Targeting pyroptosis in inflammatory bowel disease: A potentially effective therapeutic approach

Abstract

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is a chronic intestinal inflammation with complex pathogenesis. Pyroptosis a pro-inflammatory programmed cell death mediated by gasdermin D (GSDMD) cleavage plays a pivotal role in disease progression through nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3)/caspase-1 classical and caspase-4/5/11 non-classical pathways. Targeting pyroptosis has emerged as a promising therapeutic strategy, with recent advances highlighting the potential of pyroptosis inhibitors such as small-molecule compounds, biologics, and repurposed drugs that specifically target NLRP3, caspases, or GSDMD to suppress inflammasome activation, block pore formation, and mitigate downstream inflammation. This review systematically summarizes the mechanisms and therapeutic effects of these inhibitors, while addressing critical challenges including drug specificity, delivery efficiency, and long-term safety, and explores their potential in combination therapies with existing IBD treatments to enhance clinical efficacy. By integrating preclinical and clinical evidence, we provide valuable insights into the translational prospects of pyroptosis-targeted therapies for precision management of IBD.

Key Words: Inflammatory bowel disease; Pyroptosis; Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; Caspase-1; Gasdermin D; Pyroptosis inhibitors

Core Tip: This review examines pyroptosis as a pivotal mechanism in inflammatory bowel disease (IBD) pathogenesis, driving inflammation and tissue damage. It evaluates therapeutic strategies targeting pyroptosis pathways, emphasizing their potential to modulate disease progression. Key challenges including drug specificity, delivery, and safety are analyzed, alongside opportunities for combination therapies. The discussion highlights the need for precision approaches and improved translational models to advance pyroptosis-targeted treatments for IBD. Future directions focus on overcoming current limitations to optimize clinical outcomes.

INTRODUCTION

Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn’s disease (CD), represents a chronic intestinal disorder with escalating global health implications. Known as the “green cancer” for its refractory nature and propensity for lifelong recurrence, IBD imposes substantial morbidity and socioeconomic burdens. IBD is undergoing a dramatic epidemiological shift globally. Although the incidence of IBD has stabilized in early-industrialized countries, the prevalence continues to rise, with more than 1% of their populations projected to be affected within the next decade. In newly industrialized countries such as China, Malaysia, and Brazil, IBD incidence is accelerating, with annual rates ranging from 3.3 to 10.6 per 100000 people. This upward trend is expected to persist over the next two decades. The shift is closely associated with environmental triggers linked to urbanization and the adoption of Westernized diets. Consequently, IBD has transitioned from being considered a “Western disease” to a genuinely global health challenge[1]. This accelerating global burden underscores the imperative for innovative therapeutic paradigms.

The management of IBD continues to face significant challenges. Although the introduction of biologics [e.g., anti-tumor necrosis factor (TNF) agents, anti-integrins, and anti-interleukin (IL)-12/23 therapies] and small-molecule drugs [e.g., Janus tyrosine kinase (JAK) inhibitors] has markedly improved patient outcomes, therapeutic efficacy exhibits substantial interindividual variability. Approximately 30%-50% of patients fail to respond to initial treatment (primary nonresponse), while only about 40% of those who initially respond maintain clinical remission for one year. Furthermore, a “ceiling effect” of current therapies exists, with only a subset of patients achieving mucosal healing or long-term steroid-free remission. Additional limitations, including immunogenicity, infection risks, and drug-related toxicities, further constrain treatment options[2]. Notably, current mainstream therapies primarily target downstream inflammatory cytokines (e.g., TNF-α, IL-12/23) or intracellular signaling pathways (e.g., JAK-signal transducer and activator of transcription) in immune cells. However, these approaches do not adequately address upstream programmed cell death events, which play a critical role in driving tissue damage and sustaining inflammatory responses. Consequently, there is an urgent need to explore novel therapeutic targets to overcome these limitations.

Pyroptosis, a lytic cell death mechanism mediated by gasdermin family proteins, has emerged as a pivotal driver of IBD pathogenesis. It acts upstream of the inflammatory cascades targeted by current therapies, addressing the critical cell death events they fail to sufficiently inhibit. This programmed cell death process is typically triggered by inflammasomes and executed by gasdermin proteins, leading to characteristic features such as cell swelling, membrane pore formation, and the release of cellular contents. Pyroptosis is essential for host defense, but its dysregulation can trigger persistent inflammation and contributing to the progression of inflammatory diseases[3]. This detrimental role is starkly highlighted in IBD. Growing evidence demonstrates that aberrant activation of the gasdermin-mediated pyroptotic pathway contributes to intestinal epithelial barrier dysfunction, excessive inflammation, and disease progression. However, the precise regulatory mechanisms and therapeutic potential of targeting pyroptosis in IBD remain incompletely understood.

This review first outlines the molecular mechanisms of pyroptosis and its role in IBD development. It then analyzes the clinical evidence connecting pyroptosis to IBD features and finally evaluates emerging therapies, ranging from small-molecule inhibitors to combination strategies. By integrating preclinical and clinical data, we highlight challenges in drug development while proposing future directions for precision IBD treatment.

PYROPTOSIS PATHWAYS

Canonical pyroptosis pathway

The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3)-caspase-1-gasdermin D (GSDMD) pathway is the most extensively studied pyroptosis pathway. In the classical pathway, the process begins when Toll-like receptor 4 (TLR4) and NLRP3 detect pathogen-associated molecular patterns [e.g., bacterial lipopolysaccharide (LPS)], damage-associated molecular patterns (e.g., extracellular adenosine triphosphate), or homeostasis-altering molecular processes (e.g., lysosomal rupture). TLR4 then initiates nuclear factor kappa-B (NF-κB)-dependent transcriptional upregulation of NLRP3 and pro-IL-1β. Following activation, NLRP3 oligomerizes and recruits the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC), forming a large multiprotein complex known as the NLRP3 inflammasome. ASC subsequently recruits and activates caspase-1. Within the inflammasome, the precursor of caspase-1 undergoes autocatalytic cleavage to form activated caspase-1, which then exerts protease activity by cleaving multiple substrates, including the precursors of IL-1β and IL-18 (pro-IL-1β and pro-IL-18), as well as GSDMD. Cleavage of GSDMD releases its N-terminal fragment, which oligomerizes and interacts electrostatically with anionic phospholipids (like phosphatidylserine) in the plasma membrane, forming transmembrane pores. These pores disrupt osmotic balance and facilitate the release of mature IL-1β and IL-18 into the extracellular environment. Once released, these cytokines act to amplify the inflammatory response, recruit immune cells and enhance the host’s defense against infection[4] (Figure 1).

