Progress in the Treatment of Ulcerative Colitis by Targeting NLRP3 Inflammasome

Introduction

Ulcerative colitis (UC) is a prolonged and progressive inflammatory disease characterized by ulceration of colorectal mucosa. Generally, UC patients have persistent and recurrent diarrhea, accompanying abdominal pain, mucopurulent bloody stool, and tenesmus.¹ Extraintestinal manifestations occur in 20–35% of patients with UC, including arthritis, erythema nodosum, and iritis. And up to 20% of cases, these manifestations may precede intestinal symptoms.² Additionally, the risk of venous thrombosis may increase by 2- to 4-fold in patients with UC, which further elevates when patients receive glucocorticoid therapy.^3,4 Persistent stimulation of colonic tissue by chronic inflammatory responses associated with ulceration may induce malignant transformation of colonic epithelial cells, leading to colorectal cancer. The risk of colorectal cancer is significantly higher in patients with UC for more than 10 years compared with the normal.⁵ The American College of Gastroenterology (ACG) states that the treatment of UC mainly relies on non-steroidal anti-inflammatory drugs represented by 5-aminosalicylic acid derivatives, biological agents including tumor necrosis factor (TNF)-α monoclonal antibodies, and immunosuppressants such as thiopurine.⁶

The pathogenesis of UC is complex and has not yet been fully elucidated. Most studies hold that the onset of UC is jointly induced by immune, genetic, infectious and other factors. Inflammatory mediators, human leukocyte antigen (HLA), interleukin 23 receptor gene (IL23R), pathogenic microorganisms, and food antigens are all triggers for UC.^7,8 Its pathogenesis can be briefly summarized as genetically susceptible individuals developing an aberrant intestinal immune response in response to exogenous factors, ultimately resulting in intestinal mucosal damage.¹ Excessive inflammatory response contributes a lot to the pathogenesis of UC. The NOD-like receptor (NLR) family has attracted considerable attention in recent years, owing to its pivotal role in intestinal inflammatory responses. The NOD-like receptor pyrin domain-containing 3 (NLRP3) and its assembled inflammasome are the most widely studied inflammasomes.⁹ After activated, NLRP3 inflammasome can release inflammatory cytokines such as interleukin-1 beta (IL-1β) and interleukin-18 (IL-18). These inflammatory cytokines are involved in UC initiation and progression.

Mesalazine is a frontline drug for acute mild-to-moderate UC. However, reports have indicated that it might induce interstitial nephritis.¹⁰ Oral corticosteroids can be used in patients with mild-to-moderate UC who do not respond to 5-aminosalicylates or experience relapse during maintenance therapy with 5-aminosalicylates. Nevertheless, glucocorticoid therapy increases the risk of infection, gastrointestinal reaction and fractures.¹¹ UC is characterized by a protracted and refractory course. Approximately 37% of UC patients experience chronic intermittent symptoms, and 6% suffer from chronic persistent symptoms.¹² Despite expanding therapeutic options, 10–20% of patients still require panproctocolectomy due to ineffective treatment. To overcome these obstacles, the key lies in combining pharmacotherapy with precision therapies.¹ Biologics, represented by TNF-α antibodies, and small molecule drugs, represented by Janus kinase (JAK) inhibitors, have ushered in a new era of precision treatment for UC. Unfortunately, nearly 50% of patients exhibit primary non-response or loss response to TNF-α antibodies, and JAK inhibitors may increase the risk of major adverse cardiovascular events and cancer.^13,14 Therefore, exploring safe and effective novel therapeutic strategies and drug targets has become a focal point in UC research. Aberrant overactivation of NLRP3 inflammasome has been proved to be closely associated with the onset and progression of UC.¹⁵ Upon activation, NLRP3 inflammasome triggers secretion of IL-1β and IL-18, which further induces inflammatory cell infiltration and disrupts intestinal epithelial tight junctions. Multiple NLRP3 inhibitors for UC treatment have entered clinical trials, such as selnoflast and N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger. Based on existing scaffolds, novel targeted molecules have been synthesized to enhance the specificity and efficacy of NLRP3 inflammasome inhibition. Although previous reviews have discussed therapeutic feasibility of targeting NLRP3 inflammasome for UC treatment, they mainly summarized inhibiting NLRP3 inflammasome-related pathways in UC. Several studies have investigated and estimated new inhibition avenues for NLRP3 inflammasome in recent years, which have evolved rapidly and warrant update.^16,17 Therefore, we retrospect research progress on targeting NLRP3 inflammasome for treating UC. Compounds are categorized into those that inhibit NLRP3 inflammasome activation, block assembly, and suppress related proteins. Furthermore, we discuss the structure activity relationships (SAR) and core scaffolds as well as summarize current clinical drug development progress related to targeting NLRP3 for UC therapy, aiming to provide a reference for the future clinical translation of novel NLRP3 inhibitors.

