Targeting the NLRP3 inflammasome for inflammatory disease therapy

Abstract

The NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is a megadalton complex implicated in numerous inflammation-driven diseases including COVID-19, Alzheimer’s disease, and gout. Although past efforts have focused on inhibiting IL-1β downstream of NLRP3 activation using drugs such as canakinumab, no FDA-approved NLRP3-targeted inhibitors are currently available. MCC950, a direct NLRP3 inhibitor, showed promise but exhibited off-target effects. Recent research has focused on optimizing the sulfonylurea-based MCC950 scaffold by leveraging recent structural and medicinal chemistry insights into the NLRP3 nucleotide-binding and oligomerization (NACHT) domain to improve solubility and clinical efficacy. In addition, oxidized DNA (oxDNA) has emerged as a key inflammasome trigger, and molecules targeting the pyrin domain have shown promise in inhibiting NLRP3 activation. This review discusses the role of NLRP3 in inflammation-related diseases, the status of ongoing clinical trials, and emerging small-molecule therapeutics targeting NLRP3.

NLRP3: a promising therapeutic target for inflammatory diseases

The NLRP3 inflammasome (Box 1) assembles into a large protein complex which triggers the release of proinflammatory cytokines, such as IL-1β and IL-18, that contribute to inflammation and immune responses [1] (Figure 1). However, dysregulated activation of NLRP3 is linked to several inflammatory diseases. Despite efforts to design specific NLRP3 small-molecule inhibitors [2], none has been FDA-approved, raising concerns about their clinical efficacy and potential off-target effects (Box 2). Although biologics including IL-1β blockers can help to reduce inflammation, they do not directly prevent NLRP3 activation and allow continued production of IL-1β. In some diseases it may be desirable to inhibit upstream activation of NLRP3 to more directly control inflammation [3].

Box 1. Biphasic activation of the NLRP3 inflammasome results in gasdermin D pore formation and cell death.

The NLRP3 inflammasome is a multi-domain protein complex that is crucial for immune responses by detecting infections and initiating inflammation. Although NLRP3, part of the nucleotide-binding oligomerization domain (NOD)-like receptor family, is well studied, uncontrolled activation can lead to sepsis, organ failure, autoimmune disorders, and cancer. The 1.2 MDa NLRP3 inflammasome comprises NLRP3 monomers (pyrin, NACHT, and LRR domains), NEK7, ASC, and pro-caspase-1 [71]. Activation occurs in two steps: priming and activation (see Figure 1 in the main text) [72]. Priming is initiated when pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) bind to TLR2/4 receptors to activate MyD88 [73] and trigger NF-κB nuclear translocation. NF-κB upregulates the IL1B, IL18, and NLRP3 genes [74]. ASC, NEK7, and pro-caspase-1 are constitutively expressed. External IL-1β amplifies this process via IL-1R signaling [75]. Activation involves damage-associated molecular patterns (DAMPs) such as extracellular ATP, mitochondrial/nuclear DNA, uric acid crystals, and environmental toxicants {e.g., monosodium urate (MSU) [76], amyloid-β, and asbestos}. ATP binds to P2X7, causing K⁺ efflux and Ca²⁺ influx [77,78]. Nigericin, a potassium ionophore, mimics this effect. Mitochondrial calcium overload, via the mitochondrial calcium uniporter (MCU), disrupts membrane potential, leading to ROS accumulation and oxidation of guanine-rich D-loop mtDNA, forming ox-mtDNA [79]. Low levels of this DNA damage are mitigated by DNA repair via the human DNA glycosylase (hOGG1). Macrophages also treat their mitochondria as expendable and may undergo mitophagy, thereby inhibiting inflammasome activation [80,81]. Ox-mtDNA may undergo cleavage into smaller fragments by Flap endonuclease 1 (FEN1) [82]. Cleaved oxDNA exits mitochondria via the mitochondrial permeability transition pore (mPTP) and voltage-dependent anion channels (VDACs) [82,83]. OxDNA binds to cytosolic NLRP3, and triggers conformational changes [84] and inflammasome assembly with pro-caspase-1, NEK7, and ASC [71,85,86]. Cardiolipin may also activate NLRP3 [87], although its dependence on ROS is debated [88]. Phagocytosed particles can rupture lysosomes and release cathepsins that promote NLRP3 activation. NLRP3 activation triggers caspase-1 that cleaves pro-IL-1β/IL-18 into active cytokines. Caspase-1 also cleaves gasdermin D (GSDMD) which forms pores that enable secretion of IL-1β and IL-18 [89]. In addition to driving IL-1β secretion, inflammasome activation induces pyroptotic cell death, a process mediated by gasdermin D pores that disrupt membrane integrity and the release of inflammatory signals. This signaling also promotes the formation of F-actin-rich filopodia which persist after pyroptotic cell rupture and facilitate antigen recognition by CLEC9A-bearing cDC1 dendritic cells [90].

Figure 1. Mechanisms of NLRP3 inflammasome activation and inhibition.

In priming, lipopolysaccharide (LPS) or IL-1β activates NF-κB and induces the expression of proinflammatory cytokines and NLRP3. Activation mediated by ATP, nigericin, and particulate matter causes ion fluxes, mitochondrial dysfunction, reactive oxygen species (ROS) generation, and DNA damage. NLRP3 binds to oxidized mitochondrial DNA (ox-mtDNA) released through the mitochondrial permeability transition pore (mPTP), leading to inflammasome assembly. Inhibition mechanisms are shown for drugs that prevent NLRP3 activation or inflammasome formation (red boxes). Abbreviations: CARD, caspase activation and recruitment domain; LRR, leucine-rich repeat domain; MCU, mitochondrial calcium uniporter; MSU, monosodium urate; NACHT, nucleotide-binding and oligomerization domain.