Figure 1 Canonical and non-canonical activation pathways of pyroptosis. NLRP3: Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; PAMP: Pathogen-associated molecular pattern; DAMP: Damage-associated molecular pattern; LPS: Lipopolysaccharide; TLR: Toll-like receptor; NF-κB: Nuclear factor kappa-B; ASC: Adapter protein apoptosis-associated speck-like protein containing a CARD; HAMP: Homeostasis-altering molecular processes; ROS: Reactive oxygen species; mtDNA: Mitochondrial DNA; IL: Interleukin.

Non-canonical pyroptosis pathway

The non-canonical pyroptosis pathway can be activated through an atypical inflammasome pathway. Caspase-11 in mice (caspase-4/5 in humans) can directly recognize intracellular bacterial LPS and subsequently cleave GSDMD, leading to pyroptosis. This pathway does not rely on traditional inflammasomes[5] (Figure 1). Emerging evidence indicates that caspase-8 serves as a critical regulator of pyroptosis under specific pathological conditions, such as infection with Yersinia or cellular stress[6]. Notably, caspase-8 can be recruited to atypical inflammasome complexes and directly cleave both GSDMD and gasdermin E (GSDME), thereby triggering lytic cell death. Recent studies further reveal that caspase-8-mediated pyroptosis depends on non-canonical signaling pathways, including the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes axis and unconventional inflammasomes such as NLRP12-ASC[7,8].

PYROPTOSIS IN IBD

The pathogenic role of pyroptosis in IBD

IBD is primarily characterized by intestinal inflammation and epithelial damage. Although its exact etiology is unknown, IBD is thought to originate from a complex interaction of genetic susceptibility and environmental factors that trigger an aberrant immune response[9]. A growing body of evidence indicates that pyroptosis significantly contributes to IBD pathogenesis[10-12]. Emerging research now positions pyroptosis as a central driver of intestinal inflammation through three interconnected mechanisms: Epithelial barrier disruption, immune dysregulation, and microbiota imbalance. Nevertheless, a more comprehensive understanding of the pathogenic role of pyroptosis in intestinal epithelial cells (IECs) is required to fully elucidate its impact on IBD progression.

The impact of pyroptosis on intestinal barrier function: The barrier formed by IECs is crucial for preventing the invasion of harmful substances, pathogens, and antigens. Pyroptosis leads to the death of IECs, directly compromising intestinal barrier integrity (Figure 2). Inflammatory mediators produced during pyroptosis, such as IL-1β and IL-18, initiate and amplify inflammatory responses, resulting in further epithelial cell death and barrier disruption[13,14]. The study found that the NLRP3 inhibitor MCC950 significantly alleviates colonic inflammation in the Winnie spontaneous chronic colitis mouse model and mitigates the spontaneous reduction in goblet cells, crypt damage, and epithelial barrier disruption[15]. Notably, the ZAKα-activated non-canonical pyroptosis pathway contributes to colonic epithelial injury, via the caspase-11/GSDMD axis to aggravate colitis[16]. Furthermore, tight junction proteins including occludin, claudin, and zonula occludens-1 (ZO-1) are essential for intestinal barrier integrity. Inflammatory pathways activated during pyroptosis inhibit the expression of ZO-1 and occludin (Figure 2), thereby weakening epithelial barrier function[17]. Treatment with natural products such as artemisinin analogs, dioscin, and evodiamine, which inhibit pyroptosis signaling pathways, has been shown to increase the expression of ZO-1 and occludin[18-20]. The compromised epithelial barrier facilitates microbial translocation and promotes immune cell activation, thereby linking epithelial pyroptosis to mucosal immune dysregulation.

Figure 2 The pathogenic role of pyroptosis in inflammatory bowel disease. DAMP: Damage-associated molecular pattern; IL: Interleukin; ZO-1: Zonula occludens-1; NE: Neutrophil elastase cell; M: Membranous/microfold cell; DC: Dendritic cell; IEC: Intestinal epithelial cell.

Regulation of intestinal immune cells by pyroptosis: Pyroptosis significantly influences the development of IBD by modulating immune responses. The activation of inflammasomes such as NLRP3 and absent in melanoma 2 (AIM2) during pyroptosis triggers the production and release of pro-inflammatory cytokines, particularly IL-1β and IL-18[4]. These cytokines enhance intestinal inflammation by promoting the recruitment and activation of immune cells (Figure 2)[21]. Pyroptosis-related cytokines also regulate macrophage polarization (Figure 2). IL-1β, for example, promotes the differentiation of macrophages toward pro-inflammatory M1 phenotypes, which release additional inflammatory mediators and exacerbate intestinal damage[22]. Consistent with this, studies using the dextran sulfate sodium (DSS)-induced colitis model have shown that inhibiting pyroptosis signaling pathways suppresses M1 polarization and ameliorates colitis symptoms[23-25]. Furthermore, pyroptosis shapes the intestinal immune microenvironment by modulating interactions between dendritic cells (DCs) and T cells (Figure 2). Pyroptosis-stimulated DCs exhibit enhanced antigen presentation capacity, promoting the activation and expansion of pro-inflammatory T cell subsets such as Th1 and Th17 cells, which contribute to chronic inflammation in IBD[26]. Concurrently, the pyroptosis-induced inflammatory milieu can impair regulatory T cell function, further disrupting immune homeostasis[27]. A recent study revealed that the NLRP3/IL-1β pathway regulates the activation and effector functions of γδT17 cells in UC patients[28]. Inhibition of this pathway significantly reduces Th17 cell differentiation, decreases levels of pro-inflammatory factors in the colons of DSS-induced mice, and ameliorates colitis symptoms[29,30]. The immune activation orchestrated by pyroptosis not only aggravates tissue inflammation but also disrupts the symbiotic relationship with the gut microbiota, shaping a dysbiotic microenvironment.