NLRP3 Inflammasome and the Pathogenesis of UC

Composition and Functions of NLRP3 Inflammasome

NLRP3 inflammasome is a polyprotein complex composed of NLRP3 as the sensor, adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and effector pro-caspase-1.¹⁸ NLRP3 is the core protein of NLRP3 inflammasome and consists of a central nucleotide-binding oligomerization domain (NACHT), N-terminal pyrin domain (PYD), and C-terminal leucine-rich repeat (LRR) domain. NACHT possesses adenosine triphosphate (ATPase) activity and PYD mediates its interaction with the adaptor protein ASC. LRR regulates NLRP3 activity and senses endogenous danger signals. ASC contains an N-terminal PYD, which connects to upstream components, and a caspase activation and recruitment domain (CARD), which links to caspase-1.¹⁹ NLRP3 inflammasome pathway can be categorized into canonical, non-canonical, and alternative. The structure and process are shown in Figure 1.

Figure 1 NLRP3 inflammasome structure and signaling pathway. The NLRP3 inflammasome contains NLRP3, ASC and pro-caspase-1. It will assemble after priming and activation process. NLRP3 pathway can be divided into the canonical, the non-canonical and the alternative. In the canonical pathway, priming steps involve PAMPS and DAMPS causing NF-κB upregulation, subsequently inducing NLRP3 related protein expression. ATP, K⁺, Cl− and other stimulation work as triggers for NLRP3 assembly, along with caspase-1 formation. ATP also activates the P2X7. Caspase-1 brings about GSDMD cleavage, IL-1β and IL-18 maturity, which leads pyroptosis. Different from the canonical, LPS in the non-canonical pathway directly binds to pro-caspase-4/5/11. NLRP3 is activated through K+ efflux via IL-1β and IL-18 release. The alternative, however requires TLR4-TRIF-RIPK1-FADD-CASP8 signaling to cause NLRP3 assembly. Thereafter caspase-1 forms and leads inflammatory cytokines release. Created with BioGDP.com.20 Abbreviations: NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; LPS, lipopolysaccharide; IL-1β, interleukin-1 beta; IL-18, interleukin-18; NACHT, nucleotide-binding oligomerization domain; PYD, N-terminal pyrin domain; LRR, C-terminal leucine-rich repeat; pro-IL-1β, pro-interleukin-1β; pro-IL-18, pro-interleukin-18; PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns; TLR4, Toll-like receptor 4; TNF, tumor necrosis factor; ATP, adenosine triphosphate; NF-κB, nuclear factor-κB; CatB, cathepsin B; P2X7, P2X purinoceptor 7; ROS, reactive oxygen species; GSDMD, Gasdermin D; N-GSDMD, Gasdermin D N-terminal domain.