Box 2. Limitations of existing NLRP3 inhibitors in clinical and preclinical settings.

MCC950

In 2001, Pfizer introduced a class of diarylsulfonylurea compounds called cytokine release inhibitory drugs (CRIDs) which inhibit IL-1β maturation [91]. In 2015, Luke O’Neill and colleagues identified CRID3 (CP-456,773), later renamed MCC950, as a specific NLRP3 inhibitor [2]. Studies in mouse bone marrow-derived macrophages (BMDMs), human macrophages, and peripheral blood monocytes (PBMCs) showed that MCC950 dose-dependently inhibits IL-1β secretion [2]. MCC950 also blocks inflammasome activation by inhibiting ASC speck formation [2], a crucial step in inflammasome oligomerization. NLRP3 must undergo a conformation change to become active. The inactive structure of NLRP3 bound to MCC950 has been solved, suggesting that MCC950 traps NLRP3 in an inactive state.

However, the clinical development of MCC950 was halted during a Phase 2 rheumatoid arthritis (RA) trial, primarily owing to liver toxicity associated with carbonic anhydrase inhibition [92]. Because MCC950 has served as the basis for the development of other NLRP3 inflammasome inhibitors targeting the NACHT domain, it is likely that these molecules could also exhibit additional off-target effects.

Oridonin (Ori)

Ori, derived from Isodon japonicus and Isodon trichocarpus [93], is a traditional Chinese herbal medicine that is used to reduce body heat, alleviate pain, and improve circulation. It has anticancer [94–96] and anti-inflammatory [97] properties, and showed efficacy in preclinical models of colon [98], lung [99], breast [100], pancreatic [101], prostate [102], and hepatocellular cancers [103]. Ori is a covalent inhibitor of the NLRP3 NACHT domain and prevents the NLRP3–NEK7 interaction in BMDMs. It also blocks nigericin-induced ASC oligomerization but does not inhibit damage to mitochondria, suggesting that Ori must act downstream of initial NLRP3 assembly [104]. Oridonin has also been reported to inhibit NF-κB activation [105] which impacts on the expression of NLRP3 and proinflammatory cytokines. In 2020, Ori was shown to protect against myocardial injury by reducing creatine kinase MB (CK-MB) and cardiac troponin I (cTnI), that are markers of cardiac injury, in mice with induced myocardial ischemia–reperfusion (I/R) injury [97]. Pretreatment with Ori reduced levels of NLRP3, ASC, and cleaved caspase-1/pro-caspase-1 in myocardial I/R injury models [97].

Although the anti-inflammatory and anticancer properties of Ori are promising, there are concerns regarding its cytotoxicity [80] and potential for drug–drug interactions [90]. The observed cytotoxicity in preclinical models limits its therapeutic potential in clinical trials. Furthermore, the possibility of drug–drug interactions poses a challenge because many patients with inflammatory diseases are already on multiple medications, and this complicates the use of Ori in clinical settings.

NLRP3 activation mechanisms, post-translational modifications, its involvement in autoinflammatory and neurodegenerative disorders, activators, and several inhibitors based on the MCC950 scaffold, as well as gasdermin D inhibitors, have recently been reviewed [4,5]. However, comprehensive analysis of clinical trial data, structural insights into key amino acid interactions in inflammasome inhibition, and comparative evaluation of direct NLRP3 inhibitors is necessary to advance drug design.

Structural studies of NLRP3 using inhibitors such as MCC950 [6], NP3-146 [7], and GDC-2394 [8] have advanced drug design and have led to optimization of compounds with better specificity within the NACHT nucleotide-binding domain of NLRP3. In addition to designing molecules based on residues such as Arg578 and Ala228 in the NACHT domain, disrupting reactive oxygen species (ROS) activation pathways or the NLRP3 interaction with oxDNA are promising alternative therapeutic strategies. Indeed, a recent study highlighted the role of oxDNA in NLRP3 activation and demonstrated that interfering with this interaction decreases inflammasome activity [9].

In this review we provide an update on the role of NLRP3 in inflammatory diseases and the current state of NLRP3 inhibitors in clinical trials. Moreover, we present preclinical studies of emerging NLRP3 inhibitors that leverage medicinal chemistry and deep learning (DL)-driven molecular design to optimize their binding affinity for the NACHT domain, as well as repurposed inhibitors based on NLRP3 activation by oxDNA, and describe their unique structural features.

Aberrant upregulation of NLRP3 mediates inflammatory pathogenesis

The NLRP3 inflammasome plays a central role in various inflammatory diseases ranging from cryopyrin-associated periodic syndrome (CAPS) (see Glossary) to neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Beyond CAPS, NLRP3 is implicated in conditions such as gout, cardiovascular disease (CVD), and several types of cancer where its dysregulation contributes to disease progression.

Cryopyrin-associated periodic syndrome

CAPS is caused by point mutations in the NLRP3 gene [10], leading to overactive inflammasome responses [11]. CAPS varies in severity from familial cold autoinflammatory syndrome (FCAS) to Muckle–Wells syndrome (MWS) and the most severe, neonatal-onset multisystem inflammatory disease (NOMID), whose symptoms include chronic inflammation, hearing loss [12], ocular, and neurological complications [13]. Our growing understanding of NLRP3 activation mechanisms, including its interaction with oxDNA, offers important implications for drug development.