The impact of pyroptosis on intestinal microbiota homeostasis: The inflammatory response induced by pyroptosis can alter the composition and diversity of the intestinal microbiota (Figure 2). In DSS-induced IBD animal models and IBD patients, the gut microbiota often exhibits a dysbiotic state characterized by a decrease in beneficial bacteria and an increase in harmful bacteria[31,32]. Mechanistically, pyroptotic IECs release nucleotides and amino acids that selectively fuel the expansion of enteric pathogens, such as Salmonella[33]. Therapeutic modulation of this crosstalk is achievable: The NLRP3 inhibitor MCC950 restores microbial homeostasis by increasing the Firmicutes/Bacteroidetes ratio, thereby counteracting inflammation-associated dysbiosis[31].

Molecular functions of pyroptosis induction in IBD models

NLRP3: The NLRP3 inflammasome, a cytosolic multiprotein complex comprising NLRP3, ASC, and caspase-1, functions as a pattern recognition receptor central to the innate immune system. It mediates caspase-1 activation and the secretion of pro-inflammatory cytokines IL-1β and IL-18 in response to microbial infections and cellular damage[3,4]. The normal expression of NLRP3 is crucial for the host’s innate defense against bacterial, fungal, and viral infections[3,11]. However, its dysregulation is associated with the pathogenesis of many inflammation-related diseases, including Alzheimer’s disease, diabetes, and atherosclerosis[34]. The NLRP3 inflammasome is considered a key mechanism of intestinal inflammation in the DSS colitis model[10]. Selective small-molecule inhibitors of NLRP3, such as MCC950 and OLT1177, have shown promising therapeutic potential in IBD animal models[15,35]. Targeting the regulation of NLRP3 inflammasome activation offers new avenues and strategies for the treatment of IBD.

Caspase protein family: Caspase-1, the inflammatory caspase, exacerbates IBD through GSDMD-dependent pyroptosis and IL-1β/IL-18 hyperactivation[4,10]. Preclinical studies demonstrate that caspase-1 inhibitor, belnacasan (VX-765), reduces weight loss, colon shortening, and colonic pathological damage in DSS-induced colitis mice[36]. Non-canonical caspases (human caspase-4/5, murine caspase-11) are critical components of the innate immune pathway. They can sense and respond to intracellular LPS, and also trigger pyroptosis by inducing cell death, leading to a potent inflammatory response. In patients with UC and CD exhibit upregulated caspase-4/5, mirroring caspase-11 overexpression observed in DSS-induced colitis models[5,37]. Inhibition of caspase-11/4-induced macrophage pyroptosis has been shown to alleviate colitis in mice[38]. Caspase-11 not only drives inflammation but may also paradoxically maintain intestinal barrier integrity and immune homeostasis under specific conditions, highlighting its complex dual role in IBD[39]. Furthermore, apoptotic caspases caspase-3/8 have been implicated in mediating pyroptosis through the activation of GSDME[7,40,41].

GSDM protein family: In the intestinal epithelium, gasdermin family proteins play pivotal but divergent roles in IBD. GSDMD drives pyroptosis through caspase-mediated pore formation to amplify intestinal inflammation and, beyond this role, also mediates a novel nonpyroptotic pathway for IL-1β release via small extracellular vesicles in IBD[12]. Pharmacological inhibition of GSDMD attenuates this inflammatory response[42,43]. GSDME, another gasdermin member, similarly executes pyroptosis upon caspase-3/8 activation and exacerbates IBD progression by disrupting the epithelial barrier; studies show that targeting GSDME-mediated pyroptosis mitigates mucosal damage in IBD models[40,42,43]. In contrast, gasdermin B (GSDMB) is genetically associated with IBD but primarily promotes epithelial repair and regeneration instead of causing pyroptosis. According to Zhu et al[44] GSDMB generally does not trigger pyroptosis and may aid mucosal healing by improving cell migration and repair. Although it can be cleaved by some caspases under specific conditions, this usually does not result in pyroptosis and may affect other immune responses. This functional duality presents a therapeutic challenge: Inhibiting GSDMD or GSDME to reduce damaging inflammation may inadvertently interfere with GSDMB’s healing role. This is particularly concerning in the gut, where epithelial barrier integrity is crucial. Such a dichotomy underscores the critical need for patient stratification based on dominant gasdermin pathways. For instance, patients with high levels of GSDMD-N might be ideal candidates for GSDMD-targeted therapy, whereas those with elevated GSDMB expression might benefit from alternative strategies that preserve its healing function.

Dual challenges in targeting pyroptosis: Targeting key molecular components of pyroptosis, such as NLRP3, caspases, and GSDMD, represents a promising yet challenging therapeutic strategy for IBD. However, the dual roles of certain molecules introduce considerable complexity into treatment design. For instance, caspase-11 contributes not only to inflammatory responses but also to the maintenance of intestinal homeostasis[39]. Similarly, while GSDMD and GSDME primarily promote inflammation, GSDMB exhibits protective functions in epithelial repair[44]. Moreover, systemic targeting of pyroptosis carries broader safety concerns, including an increased risk of infections due to suppressed pathogen clearance and potential off-target effects that may lead to unintended immune modulation[3,11,45]. Therefore, a nuanced therapeutic approach is essential to selectively inhibit detrimental pathways without compromising protective mechanisms.

To enable such precision, the development and implementation of pyroptosis-specific biomarkers are critical. Promising candidates include: (1) Executor markers, such as circulating GSDMD-N, which correlates with disease activity in UC and allows for non-invasive monitoring[46]; (2) Effector markers, including cytokines IL-1β and IL-18 that are directly released during pyroptosis[47]; and (3) Transcriptomic markers (e.g., IL1B, GZMB), which are upregulated in active UC and are associated with macrophage pyroptosis[48]. By identifying pyroptosis-driven patient subgroups and monitoring treatment responses, these biomarkers can help reduce off-target risks and guide personalized interventions, advancing precision IBD medicine.