The canonical process contains two steps: priming and activation. In the priming stage, expression of NLRP3 and pro-interleukin-1β (pro-IL-1β) is upregulated, while the activation stage triggers the assembly of NLRP3 inflammasome.²¹ Pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) initiate the priming stage. These signals are transduced through receptors such as Toll-like receptor 4 (TLR4) or TNF receptors, leading to the activation of nuclear factor-κB (NF-κB) signaling pathway, subsequently promoting gene transcription associated with NLRP3 inflammasome.²² A binding partner of S-phase kinase-associated protein 1 (SKP1) is suppressor of the G2 allele of SKP1 (SGT1). To form a complex with NLRP3 inflammasome, heat shock protein 90 (HSP90) synergizes with SGT1. This complex maintains NLRP3 inflammasome in an inactive, but activation-competent state prior to the reception of priming signals. If this complex fails to assemble, NLRP3 protein undergoes degradation.²³

NLRP3 inflammasome activation has been the major focus of recent studies. Its signals can be triggered by a variety of molecules and cellular events, including variations in extracellular adenosine triphosphate (ATP), efflux of K⁺ and Cl⁻, Ca²⁺ mobilization, release of cathepsin B (CatB) resulting from lysosomal damage, and production of ROS induced by mitochondrial dysfunction.²⁴ During the activation steps, extracellular ATP activates P2X purinoceptor 7(P2X7) and induces K⁺ efflux.²⁵ Upon ATP stimulation, Ca²⁺ signaling triggers mitochondrial damage to activate NLRP3 inflammasome. NLRP3 inflammasome has also been reported to cause mitochondrial dysfunction. Inactive NLRP3 inflammasome localizes to the endoplasmic reticulum (ER), whereas activated NLRP3 inflammasome and its adaptor protein ASC redistribute to the perinuclear region, co-localizing with the ER and mitochondria.²⁶ Lysosome rupture works as an endogenous danger signal and NLRP3 inflammasome can respond to the stimuli. Inhibition of CatB has been observed to reduce NLRP3 inflammasome activation.²⁷ In the inflammatory response, accumulation of ROS leads to oxidative stress. A previous study revealed that ROS could induce Ca²⁺ influx through the transient receptor potential melastatin 2 (TRPM2) channel.²⁸ ROS also act as a messenger to upregulate the NF-κB pathway.²⁹ Nevertheless, NLRP3 inflammasome activators, including nigericin, ATP and ROS, were not required for NLRP3 inflammasome activation. Conversely, a decreased intracellular K⁺ concentration is necessary for activating NLRP3 inflammasome.³⁰ After activated, the PYD of NLRP3 inflammasome interacts with the PYD of ASC. Then it recruits and oligomerizes pro-caspase-1 via CARD–CARD interactions, generating caspase-1 that amplifies inflammatory response.^31,32 Caspase-1 cleaves Gasdermin D (GSDMD) into Gasdermin D N-terminal domain (N-GSDMD) to form pores in the plasma membrane, leading to pyroptosis and inflammatory cytokines secretion.^33,34

Lipopolysaccharide (LPS) from gram-negative bacteria mediates the non-canonical pathway. LPS directly enters cytosol via phagocytosis, independent of TLR4, and then binds to pro-caspase-11 (caspase-11 in mice and caspase-4/5 in humans), inducing the cleavage of GSDMD.^35–37 IL-1β and IL-18 are released, which causes K⁺ efflux and pyroptosis, thereby triggering NLRP3 inflammasome activation. Neuron-derived clone 77 (Nur77), a nuclear orphan receptor, is an intracellular LPS-binding protein that binds to NLRP3 inflammasome. To connect NLRP3 inflammasome, Nur77 requires LPS and dsDNA released from GSDMD pores.³⁸ The alternative pathway involves TLR4-TRIF-RIPK1-FADD-CASP8 signaling. NLRP3 transcription is upregulated by LPS and TLR4 interactions, driving the immune response modulated by IL-1β. This pathway is independent of K⁺ efflux and pyroptosis.³⁹