Neurodegenerative diseases

According to the US Centers for Disease Control (CDC), AD affects 6.7 million Americans over 65, and numbers are expected to rise to 13.8 million by 2060. PD affects 1 million Americans, including undiagnosed cases. Unregulated NLRP3 inflammasome activity is also implicated in neurodegenerative diseases. Disease associated amyloid-β (Aβ) plaques and α-synuclein aggregates that are associated with these diseases activate the NLRP3 inflammasome. In 2021, Aβ peptide was reported to activate the NLRP3 inflammasome [14], and elevated NLRP3 expression is observed in patients with severe neurodegenerative disorders. Inhibition of NLRP3 with MCC950, a popular NACHT-targeting inhibitor, can enhance the clearance of α-synuclein oligomers [15].

Metabolic and inflammation-driven diseases

Dysregulated NLRP3 activation contributes to several metabolic and inflammation-driven diseases including gout, CVD, and cancer. Gout arises from monosodium urate (MSU) crystal deposition [16] that triggers NLRP3 activation and IL-1β release. In recent years, small molecules such as Z1456467176 [17] and gallic acid [18] have shown potential in reducing gout symptoms by blocking NLRP3 activation. Briefly, Z1456467176 was designed as an allosteric P2X7 receptor inhibitor that exploits possible protein conformation remodeling [17]. Gallic acid may inhibit the NLRP3–NEK7 interaction [18]. Human pathology studies provide evidence of NLRP3 inflammasome activation during acute myocardial infarction (AMI) [19] in which patients show elevated plasma levels of NLRP3 and caspase-1 [20]. Dysregulated NLRP3 expression is associated with 15 of 24 cancer types [21] including lung, prostate [22], breast, colorectal, and bladder cancers. NLRP3 is also increased in plasmacytoid dendritic cells from non-small cell lung cancer (NSCLC) patients compared to healthy patients [23], promotes breast cancer growth and metastasis via IL-1β induction [24], and is linked to increased risk of bladder cancer [25] through polymorphisms.

NLRP3 inhibitors in clinical studies

Several NLRP3-targeting drugs are or have been in clinical trials (Table 1, Key table). These are reviewed below.

Table 1.

Key table. Summary of NLRP3 inhibitors across clinical trials, disease models, and efficacy

NLRP3 inhibitor	NLRP3-associated disease target	Cellular target	Clinical stage(trial name and indication)	Adverse effects	Results	Clinical trial identifier (indication)	Off-target
MCC950	RA	NLRP3 NACHT domain (Walker B)	Terminated Phase 2	Liver toxicity			Carbonic anhydrase
Glyburide	TBI	Direct mechanism unknown	Phase 2	Not reported	TBI: reduction of lesion volume and hemorrhage volume	NCT01454154 [28]	ATP-sensitive potassium channels (original target) CFTR
OLT1177	Gout, OA, CVD, T2D	Disruption of ATPase, blocks interaction with ASC	Phase 2a, 2/3 (PODAGRA; gout, recruiting) Phase 1/2 (OA) Phase 1b (CVD) Phase 2 (Dapan-Dia; diabetes, recruiting)	Not reported	Gout: reduced joint pain OA: reduced pain CVD: well tolerated	NCT05658575 (gout) NCT01768975 (OA) NCT03534297 (CVD) NCT06047262 (diabetes)
Canakinumab	CVD, CAPS, NSCLC, RA, idiopathic arthritis	IL-1β neutralization	Phase 3 (CANTOS; CVD) Phase 3 (REMITTER; CAPS)Terminated Phase 3 (CANOPY-2; NSCLC) Phase 3 (CANOPY-A; NSCLC) Phase 3 (RA) Phase 2 (gout)	Risk of thrombocytopenia and neutropenia (CVD)	CAPS: remission of symptoms Gout/RA: well tolerated and improved symptoms NSCLC: terminated owing to lack of efficacy (CANOPY-2) No benefits following treatment with cisplatin (CANOPY-A)	CANTOS NCT01327846 (CVD) REMITTER NCT01576367 (CAPS) CANOPY-2 NCT03626545 CANOPY-A NCT03447769 (NSCLC) (RA) [56] (gout) [57]
RRx-001	SCLC and SOM	Interaction with NLRP3 Cys409 to block NEK7/NLRP3	Phase 1 (PRIMETIME) Phase 2 (QUADRUPLE THREAT) Phase 3 (SCLC; active) Phase 2 (PREVLAR/KEVLARx; SOM)	Risk of infusion reaction, pneumonitis, hyperthyroidism (PRIMETIME)	SCLC: decreased PD-L1 expression correlates with response to reintroduced platinum doublet SOM: decreased duration Metastatic cancer: well tolerated, anticancer evidence	QUADRUPLE THREAT NCT02489903 REPLATINUM NCT03699956/NCT05566041 (SCLC) PREVLAR/KEVLARx NCT03515538/NCT05966194 (SOM) PRIMETIME NCT02518958	DNA MTases HDAC NRF2
Inzomelid	CAPS	NLRP3	Phase 1	Not reported	CAPS: well tolerated (one patient showed improvement)	NCT04015076
ZYIL1	CAPS, ALS, ulcerative colitis	NLRP3	Phase 2 (CAPS) Phase 2 (ALS; active) Phase 2 (ulcerative colitis)	Not reported	CAPS: reduced inflammatory markers, disease activity improvement	NCT05186051 (CAPS) NCT06398808 (ulcerative colitis) NCT05981040 (ALS)
HT-6184	Post-operative oral surgery inflammation	NLRP3 interaction with NEK7	Phase 2	Not reported		NCT06241742
VTX2735	CAPS (FCAS)	NLRP3	Phase 2	Not reported	CAPS: improved disease activity, reduced key symptoms score, decreased inflammatory biomarkers	NCT05812781
NT-0796	PD	NLRP3 NACHT domain	Phase 1/2	Not reported	PD: reduced IL-1β, IL-6, CCL2, and CXCL8 in CSF, decreased levels of fibrinogen Healthy volunteers: passes the blood–brain barrier	NodThera
DFV-890	COVID-19 pneumonia, knee OA	NLRP3 NACHT domain	Phase 2 (OA) Phase 2 (COVID-19)	Not reported	COVID-19: early clearance, improved clinical status, fewer fatalities	NCT04382053 (COVID-19) NCT04886258 (OA)
Rilonacept	CAPS (MWS)/SchS and gout	IL-1β/IL-1α neutralization	Phase 3 (PRESURGE-2; gout)	Not reported	Gout: reduced gout flares and safe/tolerable MWS/SchS: decrease in daily health assessment; reduced CRP and serum amyloid A levels	NCT01045772 (MWS/SchS) NCT00958438 (gout)
Goflikicept	STEMI and IRP	IL-1β/IL-1α neutralization	Phase 2a (STEMI) Phase 2/3 (IRP)	Not reported	STEMI: reduced systemic inflammation and well tolerated IRP: reduced CRP and decreased chest pain	NCT04463251 (STEMI) NCT04692766 (IRP)