PYROPTOSIS-REGULATING DRUGS IN IBD

Small-molecule inhibitors: Precision targeting of key pyroptotic nodes

The pathological hyperactivation of pyroptosis in both IBD animal models and patients underscores its potential as a therapeutic target. Small-molecule inhibitors and repurposed drugs targeting key pyroptotic nodes have demonstrated anti-inflammatory efficacy in preclinical and clinical studies (Table 1).

Table 1 Small-molecule inhibitors and repurposed drugs of pyroptosis-related proteins.

Name	Target protein	Pharmacological mechanism	IBD disease model	Clinical stage	Ref.
MCC950 (CP-456773)	NLRP3	Binds to the NACHT domain of NLRP3, preventing ATP hydrolysis and oligomerization	Winnie spontaneous colitis, DSS-induced acute colitis	Phase II discontinued (rheumatoid arthritis)	Perera et al[15]; Wang et al[31]; Coll et al[49]; Saber et al[50]; Saber and El-Kader[51]; Li et al[52]; Liu et al[53]
OLT1177 (dapansutrile)	NLRP3	Binds NLRP3 LRR domain, blocks NLRP3 inflammasome assembly	DSS-induced acute colitis	Phase II completed (osteoarthritis; No. NCT02104050); phase III planned (gout; No. NCT05658575)	Oizumi et al[35]; Marchetti et al[54]; Saber et al[55]
INF39	NLRP3	Irreversibly inhibits NLRP3 ATP activity	DNBS-induced colitis	Preclinical	Shi et al[56]; Pellegrini et al[57]
VI-16	TXNIP-NLRP3	Disrupts TXNIP-NLRP3 interaction, reduces oxidative stress	DSS-induced colitis, LPS-stimulated THP-1/BMDM	Preclinical	Zhao et al[58]
CY-09	NLRP3	Binds NLRP3 NACHT domain to block ATP activity	LPS-stimulated BMDM	Preclinical	Jiang et al[59]
VX-740 (pralnacasan)	Caspase-1	Reversible competitive caspase-1 inhibitor	DSS-induced acute colitis	Phase II discontinued (rheumatoid arthritis)	Bauer et al[60]; Dhani et al[61]
VX-765 (belnacasa)	Caspase-1	Prodrug of VRT-043198, a reversible caspase-1 inhibitor	DSS-induced acute colitis	Phase II discontinued (epilepsy; No. NCT01501383)	Wang et al[36]; Dhani et al[61]
Ac-YVAD-CMK	Caspase-1	Irreversible covalent inhibitor (chloromethylketone modification of Cys285)	DNBS-induced colitis	Preclinical	Pellegrini et al[57]
Necrosulfonamide	GSDMD	Alkylates GSDMD to prevent pore formation	DSS-induced acute colitis	Preclinical	Rathkey et al[62]; Yang et al[63]
Metformin	AMPK/NLRP3	AMPK activation inhibits NLRP3 inflammasome assembly	DSS-induced acute colitis	Marketed (T2DM); IBD trials ongoing (No. NCT05574387)	Hosseini et al[64]; Cao et al[65]; El-Haggar et al[66]
Tranilast	NLRP3	Inhibits NLRP3 oligomerization and ASC speck formation	DSS/TNBS-induced colitis	Marketed (anti-allergic)	Huang et al[67]; Seto et al[68]; Oshitani et al[69]
Disulfiram	GSDMD	Covalent modification of GSDMD to inhibit pore formation	DSS/TNBS-induced colitis	Marketed (alcohol addiction)	Chi et al[70]; Lei et al[71]; Zhou et al[72]; Ou et al[73]; Luo et al[74]
Dimethyl fumarate	GSDMD	Covalent modification of GSDMD to inhibit pore formation	DSS-induced colitis	Marketed (multiple sclerosis)	Humphries et al[75]; Li et al[76]; Patel et al[77]; Buscarinu et al[78]; Shah et al[79]

Detailed information regarding primary endpoints and adverse reactions for these candidate drugs is selectively summarized in the corresponding drug profiles within the “small-molecule inhibitors: Precision targeting of key pyroptotic nodes” subsection of the main text. NLRP3: Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; GSDMD: Gasdermin D; AMPK: Adenosine 5’-monophosphate-activated protein kinase; ATP: Adenosine triphosphate; LRR: Leucine-rich repeat; ASC: Adapter protein apoptosis-associated speck-like protein containing a CARD; DSS: Dextran sulfate sodium; DNBS: Dinitrobenzene sulfonic acid; TNBS: 2,4,6-trinitrobenzenesulfonic acid solution; T2DM: Type 2 diabetes mellitus; IBD: Inflammatory bowel disease.

Development of NLRP3 inhibitors: MCC950 (CP-456773) is a selective NLRP3 inhibitor that targets the NLRP3 adenosine triphosphatase domain, blocking inflammasome activation and inhibiting IL-1β/IL-18 release[49]. Its broad efficacy has been shown in preclinical IBD models. In IL-10 -/- mice, it attenuates spontaneous colitis[15]; additionally, in the DSS-colitis model, it restores microbial balance by enriching Lactobacillus while suppressing Escherichia-Shigella[31]. MCC950 also acts synergistically with celastrol or metformin via HSP90 modulation and autophagy induction[50,51]. However, its clinical development was discontinued due to hepatotoxicity[52] (Figure 3). In the pursuit of safer therapeutics, researchers developed a series of sulfonylurea derivatives based on the structure of MCC950. Among them, compound 15 emerged as a potent NLRP3 inhibitor, demonstrating superior efficacy to MCC950 in mouse models of acute peritonitis and diabetic nephropathy[53]. Although MCC950 remains a key tool compound for NLRP3 research, its clinical application in IBD is limited by toxicity, necessitating strategies such as structural optimization or targeted delivery.

Figure 3 Research progress on pyroptosis inhibitors for inflammatory bowel disease treatment. IBD: Inflammatory bowel disease; siRNA: Small interfering RNA.