NLRP3 Inflammasome and UC

Multiple studies have demonstrated that NLRP3 inflammasome and its related signaling pathways are involved in UC. A prospective observational study indicated that NLRP3 serum levels and protein expression of NLRP3 inflammasome components in the colonic mucosa were upregulated, which was positively correlated with disease severity. Additionally, an increased number of γδT17 cells was also observed.^15,40,41 In both UC patients and experimental models, the protein expression of TLR4, NF-κB, and NLRP3 was higher than the control, indicating that the TLR4/NF-κB/NLRP3 pathway contributed to UC progression through inflammatory responses and mucosal damage.^42,43 In dextran sulfate sodium (DSS)-induced UC mouse models, mRNA expressions of NIMA-related kinase 7 (NEK7), caspase-1, NLRP3 and GSDMD were significantly increased. Furthermore, interactions between NEK7-NLRP3 modulate UC progression via pyroptosis.^44,45 Orally administered DSS, Nlrp3-knockout (Nlrp3^−/−) mice showed less severe colitis. These results emphasize that the NLRP3 inflammasome is critical for intestinal inflammation.⁴⁶ At the cellular level, the imbalance between M1 and M2 macrophages during inflammation contributes to the pathogenesis of UC. M1 macrophages act as drivers of tissue damage due to their potent pro-inflammatory functions, whereas M2 macrophages facilitate tissue repair and maintain intestinal homeostasis by producing growth factors.⁴⁷ NLRP3 inflammasome in M1 macrophages can release IL-18 and IL-1β after activated, which exacerbates colonic inflammation. Specifically, IL-1β induces expression of chemokines and adhesion molecules, promoting inflammatory cell infiltration and increasing intestinal epithelial permeability.⁴⁸ Additionally, hyperactive IL-18 signaling hinders goblet cell maturation, thereby promoting colitis.⁴⁹ Inhibiting NLRP3/caspase-1/IL-1β pathway or neutralizing IL-18 can alleviate UC symptoms and reduce gene expression of pro-inflammatory cytokines.^44,50 However, under specific conditions, overexpression of NLRP3 inflammasome in phagocytes can protect the host from colitis. Nlrp3^R258W mice, characterized with hyper activation of NLRP3 inflammasome, exhibited high expression of NLRP3 in lamina propria CD11b⁺/CD11C⁺ mononuclear phagocytes and were more resistant to DSS-induced colitis. The mechanism might be that NLRP3 promoted secretion of IL-1β and increases antimicrobial peptides from the intestinal epithelia, which maintained homeostasis via an alternative regulatory T cell pathway.⁵¹ The gut microbiota also influences NLRP3 inflammasome in UC. Co-culture of mouse macrophages (Raw264.7) and bone marrow-derived macrophages (BMDM) with A. muciniphila led to upregulation of NLRP3 transcription. Further experiments confirmed that A. muciniphila alleviated colitis symptoms. However, in the absence of Nlrp3, A. muciniphila failed to protect the colon from inflammatory injury.⁵² These findings suggest NLRP3 inflammasome is complex in regulation of intestinal homeostasis.

NLRP3 inflammasome is expressed in both colonic macrophages and colonic epithelial cells, indicating that these two cell types are both crucial in chronic colitis mediated by NLRP3 inflammasome.¹⁵

The intestinal mucosa plays a pivotal role in sensing pathogens and danger signals, and this protective mechanism mainly occurs in intestinal epithelial cells (IECs) rather than immune cells. At the early stage of infection, signaling transduction of NLRP3 and ASC in IECs can restrict pathogen colonization and prevent pathological injury.⁵³ In vitro studies have confirmed that, compared to wild-type UC mice, Nlrp3^−/− UC mice exhibited more severe pathological injury in intestinal mucosa, with significantly reduced tight junction proteins and higher release of inflammatory cytokines.^54,55 In addition, Nlrp3^−/− UC mice induced by oxazolone exhibited severe colitis with increased Th2 cytokines and reduced IL-1β and IL-18. This phenomenon was mitigated by exogenous IL-1β or IL-18.⁵⁶ Notably, IL‑18 is mainly derived from IECs in the early stage of inflammatory bowel disease (IBD), whereas it is predominantly secreted by immune cells in the later stage of chronic inflammation, thereby driving Th1 immune response.⁵⁷ Upon activation, IECs can secrete inflammatory cytokines, recruit immune cells from the underlying lamina propria, and initiate local inflammatory responses. NLRP3 inflammasome also mediates pro-inflammatory effects of IECs. Another study demonstrated that Na⁺‑K⁺‑2Cl⁻ cotransporter knockout in IECs enhanced sensitivity of intestinal epithelial cells to alterations in intracellular K⁺ concentration and downregulated activation threshold of NLRP3 inflammasome in IECs, which facilitated secretion of IL‑1β and IL‑18.⁵⁸ This provides a potential target for the future development of targeted therapeutic strategies.