Glyburide

Glyburide is a sulfonylurea drug used to treat type 2 diabetes (T2D) by inhibiting ATP-sensitive K⁺ channels in pancreatic β cells and stimulating insulin secretion. Typical doses range from 2.5 to 5 mg daily. Glyburide consists of a cyclohexylurea group, a benzamido group, and a sulfonylurea group. Notably, the cyclohexylurea group is not essential for inhibiting lipopolysaccharide (LPS)/ATP-induced caspase-1 activation [26]. However, the sulfonyl and benzamido groups are required for inhibition. Glyburide also inhibits induction of IL-1β and IL-18 secretion by nigericin [27] in LPS-primed human and mouse macrophages. NLRP3-deficient mice treated with 500 mg/kg glyburide survived LPS-induced lethality [26], but this high dose causes hypoglycemia, limiting its therapeutic use for inflammasome diseases.

In a Phase 2 clinical trial, intravenous glyburide was examined in traumatic brain injury (TBI) patients to assess efficacy and safety. Patient lesion volumes increased by 1036% with placebo treatment compared to 136% with glyburide. Similarly, hemorrhage volume increased by 11.6% with placebo treatment and decreased by 29.6% with glyburide [28]. Despite its potential benefits, glyburide has been reported to inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) [29] and ATP-sensitive potassium channels, which may contribute to fluid retention and hypoglycemia in diabetic patients.

RRx-001

RRx-001 is an anticancer agent that inhibits NLRP3 by covalently binding to Cys409 and blocking NEK7 interaction [30] and inflammasome formation. In the PRIMETIME Phase 1 trial (NCT02518958), RRx-001 combined with nivolumab was well tolerated and there was evidence of anticancer activity. The main adverse events included transfusion reaction, pneumonitis, and hyperthyroidism [31]. In the Phase 2 clinical trial QUADRUPLE THREAT (NCT02489903), RRx-001 reduced programmed death ligand 1 (PD-L1) on circulating tumor cells (CTCs) and promoted the response to reintroduced platinum therapy in small-cell lung cancer (SCLC) patients [32]. An active Phase 3 trial is currently examining whether RRx-001 and platinum chemotherapy are more effective than chemotherapy alone in SCLC (NCT05566041).

In addition, the Phase 2a PREVLAR trial (NCT03515538) evaluated the safety and efficacy of RRx-001 for oral mucositis (OM) in patients undergoing chemoradiotherapy for head and neck cancer. This trial reported no adverse events, and treatment cohorts showed similar or reduced OM duration compared to controls [33]. The ongoing KEVLARx study (NCT05966194) is examining the effects of RRx-001 in combination with cisplatin and radiation on severe OM reduction. Furthermore, a preclinical study is assessing RRx-001 as a potential treatment for neurodegenerative diseases including PD and amyotrophic lateral sclerosis (ALS).

Mechanistically, RRx-001 exerts its anticancer effects by modulating gene expression through inhibition of DNA methyltransferases (MTases) and histone deacetylases (HDACs) [34]. It also activates the nuclear factor erythroid 2-related factor (NRF2) pathway which regulates antioxidant gene expression [35]. Although these off-target effects contribute to the efficacy of RRx-001, they may influence its long-term safety and clinical applications.

Inzomelid

Inflazome Ltd conducted a Phase 1 clinical trial of inzomelid involving 80 participants, including patients with CAPS. In March 2020 they reported a linear relationship between drug dosage and blood concentration levels, which correlated with NLRP3 activity. The drug was described as tolerable, and one CAPS patient reportedly showed rapid improvement; however, additional details were not disclosed (NCT04015076).