OLT1177 (dapansutrile) is a clinically safe, selective NLRP3 inflammasome inhibitor that binds the NLRP3 leucine-rich repeats domain, preventing ASC oligomerization and inflammasome assembly without altering NLRP3 expression or adenosine triphosphate activity[54]. In IBD models, it reduces IL-1β and protects intestinal barrier integrity in DSS-induced colitis[35], and exhibits synergistic efficacy when combined with the P2X7 antagonist brilliant blue G via dual suppression of NLRP3 and MyD88/NF-κB pathways[55]. With a favorable clinical safety profile, OLT1177 has completed phase I (No. NCT02134964) and phase II osteoarthritis trials (No. NCT02104050), and is currently in phase III for gout (No. NCT05658575). Its robust preclinical data, pharmacokinetic properties, and distinct mechanism free from hepatotoxicity risks associated with analogs like MCC950 support its promise as a next-generation IBD therapeutic. Further human studies in IBD are warranted.

The acrylate derivative INF39 irreversibly inhibits NLRP3 by covalently modifying Cys409 in its NACHT domain. Administered at 25 mg/kg, it completely suppresses dinitrobenzene sulfonic acid-induced colitis with no observed toxicity, due to its rapid plasma clearance[56,57]. In contrast, the flavonoid-based VI-16 employs a novel redox mechanism. It disrupts the thioredoxin-interacting protein-NLRP3 interaction and reduces mitochondrial reactive oxygen species by 75% in THP-1 macrophages. This mechanism coffers superior efficacy to MCC950 in chronic DSS models, particularly for preventing fibrosis progression[58]. CY-09 selectively stabilizes NLRP3’s inactive NACHT domain, inhibiting NLRP3 (not NLRC4/AIM2) and suppressing IL-1β/IL-18 in macrophages. CY-09 exhibits exciting therapeutic potential in different types of diseases, including cryopyrin-associated autoinflammatory syndrome (CAPS) mouse model and type 2 diabetes[59]. Currently in preclinical development, the NLRP3 inhibitors INF39, VI-16 and CY-09 have not yet entered human clinical trials (Figure 3).

Limitations of caspase-1 inhibitors: Inhibition of caspase-1 by VX-740 and VX-765 has been shown to attenuate DSS-induced colitis in mice[36,60]. However, both compounds face challenges related to metabolic instability and hepatotoxicity. Structurally, they incorporate a difluorophenol warhead that enables potent caspase-1 binding but is susceptible to extensive glucuronidation, leading to poor metabolic stability. These pharmacokinetic drawbacks have significantly limited their clinical translation. Specifically, VX-740 was discontinued in phase II trials for rheumatoid arthritis due to hepatotoxicity and observations of hepatic fibrosis in primate studies. Similarly, VX-765 failed to achieve efficacy endpoints in a clinical trial for epilepsy (No. NCT01501383), despite evidence of sufficient target engagement[61] (Figure 3). Ac-YVAD-CMK (caspase-1 inhibitor II) irreversibly inhibits caspase-1, reducing IL-1β and myeloperoxidase (MPO) activity in colitis models, though its effects on downstream cytokines remain limited compared to NLRP3 or IL-1-targeted therapies[57].

Pyroptosis execution protein inhibitors: Necrosulfonamide (NSA) represents a mechanistically distinct class of small-molecule pyroptosis inhibitors that directly targets the executioner protein GSDMD. This synthetic sulfonamide compound (molecular weight: 427.5 Da) exerts its therapeutic effects through covalent modification of human GSDMD at Cys191 within the N-terminal pore-forming domain, effectively blocking plasma membrane pore formation (half maximal inhibitory concentration = 1.8 μM)[62]. In DSS-induced murine colitis models, daily NSA administration attenuated disease severity, evidenced by improved disease activity index scores and reduced histopathological inflammation compared to controls. Beyond inhibiting GSDMD-mediated pyroptosis, NSA also directly suppresses MLKL to block necroptosis, reduces macrophage infiltration and T-cell activation, and helps maintain intestinal barrier integrity to prevent bacterial translocation. These pleiotropic immunomodulatory effects collectively contribute to the amelioration of IBD[63].

Small-molecule pyroptosis inhibitors demonstrate considerable therapeutic potential for IBD. Preclinical studies have shown that NLRP3 antagonists (e.g., MCC950, OLT1177) and GSDMD inhibitors (e.g., NSA) effectively attenuate colitis, restore microbial balance, maintain epithelial barrier integrity, and exert pleiotropic immunomodulatory effects. However, their clinical translation has encountered significant obstacles. Although NLRP3 inhibitor MCC950 and caspase-1 inhibitors VX-740/VX-765 exhibited robust efficacy in preclinical models, their development was halted due to safety and pharmacokinetic concerns. MCC950 was discontinued owing to hepatotoxicity, while the VX compounds failed because of metabolic instability resulting from rapid glucuronidation of their difluorophenol warhead[52,61]. These setbacks underscore a critical translational challenge: Strong target engagement and efficacy in animal models are insufficient to ensure clinical success. Major barriers include unforeseen off-target organ toxicity, inadequate predictive toxicity models, poor metabolic stability of compounds, and a lack of biomarkers for patient stratification. Therefore, future research should prioritize comprehensive safety profiling, structural optimization to improve metabolic stability, and biomarker-driven clinical trial designs to facilitate the successful application of pyroptosis inhibitors in IBD treatment.

Drug repurposing (accelerated clinical translation pathways): Beyond novel small-molecule inhibitors, several repurposed pharmacological agents targeting pyroptosis-related proteins have exhibited significant anti-inflammatory efficacy in both in vitro studies and experimental models of IBD.

Metformin exerts anti-inflammatory effects primarily through adenosine 5’-monophosphate-activated protein kinase-dependent inhibition of the NLRP3 inflammasome, leading to reduced IL-1β secretion, oxidative stress, NF-κB activation, and ASC speck formation[64,65]. It also enhances intestinal barrier integrity and attenuates pyroptosis via upregulation of UCP2 and NCF1, thereby suppressing reactive oxygen species[65]. Clinically, as an adjunct to mesalamine, metformin has been shown to improve Mayo scores, pain, inflammatory markers, and zonulin levels in UC patients in a randomized trial (No. NCT05553704)[66]. With an ongoing IBD clinical investigation (No. NCT05574387) and a well-established safety profile, metformin represents a promising repurposed therapeutic for UC.