Current studies suggest that NLRP3 inflammasome plays a protective role against colonic inflammation during the initial phase. But its function in different stages and microenvironments of chronic inflammation has not been fully elucidated. Although TNF‑α inhibitor infliximab has been utilized for UC, patients with poor response to monotherapy require combination therapy with TNF-α and IL-23 inhibitors to alleviate symptoms.⁵⁹ However, TNF‑α is only expressed in immune cells, whereas NLRP3 inflammasome is expressed in both IECs and immune cells. Therefore, NLRP3 inflammasome inhibitors have significant advantages in UC treatment and development of drug delivery systems targeting NLRP3 is also a new direction. Considerable research has focused on inventing compounds that can selectively modulate NLRP3 inflammasome activity. In the following section, we summarize unique NLRP3 inflammasome inhibitors and highlight their potential as UC treatment options.

Small Molecule Compounds Targeting NLRP3 Inflammasome Treating UC

Inhibition of NLRP3 Inflammasome Assembly

Directly binding relevant structures of NLRP3 inflammasome can specifically inhibit its function. Designing small molecule compounds based on associated domains has become a cutting-edge area of research owing to their definite targeting properties.

The NACHT domain activates NLRP3 via ATP hydrolysis. Tetrahydroquinoline analog compound 6, triazinone derivative compound L38, non-sulfonylurea compound B6, all of them could bind to NACHT domain, thereby inhibiting the assembly NLRP3 inflammasome, and exhibited anti-inflammatory ability in DSS-induced UC models.^60–62 The steric properties of R-substituent on tetrahydroquinoline derivatives significantly impacted inhibiting IL-1β triggered by NLRP3 inflammasome. Long-chain and polyaromatic substituents at R-position were conducive to improving anti-inflammatory ability of compounds with a tetrahydroquinoline core. Compound 6 with a p-acetylphenyl group at R-position showed the most potent anti-inflammatory effect.⁶⁰ The thieno[2′,3′:4,5]pyrrolo[1,2-d][1,2,4]triazine scaffold possesses anti-inflammatory and antitumor properties, thus regarded as a highly promising structure.⁶³ Nevertheless, replacement of amide moiety in side chain region A with 1,3,4-oxadiazole scaffold yielded favorable inhibitory potency. Substituted pyridine at the R group of 1,3,4-oxadiazole achieved satisfactory inhibitory effects. In contrast, modification of methylene group in region L led to compound activity or metabolic stability decline, indicating that this methylene was critical for maintaining biological activity and metabolic stability.⁶¹ Ex2AH (WO 2018167468 A1) has been identified as the first NLRP3 inhibitor without sulfonylurea scaffold. Molecular docking revealed that the nitrogen atom on the right side of the ureido group does not form any hydrogen bond with the NLRP3 protein.⁶⁴ Shifting this nitrogen atom to form a new carbamate, and introducing a six-membered aromatic heterocycle at its R substituent, significantly improved the compound’s activity. The number or position of nitrogen atoms in the six‑membered aromatic heterocycle also influenced activity. While introduction of a sterically hindered methyl group or substituents on the benzene ring markedly reduced the inhibitory capacity of these compounds. Compound B6 displayed the best efficacy. Further SAR studies of B6 suggested that the hydrophobic pocket accommodating R3 was relatively small, requiring a small volume substituent.⁶²