ZYIL1

ZYIL1 has shown promise in treating inflammatory conditions associated with NLRP3 activation. In a Phase 1 trial in healthy subjects, ZYIL1 was well tolerated and showed rapid oral absorption. In addition, ZYIL1 demonstrated ex vivo inhibition of LPS-induced IL-1β production, and >90% suppression was observed across all dose groups [36]. In a Phase 2 clinical trial (NCT05186051), CAPS patients demonstrated improved disease activity and reduced inflammatory biomarkers [37]. Another Phase 2 trial is evaluating the efficacy, safety, tolerability, pharmacokinetics, and pharmacodynamics of ZYIL1 in patients with ALS (NCT05981040), expanding its potential therapeutic applications. Furthermore, this company is sponsoring a study to examine the safety of ZYIL1 for ulcerative colitis patients (NCT06398808).

HT-6184

Halia Therapeutics HT-6184 has been shown to inhibit the interaction between NEK7 and NLRP3 and prevent inflammasome formationⁱ. A Phase 1 trial (NCT05447546) in healthy volunteers reported no severe adverse events up to 4 mg dose and a 90% suppression of NLRP3-mediated cytokines. Halia Therapeutics is pursuing a Phase 2 study to evaluate HT-6184 for managing post-procedural inflammation (NC06241742).

VTX2735

VTX2735, developed by Ventxy BioSciences, completed a Phase 1 trial and showed good tolerability across all dose groups with no severe adverse events [38]. The treatment also suppressed IL-1β release compared to baseline. In a Phase 2 trial in CAPS (FCAS) patients (NCT05812781), treatment led to an 85% reduction in symptom scores and decreased inflammatory biomarkersⁱⁱ. Ventex is also developing VTX3232, a central nervous system (CNS)-penetrant NLRP3 inhibitor, and is currently recruiting patients for a Phase 2a clinical trial in PD (NCT06556173)ⁱⁱⁱ.

OLT1177

In a Phase 1/2 clinical study on OLT1177 (NCT01768975), patients with moderate-to-severe knee osteoarthritis (OA) pain were treated with OLT1177 gel administered topically three times a day for 13 days and once on day 14. The treatment was found to be effective in reducing pain. A Phase 1b study (NCT03534297) examined the activity of oral OLT1177 capsules in CVD, and found that the treatment was safe and well tolerated [39]. The Phase 2/3 PODAGRA trial (NCT05658575) is recruiting and is exploring the efficacy and safety of OLT1177 in the treatment of acute gout flares in view of previous positive results of reduced joint pain [40]. In addition, the Phase 2 Dapan-Dia trial is recruiting patients to examine OLT1177 in patients with diabetes/diabetes complications (NCT06047262). OLT1177 has been reported to cross the blood–brain barrier in PD mouse model [41] and is also planned to enter a Phase 2 clinical study in subjects with PD. These trials represent the wide therapeutic potential of OLT1177 across various indications.

NT-0796 and NT-0249

NT-0796 and NT-0249 from NodThera demonstrate promising advances in inflammation-targeting therapies. NT-0796, which converts to NDT-19795 in vivo, binds to the NACHT domain and effectively inhibits IL-1β and IL-18 in ex vivo blood samples. In a Phase 1 trial (ACTRN12621001082897), it showed no liver-related toxicities and demonstrated blood–brain barrier penetration. In Phase 1b/2a trials, NT-0796 has shown reductions in inflammatory and disease biomarkers in the blood of PD patients, suggesting potential therapeutic benefits^iv. NT-0249, another NodThera candidate in Phase 1 trials, demonstrated tolerability but does not cross the blood–brain barrier (ACTRN12622000195752)^v. These findings highlight the focus of NodThera on innovative strategies for treating inflammation-driven diseases.

DFV-890

DFV-890, developed by Novartis Pharmaceuticals, has been investigated for various inflammatory conditions and was reported to bind directly to the NLRP3 NACHT domain [42]. In a Phase 2 trial with COVID-19 pneumonia patients (NCT04382053), DFV-890 showed promise by accelerating infection clearance, improving clinical outcomes, and reducing fatalities compared to the control group [43]. The drug is also currently being studied in a Phase 2 trail for its efficacy in managing knee OA (NCT04886258). These studies highlight the broad therapeutic potential of DFV-890 across inflammatory diseases, although further large-scale trials are necessary.

Emerging therapies for NLRP3-related diseases

NP3-562/NP3-146

NP3-146 and NP3-562 are analogs of MCC950 (Figure 2A,B), and NP3-562 was identified through screening using a fluorescent sulfonylurea probe derived from NP3-146 that binds to the MCC950 binding pocket [44]. The crystal structures of NP3-146 and NP3-562 bound to the NLRP3 NACHT domain reveal key interactions with Arg578, Arg351, and Ala228 for NP3-146 [7]. However, NP3-562 binds differently, and avoids Arg351 while interacting with Arg578 and forming polar interactions via its amide carbonyl. Both drugs bind to the NACHT domain and trap NLRP3 in the inactive state [7,44] (Figure 2A,B). NP3-562 demonstrated complete inhibition of IL-1β secretion at 30 mg/kg in mice. In addition, NP3-562 can cross the blood–brain barrier and reach the CNS [44], suggesting that it may have potential as a therapy for neurodegenerative diseases linked to NLRP3. However, further human clinical testing is needed.

Figure 2. Structures of the NLRP3 nucleotide-binding and oligomerization (NACHT) active site bound to small molecules.