Originally developed as an anti-allergic agent, tranilast (TL) has been repurposed as a selective NLRP3 inflammasome inhibitor that directly targets the NACHT domain to prevent oligomerization, demonstrating therapeutic potential across multiple NLRP3-driven conditions including gout and autoinflammatory diseases[67]. A study demonstrates that TL alleviates 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced experimental colitis by inhibiting MPO activity and inflammatory cell infiltration. However, pharmacokinetic analysis reveals that TL exhibits poor oral bioavailability (approximately 6.5%) in TNBS-induced colitis rats, suggesting its limited intestinal absorption may restrict clinical efficacy. These findings indicate the need to improve TL’s bioavailability for enhanced therapeutic outcomes[68]. Its clinical application for IBD awaits formal trials despite preliminary evidence of efficacy in CD cases[69].

Disulfiram, an anti-alcoholism drug repurposed for IBD, has demonstrated robust efficacy in preclinical studies by exerting pleiotropic effects. It not only directly inhibits GSDMD-mediated pyroptosis but also activates the nuclear respiratoty factor 2 (Nrf2)/heme oxygenase 1 antioxidant pathway via GSK-3β downregulation, elevating the glutathione/glutathione, oxydized ratio and reducing oxidative stress[70]. Additionally, it suppresses inflammatory cytokine release and modulates gut microbiota and bile acid metabolism[71]. Its activity is potentiated by copper ions, and its targeting has been improved using lactoferrin nanoparticles to enhance bioavailability[72,73]. Despite these wide ranging benefits, the clinical use of disulfiram faces major challenges. These include significant side effects, particularly the disulfiram ethanol reaction caused by inhibition of acetaldehyde dehydrogenase, and a critical lack of data from human trials[74]. Future studies may explore strategies such as structural modification, targeted delivery, or combination therapy to circumvent alcohol-related adverse reactions and broaden its therapeutic window.

Dimethyl fumarate (DMF), an oral drug for relapsing multiple sclerosis, inhibits pyroptosis by succinylating cysteine C192 of GSDMD, thereby blocking its activation by inflammatory caspases[75]. In DSS-induced colitis models, DMF demonstrates broad efficacy by simultaneously activating Nrf2-mediated antioxidant pathways and suppressing inflammatory responses, and based on this mechanism, it shows promising potential in the treatment of gastrointestinal disorders such as gastric ulcers and UC[76,77]. A localized delivery strategy may reduce systemic exposure and mitigate adverse effects such as lymphopenia while preserving efficacy[77]. Although clinical studies in MS indicate benefits on gut barrier and microbiota[78,79], further research is needed to validate its efficacy and safety specifically in IBD contexts.

The repurposing of existing drugs such as metformin, TL, disulfiram, and DMF represents a promising approach to modulate pyroptosis in IBD, with each agent employing distinct mechanisms including NLRP3 inhibition, GSDMD suppression, or Nrf2 activation. However, the clinical application of these drugs faces several challenges: TL exhibits low oral bioavailability, disulfiram is associated with systemic side effects including the alcohol-disulfiram reaction, and DMF may lead to systemic adverse reactions such as lymphopenia. These limitations collectively underscore their shortcomings in tissue targeting, pharmacokinetic properties, and systemic safety profiles[68,74,77]. Addressing these challenges requires targeted delivery strategies that maximize drug accumulation in the colon and minimize systemic exposure. This can be achieved through platforms such as nanocarriers, as well as site-specific technologies including potential of hydrogen-responsive, enzyme-triggered, and ligand-receptor systems. Functional biomaterials like hyaluronic acid (targeting CD44) and chitosan (mucoadhesive) further enhance localized retention and penetration[80]. For example, lactoferrin-based nanoparticles loaded with disulfiram have demonstrated improved bioavailability and targeted delivery to inflamed intestinal regions, resulting in enhanced local efficacy and reduced systemic side effects[73]. Such advanced delivery systems offer a viable pathway to overcome existing limitations, enabling safer and more precise therapeutic intervention against pyroptosis-mediated intestinal inflammation.

Biologics: Next-generation targeted intervention approaches

Biologic therapies targeting pyroptosis offer precision intervention for IBD through monoclonal antibodies, nucleic acid-based agents, and engineered proteins, minimizing off-target effects compared to small molecules (Table 2).

Table 2 Biologics of pyroptosis-related proteins.

Name	Target protein	Pharmacological mechanism	Pyroptotic diseases	Clinical stage	Ref.
Canakinumab (ilaris)	IL-1β	IL-1β monoclonal antibody, inhibits NLRP3 downstream effects	Cryopyrin-associated periodic syndromes	Phase III completed (SJIA; No. NCT02396212)	Shaul et al[81]; Miyamoto et al[82]
Anakinra	IL-1	Recombinant IL-1 receptor antagonist	Rheumatoid arthritis; Winnie-TNF-KO murine model (IBD)	Phase IV (RA; No. NCT00121043)	Liso et al[83]; Dogan et al[84]; Truyens et al[85]
GSK1070806	IL-18	Humanized monoclonal antibody against IL-18	Rheumatoid arthritis; Crohn’s disease	Phase II (CD; No. NCT03681067)	Guha et al[86]
DFV890	NLRP3	Oral NLRP3 inhibitor (small molecule biologic)	Coronary heart disease; COVID-19	Phase II (COVID-19; No. NCT04382053)	Shen et al[87]; Gatlik et al[88]; Madurka et al[89]
NLRP3-ASO	NLRP3 mRNA	Antisense oligonucleotide suppressing NLRP3	Cryopyrin-associated periodic syndromes; Alzheimer’s disease	Preclinical	Kaufmann et al[90]; Braatz et al[91]
IC100 (ASC-mAb)	ASC speck	Monoclonal antibody blocking inflammasome assembly	Retinopathy of prematurity; Parkinson’s disease	Preclinical	de Rivero Vaccari et al[92]; Yuan et al[93]; Cyr et al[94]
Caspase-1 siRNA	Caspase-1 mRNA	siRNA silencing caspase-1 expression	Alzheimer’s disease	Preclinical	Han et al[95]
GSDMD-Nb	GSDMD	Nanobody binding GSDMD-N terminus to prevent pore formation	Sepsis	Preclinical	Schiffelers et al[96]

Detailed information regarding primary endpoints and adverse reactions for these biologics is selectively summarized in the corresponding agent profiles within the “biologics: Next-generation targeted intervention approaches” subsection of the main text.

