatal-onset multisystem autoinflammatory syndrome (NOMID).
      Gasdermin D (GSDMD)
      pore-forming protein that induces pyroptosis downstream of inflammasome activation.
      Inflammasome
      signaling complex that activates inflammatory caspases. Most inflammasomes are multiprotein assemblies that activate the caspase-1 protease. The so-called ‘non-canonical inflammasome’ is a protein–lipid assembly that activates the protease function of caspase-11 in mice and caspase-4 and caspase-5 in humans.
      Interleukin-1 (IL-1)
      a family of 11 cytokines that includes IL-1β and IL-18. IL-1β and IL-18 are produced as inactive precursor proteins that require caspase-1 cleavage to generate their secreted, bioactive form.
      NACHT
      NAIP (NLR family apoptosis inhibitory protein), CIITA (i.e., C2TA or MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina), and TEP1 (i.e., TP1 or telomerase-associated protein) is the central nucleotide-binding and oligomerization domain of NLR receptors, including NLRP3. This acronym reflects some of the proteins that contain this particular domain.
      Nucleotide-binding oligomerization domain, leucine-rich repeat receptor and pyrin-domain containing-protein 3 (NLRP3)
      sensor protein of the NLRP3 inflammasome.
      Purinergic receptor type 2, family X, subunit 7 (P2X7)
      ATP-gated cationic receptor that effluxes potassium from the cells, inducing NLRP3 inflammasome activation.