In each structure the color scheme for the NACHT subdomains is the same: nucleotide-binding domain (NBD) residues 131–373 are shown in purple, helical domain 1 (HD1) residues 374–434 (green), winged-helix domain (WHD) residues 435–538 (pink), and helical domain 2 (HD2) residues 539–676 (orange). (A) NP3-562 bound to the NLRP3 NACHT domain [44] (PDB: 8RI2). (B) NP3-146 bound to the NLRP3 NACHT domain [7] (PDB: 7ALV). (C) SN3-1 bound to the NLRP3 NACHT domain [45] (PDB: 8ZEM). (D) MCC950 bound to the NLRP3 NACHT domain [6] (PDB: 7PZC). (E) GDC-2394 bound to the NLRP3 NACHT domain [8] (PDB: 8ETR). (F) Compound 32 bound to the NLRP3 NACHT domain [47] (PDB: 8WSM). The ligand binds to the same active site across all structures. Images were rendered and constructed using University of California San Francisco (UCSF) ChimeraX [106].

SN3-1

Using DL-driven molecular design based on MCC950, SN3-1 was optimized for high binding affinity for the NACHT domain of NLRP3. X-ray crystallography revealed that SN3-1 stabilizes the NACHT domain in an inhibited conformation. The sulfonylurea group forms hydrogen bonds with Arg578 and Ala228, whereas the hydroxyl interacts with Glu629 via hydrogen bonding (Figure 2C) [45]. SN3-1 blocks the maturation of IL-1β and caspase-1 without affecting other inflammasomes such as AIM2 or NLRC4, and inhibits the assembly of the NLRP3 inflammasome by reducing ASC oligomerization and disrupting interactions between NLRP3 and NEK7 [45]. Importantly, SN3-1 demonstrates efficacy across various NLRP3-related disease models, including gout and AD, highlighting its potential therapeutic promise in inflammatory conditions [45].

GDC-2394

The clinical development of MCC950 for rheumatoid arthritis (RA) was halted owing to liver toxicity, likely from its furan ring structure (Figures 2D and 3). In 2022, researchers replaced the furan group with aryl and heteroaryl groups to reduce toxicity while inhibiting NLRP3. A hit compound, 3-substituted-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine, showed promise but caused renal toxicity in cynomolgus monkeys [8].

Figure 3. NLRP3 drug structures and their shared features.

NLRP3 inhibitors share a similar backbone and have many similar functional groups, including tricyclic ring systems (blue), sulfonylurea groups (green), hydroxyls (red), amides (gray), halogens (orange), urea (yellow), and sulfonyl groups (dark gray). TH5487 and SU0268 bind directly to the pyrin domain of NLRP3. TH5487 contains a 8-oxo-dG-like moiety.

The introduction of amine substituents (GDC-2394) improved solubility and eliminated renal toxicity. GDC-2394 inhibited NLRP3-induced caspase-1 activity, reduced IL-1β release, and suppressed ASC speck formation in both human and mouse blood. However, in a Phase 1 study in healthy participants, two participants experienced grade 4 drug-induced liver injury, leading to termination of the study [46]. Cryo-electron microscopy (Cryo-EM) confirmed GDC-2394 binding to the NLRP3 NACHT domain in which the urea group interacts with Ala228 and Arg578, while Arg351 and Arg578 form a pocket around the sulfonamide oxygen [8] (Figure 2E).

Compound 32

Compound 32 was designed using scaffold hopping and bioisosteric replacement to optimize potency and selectivity. Compound 32 inhibits activation of the NLRP3 inflammasome by binding directly to the NACHT domain [47] to stabilize its structure and block its interaction with other inflammasome components, the production of proinflammatory cytokines, and pyroptosis. The inhibitory mechanism involves multiple interactions such as hydrogen bonds with specific residues in the NLRP3 NACHT domain (e.g., Ala228, Arg351, and Val353) and van der Waals interactions in the binding pocket (Figure 2F). The compound also shows high selectivity for NLRP3 versus other inflammasomes such as NLRC4. In animal models, compound 32 demonstrated effective suppression of IL-1β production and improved the symptoms of glomerulonephritis, highlighting its potential as a therapeutic agent for NLRP3-related inflammatory diseases [47].

TH5487

TH5487, a selective inhibitor of the DNA repair enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG1), was identified via a fluorescence-based DNA cleavage screen. TH5487 specifically inhibits hOGG1 without affecting other glycosylases and resulted in increased levels of 8-oxo-dG [48]. ROS are known to increase hOGG1 substrates in guanine-rich promoter regions, and recruit hOGG1 and NF-κB to drive transcription of NLRP3 and other proinflammatory genes. TH5487 inhibits NF-κB binding to these promoters and reduces cytokine expression in TNF-α-challenged mouse models [48]. TH5487 also shows promise in pulmonary fibrosis. In bleomycin (BLM)-induced fibrosis models, TH5487 reduced profibrotic markers such as CTGF, fibronectin, and type 1 collagen [49].

Because oxDNA, a hOGG1 substrate, is a known NLRP3 activator, Reginald McNulty and colleagues examined the effects of TH5487 on oxDNA-driven inflammasome activation in immortalized bone marrow-derived macrophages (BMDMs). TH5487 directly binds to purified NLRP3 protein and blocks NLRP3 binding to oxDNA [9]. This drug was successfully repurposed to inhibit IL-1β secretion in macrophages [9]. This shows that OGG1-mediated DNA repair can be inhibited while simultaneously inhibiting NLRP3; however, in vivo validation is needed.