NLRP3: Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; ASC: Adapter protein apoptosis-associated speck-like protein containing a CARD; IL: Interleukin; ASO: Antisense oligonucleotide; siRNA: Small interfering RNA; GSDMD: Gasdermin D; mRNA: Messenger RNA; TNF: Tumor necrosis factor; KO: Knockout; IBD: Inflammatory bowel disease; COVID-19: Coronavirus disease 2019; SJIA: Systemic juvenile idiopathic arthritis; CD: Crohn’s disease; RA: Rheumatoid arthritis.

Canakinumab is a selective, fully humanized monoclonal antibody targeting IL-1β, which has been successfully used in the treatment of various autoinflammatory diseases. Clinically approved for CAPS, it also shows potential in very early-onset IBD (VEO-IBD)[81]. A phase III trial in patients with systemic juvenile idiopathic arthritis (No. NCT02396212; Figure 3) demonstrated high efficacy, including 100% American College of Rheumatology pediatric (ACR pedi) 30 response at week 8 and 73.7% corticosteroid tapering by week 28. At week 48, ACR pedi 50/70/90/100 responses were achieved by 100.0%/100.0%/87.5%/68.8% of patients, respectively. However, the safety profile included frequent infections (271.6 per 100 patient-years) and serious adverse events in 42.1% of patients, with two suspected cases of macrophage activation syndrome though no deaths were reported. Importantly, the safety profile of canakinumab appears to vary across indications; while it has shown a confirmed long-term safety record in CAPS[82], its applications in IBD remain investigational, requiring biomarker-driven trials to identify optimal responders.

Anakinra, a recombinant IL-1 receptor antagonist targeting IL-1 signaling, is currently approved for rheumatoid arthritis with established phase IV clinical evidence (No. NCT00121043). Emerging evidence suggests its potential therapeutic role in IBD, particularly in specific subtypes characterized by IL-1 pathway dysregulation. Preclinical studies using the Winnie-TNF-knockout murine model demonstrated that anakinra ameliorates colonic inflammation in TNF-independent UC by blocking IL-1β-mediated effects[83]. Clinically, case reports highlight efficacy in IL-10 receptor β deficiency-associated IBD and refractory UC, yet controlled trials are needed to define its role[84,85].

GSK1070806, an investigational anti-IL-18 monoclonal antibody, is currently undergoing phase II clinical evaluation for CD (No. NCT03681067), demonstrating emerging therapeutic potential in IBD (Figure 3). Notably, this agent has shown clinical efficacy in treating VEO-IBD associated with IL-18opathy[86]. The therapeutic mechanism involves targeted neutralization of IL-18-mediated inflammatory signaling, which appears particularly relevant in certain monogenic IBD forms characterized by IL-18 pathway dysregulation. While these preliminary findings suggest promise for IL-18 inhibition in selected IBD populations, particularly pediatric-onset cases with specific autoinflammatory features, further clinical development is necessary to establish its safety profile, optimal dosing, and efficacy across broader IBD patient subgroups. The ongoing phase II trial in CD may provide more definitive evidence regarding its therapeutic potential in conventional IBD presentations.

DFV890 is a highly selective oral small-molecule NLRP3 inflammasome inhibitor that directly targets the NACHT domain of NLRP3 protein through its unique sulfonimidamide structure, effectively blocking inflammasome oligomerization and activation[87]. Preclinical studies demonstrate that DFV890 significantly inhibits caspase-1 activation and subsequent maturation/release of downstream IL-1β and IL-18, while showing superior selectivity for NLRP3 over other inflammasomes (e.g., AIM2 or NLRC4). In first-in-human trials, DFV890 exhibited favorable safety and tolerability profiles with ideal pharmacokinetic characteristics supporting once-daily dosing[88]. The compound has completed evaluation in a randomized, double-blind, placebo-controlled phase II clinical trial (No. NCT04382053) for coronavirus disease 2019-associated pneumonia, demonstrating superior clinical improvement, viral clearance, and ventilator-free survival, along with a favorable safety profile[89]. Although no clinical studies have yet been conducted for IBD, DFV890 shows promising therapeutic potential in IBD treatment based on the pivotal role of NLRP3 inflammasome. Future preclinical efficacy validation and biomarker-guided clinical studies are warranted to evaluate DFV890’s potential value for IBD therapy.

NLRP3-targeted antisense oligonucleotide (NLRP3-ASO) represents a novel therapeutic modality that specifically suppresses NLRP3 expression through the principle of Watson-Crick base pairing. Preclinical evidence supports the therapeutic potential of NLRP3-ASO in inflammatory diseases[90]. Kaufmann et al[90] showed that NLRP3-ASO treatment significantly reduced IL-1β expression and improved survival in mutant NLRP3 mice, establishing proof-of-concept for this approach in systemic autoinflammatory disorders, while Braatz et al[91] confirmed suppression of microglial activation in neuroinflammatory contexts. NLRP3-ASO remains a preclinical-stage intervention whose efficacy in IBD awaits experimental assessment.

The humanized IgG4 monoclonal antibody IC100 represents a novel therapeutic approach targeting the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), a critical adaptor protein in inflammasome assembly[92]. Current preclinical studies demonstrate its broad anti-inflammatory potential. In oxygen-induced retinopathy, IC100 significantly reduced pathological angiogenesis and inflammation[93], and in Parkinson’s disease models, it effectively attenuated neuroinflammation induced by α-synuclein aggregates[94]. While current research has focused on retinopathy and neurodegenerative applications, the demonstrated ability of IC100 to block multiple inflammasome pathways suggests potential utility in other inflammatory conditions, including possible future exploration in IBD.