SU0268

SU0268, a tetrahydroquiniline biphyenyl sulfonamide derivative, is a specific and competitive hOGG1 inhibitor. It is membrane-permeable and demonstrates low toxicity in HEK293T and HeLa cells. Inhibition of hOGG1 by SU0268 leads to the accumulation of 8-oxo-dG, confirming its role in disrupting the hOGG1 repair pathway [50]. In mouse alveolar macrophages and wild-type C57BL/6 mice, SU0268 enhanced type 1 interferon responses while reducing the proinflammatory response to bacterial infection of the lung. SU0268 attenuated ROS levels and decreased the release of mtDNA in response to Pseudomonas aeruginosa, as confirmed by qPCR and immunofluorescence [51].

The hOGG1 inhibitor SU0268 has also been demonstrated to target NLRP3 [9]. It was shown that at concentrations as low as 0.01 μM SU0268 inhibited NLRP3 binding to ox-mtDNA [9]. Thermal shift assays showed direct binding of SU0268 to NLRP3 at 400 μM, evidenced by increasing melting temperatures [9], and 0.01 μM SU0268 inhibited inflammasome activation in mouse macrophages. These findings illustrate that hOGG1 and NLRP3 can be simultaneously inhibited with a single drug.

MRT-8102

Monte Rosa Therapeutics, Inc. announced a ‘molecular glue’ that targets NEK7. In non-human primates, they report degradation of NEK7 by MRT-8102, resulting in a decrease in IL-1β secretion^vi. They anticipate that MRT-8102 will be promising in treating inflammatory diseases, including gout and CVD, and potentially in neurodegenerative diseases because it can penetrate the blood–brain barrier. Monte Rosa is in the process of applying for an Investigational New Drug (IND) application, and completion is expected in 2025^vi.

IL-1β blockade as a therapeutic strategy for inflammatory disease

Although many approaches focus directly on inhibiting NLRP3, others target IL-1β downstream of inflammasome activation to mitigate inflammatory effects.

Canakinumab (Ilaris)

Canakinumab is a monoclonal antibody against IL-1β that prevents receptor interaction. It is approved for treatment of diseases such as CAPS and tumor necrosis factor receptor-associated periodic syndrome (TRAPS). In the CANTOS Phase 3 trial (NCT01327846), a 150 mg dose reduced recurrent cardiovascular events but increased thrombocytopenia and neutropenia [52]. Treatment showed symptom remission in the REMITTER trial (NCT01576367) [53], but was ineffective in NSCLC in the CANOPY-2 (NCT03626545) and CANOPY-A (NCT03447769) trials [54,55]. Canakinumab has been tested in RA [56], idiopathic arthritis, and gouty arthritis [57], and showed good tolerability.

Rilonacept

Rilonacept inhibits signaling by neutralizing IL-1β and IL-1α before they can interact with the IL-1 receptor. Rilonacept has been studied in patients with gout and MWS/Schnitzler syndrome (SchS). In a Phase 2 study in SchS patients, treatment with an initial 320 mg dose followed by weekly 160 mg for up to 1 year resulted in a significant improvement in daily health assessment scores and patient global assessment scores compared to baseline. There was also a decrease in C-reactive protein (CRP) and serum amyloid A levels. The treatment was well tolerated and there were no reported treatment-related adverse events (NCT01045772) [58]. In a Phase 3 clinical trial, PRESURGE-2, gout patients received a combination of allopurinol and either placebo or rilonacept for 16 weeks. The rilonacept-treated groups showed reduced gout flare-ups and acceptable tolerability (NCT00958438) [59].

Goflikicept

Goflikicept is an IL-1β/IL-1α trap that binds to and neutralizes active cytokines by preventing their interaction with the IL-1 receptor. It has been studied in diseases such as acute ST-segment elevation myocardial infarction (STEMI) and idiopathic recurrent pericarditis (IRP). IRP is a pericardium inflammatory disease that is defined by relapse after a symptom-free interval of at least 4–6 weeks [60]. In a Phase 2a clinical study in patients with STEMI, goflikicept reduced systemic inflammation and was well tolerated (NCT04463251) [61]. In a Phase 2/3 study in 2023, goflikicept reduced CRP levels and chest pain in patients with IRP, and there were no recurrence events, compared to recurrence in nine of ten patients in the placebo group (NCT04692766) [62].

Backbone and unique structural features of NLRP3 inflammasome inhibitors

Many NLRP3 inhibitors evaluated in preclinical or clinical studies share similar functional groups such as sulfonylurea groups, tricyclic ring systems, halogen groups, hydroxyls and tertiary alcohols, urea and amide groups, and furan groups (Figure 3). MCC950, glyburide, ZYIL1, SN3-1, NP3-146, inzomelid, and GDC-2394 all have a sulfonylurea group which may hydrogen bond with amino acids in the active site of NLRP3 [45]. In addition, MCC950, DFV-890, NT-0796, GDC-2394, and ZYIL all feature a tricyclic ring system which shapes the steric profile for interaction within the active site of NLRP3. The aromatic rings often enable π–π stacking that stabilizes the inhibitor–target complex [63].

Halogen functional groups are present in TH5487, glyburide, compound 32, NP3-146, SN3-1, NP3-562, and RRx-001. Halogens increase drug lipophilicity, reduce enzymatic degradation, and improve metabolic stability [64]. Hydroxyls and tertiary alcohols that are present in many of these compounds allow efficient hydrogen bonding and increased polarity [45]. Urea and amide groups similarly contribute to hydrogen bonding and polarity, and enhance drug solubility and binding.