A novel therapeutic approach using adeno-associated virus 9-delivered caspase-1 small interfering RNA (siRNA) demonstrated significant neuroprotective effects in APP/PS1 transgenic mice, a model of Alzheimer’s disease. The study revealed that β-amyloid-induced neuronal pyroptosis occurs through caspase-1 activation, establishing a new mechanism of neural injury in Alzheimer’s disease pathogenesis. By specifically silencing caspase-1 expression via siRNA, researchers observed decreased neuroinflammation and improved neuronal survival[95]. While currently in preclinical development for Alzheimer’s disease, this caspase-1 inhibition strategy may have broader implications for other caspase-1-dependent inflammatory conditions, pending further investigation.

Schiffelers et al[96] developed antagonistic nanobodies that specifically target the N-terminal domain of GSDMD, effectively inhibiting pore formation by blocking GSDMD oligomerization, thereby providing a novel therapeutic approach for inflammatory diseases such as sepsis. In LPS- or bacteria-induced septic mouse models, GSDMD-Nb demonstrated significant efficacy in reducing serum levels of IL-1β and IL-18, attenuating systemic inflammatory responses, and markedly improving survival rates. GSDMD-Nb demonstrates significant advantages over small-molecule inhibitors like disulfiram in terms of target specificity, mechanistic precision and sustained efficacy, albeit with higher development costs and unresolved delivery challenges.

A range of novel inhibitors including monoclonal antibodies, small molecule drugs, oligonucleotides, and nanobodies have demonstrated promising therapeutic potential in autoinflammatory diseases and IBD. These agents precisely target key nodes of the pyroptosis pathway and have shown significant efficacy in preclinical models and early clinical studies. However, current clinical evidence suggests that single-target therapies (e.g., canakinumab or anakinra), while effective in some patients, often face limitations due to disease heterogeneity, compensatory inflammatory pathway activation, or feedback mechanisms, in addition to safety concerns such as increased infection risk. These insights highlight the need for multi-target intervention strategies to achieve more robust and sustained anti-inflammatory effects in complex diseases like IBD. Promising approaches include the development of dual-target inhibitors (e.g., simultaneously inhibiting NLRP3 and caspase-1) to broadly suppress pyroptosis signaling and reduce compensatory activation, as well as rationally designed combination therapies incorporating pyroptosis inhibitors with mechanistically complementary agents such as anti-cytokine biologics (anti-TNF-α, anti-IL-23) or JAK inhibitors which may enhance efficacy in refractory cases and allow dose reduction to mitigate potential risks.

Strategic target selection in the pyroptosis pathway

Strategic target selection in the pyroptosis pathway represents a critical translational bridge, guiding the rational application of the aforementioned therapeutic agents from small molecules and biologics to repurposed drugs and advanced delivery systems into clinically viable regimens. Selecting the best target in the pyroptosis pathway involves balancing scientific potential with clinical feasibility. Inhibiting the downstream protein GSDMD represents the most promising long-term strategy, as it blocks the final step of inflammation and avoids activation of alternative pathways. Currently, targeting upstream NLRP3 is the most clinically advanced approach, with safe and effective candidates like OLT1177 already in phase II trials, making it suitable for first-line use. Midstream caspases are less ideal targets due to pharmacokinetic issues, functional redundancy, and previous clinical failures. Targeting downstream cytokines such as IL-1β and IL-18 offers an alternative strategy; however, cytokine inhibition may be limited by compensatory pathways and broader immunosuppressive effects, whereas upstream targeting of NLRP3 or executioner GSDMD may provide more comprehensive control of pyroptotic inflammation. Thus, an optimal strategy may involve a tiered approach: Starting with NLRP3 inhibitors for most patients, and adding or switching to GSDMD inhibitors for those with hard-to-treat disease, to achieve better control of gut inflammation (Figure 4).

Figure 4 Targeting the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome pathway: Mechanisms and therapeutic inhibitors. NLRP3: Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; IL: Interleukin; TLR: Toll-like receptor; NF-κB: Nuclear factor kappa-B; GSDMD: Gasdermin D; IL-1R: Interleukin-1 receptor; IL-18R: Interleukin-18 receptor; ASC: Adapter protein apoptosis-associated speck-like protein containing a CARD; ASO: Antisense oligonucleotide; siRNA: Small interfering RNA.

CONCLUSIONS

Pyroptosis has emerged as a critical pathogenic mechanism in IBD, characterized by dysregulated activation of the NLRP3 inflammasome-caspase-GSDMD axis which drives intestinal barrier dysfunction, immune dysregulation and microbial imbalance (Figure 2). Therapeutic targeting of this pathway demonstrates significant promise but faces translational barriers including hepatotoxicity risks with first-generation inhibitors (e.g., MCC950), suboptimal colonic biodistribution, and the absence of validated stratification biomarkers. Leading clinical candidates notably the NLRP3 inhibitor OLT1177 and IL-18 neutralizing antibody GSK1070806 (both in phase II trials) show dual efficacy in suppressing inflammation and promoting epithelial repair (Figure 3). Repurposed drugs including DMF, disulfiram and metformin provide complementary strategies requiring confirmatory clinical validation (Figure 3). While these approaches show considerable potential, key challenges remain in optimizing drug safety profiles, enhancing tissue-specific delivery, and developing biomarkers for patient stratification. Future progress requires a multipronged approach: Developing precision biomarkers for patient stratification, structurally optimizing safer and more selective inhibitors, and designing innovative delivery systems (e.g., colon-targeted nanoparticles) to enhance therapeutic localization. Combination strategies with existing immunomodulators may broaden efficacy in refractory cases while preserving mucosal immunity. Success demands multidisciplinary collaboration to bridge mechanistic insights with clinical innovation specifically through biomarker-stratified phase II trials of lead compounds and real-world validation of monitoring protocols. This paradigm shift toward mechanism-based intervention promises to transform IBD management by achieving durable deep remission in treatment-resistant patients through precision modulation of pyroptotic pathways.