In contrast to the other small molecules, TH5487 and SU0268 are unique in their direct binding to the pyrin domain coupled with their ability to reduce inflammasome activation [9]. TH5487 contains a group that is structurally similar to oxidized guanine, highlighting the connection between the NLRP3 pyrin domain and its interaction with damaged DNA [9]. SU0268 also contains a sulfonylamide, suggesting that it has a similar backbone to other NLRP3 inhibitors.

Concluding remarks and future perspectives

Structural studies of NLRP3 with inhibitors such as MCC950 [6], NP3-146 [7], and GDC-2394 [8] have advanced our understanding of the NACHT domain and active site of NLRP3. However, to date no NLRP3 inhibitors are FDA approved, and the full consequences of NLRP3 blockade remain unclear (see Outstanding questions).

Outstanding questions.

What are the clinical outcomes of inhibitors that directly target NLRP3?

How does the cell compensate when NLRP3 is inhibited?

Can drugs be designed to target NLRP3 pyrin or LRR domains in addition to the NACHT domain?

What off-target effects do NLRP3 inhibitors have?

A major challenge is selectivity. Because the NACHT domain binding site for MCC950 is predominantly hydrophobic [65], this contributes to off-target interactions with other proteins such as carbonic anhydrase that also possesses hydrophobic binding pockets. By contrast, repurposed drugs that bind the more hydrophilic pyrin domain may reduce off-target effects. However, pyrin and death domain-containing proteins play roles in other signaling pathways such as NF-κB activation [66], raising concerns about broader immune disruption.

Beyond domain-specific targeting, NLRP3 is closely linked to core cellular processes such as metabolism and autophagy, highlighting the need for carefully designed and selective inhibitors. The silent information regulator type 1 (SIRT1), a NAD⁺-dependent deacetylase, suppresses inflammation by deacetylating the p65 subunit of NF-κB and downregulating NLRP3 and IL1B transcription [67]. However, oxidative stress can activate poly(ADP-ribose) polymerase (PARP), leading to NAD⁺ consumption [68] and reduced SIRT1 activity. Although NLRP3 inhibition may help to restore NAD⁺ levels and promote SIRT1 activity, it may also reduce cellular stress signals, such as ROS, that are necessary to trigger the protective features of SIRT1.

Autophagy is a key mechanism for degrading damaged organelles, such as mitochondria, which can release damage signals that activate NLRP3. By clearing these damaged components, autophagy acts as a negative regulator of NLRP3 inflammasome activation. However, studies have shown that Nlrp3 knockout mice exhibit elevated autophagy levels both under basal conditions and during stress [69]. These studies show that NLRP3 is deeply connected to pathways that are necessary for cellular homeostasis.

A fundamental question that must be addressed is whether complete inhibition of NLRP3 is the optimal strategy, or whether modulating its activity in a context-dependent manner would yield better therapeutic outcomes. Chronic suppression may impair host defenses and increase infection risk [70]. In addition, the potential for inflammasome crosstalk must be considered – if NLRP3 is inhibited, other pathways (e.g., AIM2, NLRC4, cGAS) may compensate, altering immune responses in unpredictable ways.

Future studies should aim for selective and well-tolerated inhibitors. Pyrin domain inhibitors represent a promising avenue, but their long-term effects require further study. Combination therapies that modulate inflammasome activity may offer a safer and more effective treatment. The continued integration of structural biology with medicinal chemistry is likely to accelerate the development of safe and effective NLRP3-targeting therapeutics, and ultimately address significant unmet clinical needs across a broad spectrum of inflammatory diseases.

Highlights.

The NLRP3 inflammasome is a key regulator of inflammatory diseases but lacks FDA-approved inhibitors.

Oxidized DNA (oxDNA) plays a crucial role in NLRP3 activation, and thus offers a new therapeutic target.

Repurposed glycosylase inhibitors block NLRP3 pyrin binding to oxDNA and reduce IL-1β secretion.

Structural analysis reveals shared active site features among NLRP3 inhibitors that will aid drug development.

Several NLRP3 inhibitors in clinical trials have potential applications in neurodegeneration, cancer, and metabolic diseases

Glossary

Amyotrophic lateral sclerosis (ALS)

a progressive neurodegenerative disease that leads to muscle weakness and loss of voluntary movement.

Alzheimer’s disease (AD)

a neurodegenerative disorder characterized by progressive memory loss, cognitive decline, and changes in behavior.

Cardiovascular disease (CVD)

a group of disorders that affect the heart and blood vessels, including conditions such as coronary artery disease, heart attacks, stroke, and hypertension, often related to atherosclerosis.

Cystic fibrosis transmembrane conductance regulator (CFTR)

a chloride ion channel that plays a crucial role in regulating salt and water balance in epithelial cells.

Cryopyrin-associated periodic syndrome (CAPS)

a condition caused by point mutations in the NLRP3 gene that are linked to inflammation.

Neutropenia

low neutrophil count.

Parkinson’s disease (PD)

a neurodegenerative disorder that affects movement, caused by degeneration of dopamine-producing neurons in the brain.

Programmed death ligand 1 (PD-L1)

a polypeptide that binds to PD-1 receptor on T cells and inhibits their activation.

Thrombocytopenia

low platelet levels.

Traumatic brain injury (TBI)

physical damage to the brain that can trigger inflammatory responses in the brain.