As a key adaptor molecule for NLRP3 inflammasome assembly, ASC participates in NLRP3 inflammasome activation via its interaction with NLRP3 and oligomerization. However, small molecules can antagonize this process by blocking NLRP3 and ASC interaction or suppressed inhibiting ASC oligomerization, which can alleviate pathological damage of UC. These compounds include (4-((E)-3,5-dimethoxy-2-((E)-2-nitrovinyl) styryl) aniline derivative compound 27, 2,3-dihydro-1H-indene-5-sulfonamide analog compound 15z, compound Z48 with arylacrylamide scaffold, quinoline derivative W16, F14 with chalcone structure and boron-containing compound 27.^65–70 Pterostilbene-derived derivatives have been demonstrated to target NLRP3 inflammasome and interfere with its assembly process. But structural optimization is required due to their short half-life period.⁷¹ Nitrostyrene severed as the key structural moiety for binding to NLRP3. SAR analysis indicated appropriately sized rigid planar aromatic substituents adjacent to the carbonyl group facilitated occupation of the hydrophobic cavity. Compound 27 was then synthesized and exhibited potent anti-inflammatory activity, favorable absorption capacity, and satisfactory cell membrane permeability.⁶⁵ Previous studies validated that sulfonamide derivatives inhibited NLRP3 inflammasome activity and reduced side effect of hypoglycemia.⁷² Based on this scaffold, introduction of propylamine at R5 position and replacement with 2-methoxyethyl at R3 position yielded optimal activity. During optimization of R2 substituent, bromoacetic acid and cinnamic acid showed high inhibitory potency. But both of them showed non-specific inhibition and possessed cytotoxicity. Compound 15z was produced by cyclization of cinnamic acid, which displayed the highest concentration in colonic tissue.⁶⁶ The aryl acrylamide scaffold was proved holding potential for inhibiting NLRP3 inflammasome. Inspired by OLT1177, 3-sulfonylpropionitrile analogs were introduced to this scaffold. Halogen substitution at 4-position of the benzene ring was superior to that at 3-position, and molecular docking experiments proved that 4-bromo-substituted compound could form a halogen bond with NLRP3. However, halogen substitution on the benzene ring and ring-expansion strategies did not significantly improve compound activity. Therefore, the focus shifted to hydrophobic region at the bottom of NLRP3 protein pocket, and generated compound Z48. Notably, Z48 showed no significant therapeutic effect in Nlrp3^−/− mice.⁶⁷ Chloroquine and hydroxychloroquine were reported owning antagonistic effects against COVID-19. The mechanisms might lie in inhibition of NLRP3 inflammasome activity.⁷³ Removing chlorine atom at 7-position of chloroquine and introducing 2-phenyl and 4-amide groups improved compound activity. The 2-phenyl group was identified as a key moiety, and meta-fluorophenyl substitution was conducive to potency. Introduction of nitro group at 8-position generated compound W16 illustrated the highest activity.⁶⁸ Quinoline compounds are capable of binding to NACHT domain and inhibiting NLRP3-ASC interaction, making quinoline one of the most promising scaffolds. Chalcone derivatives could form hydrogen bonds and π-π stacking interactions with active sites of NLRP3. Introduction of a hydroxyl group at ortho of B ring leads to a substantial increase in compound activity. Among this series, compound F14 exhibited the best bioactivity. Molecular dynamics simulation assay demonstrated a favorable binding affinity between F14 and NLRP3, with tyrosine (Tyr)381 and phenylalanine (Phe)508 in NLRP3.⁶⁹ Drug repurposing is a method for discovering new compounds and exploring novel mechanisms. Boron-containing compounds was reported inhibiting activation of NLRP3 inflammasome, with ixazomib demonstrated the most excellent capability.⁷⁴ The hydrogen bond donors of boronic acid group and arylamide moiety were critical for inhibiting IL-1β release, while variations in aryl and amino acid residues affected compound bioactivity. In terms of spatial volume and polarity of the amino acid region, benzyl is the most suitable substituent. Boron-containing compound NIC-0102 induced NLRP3 polyubiquitination, thereby disrupting NLRP3 and ASC interaction.⁷⁰

Caspase drives the release of inflammatory cytokines and amplifies inflammatory responses. 1-ethyl-5-methyl-2-phenyl-1H-benzo[d]imidazole, also named Fc11a-2, prevented autocleavage of caspase-1, leading to a significant reduction in caspase-1 released from the ASC/NLRP3 complex, thereby reducing intestinal damage in UC.⁷⁵

Inhibition of NLRP3 Inflammasome Activation

Beyond directly targeting relevant domains of NLRP3, multi-target regulatory strategies focusing on its activation phase represent another vital area of inhibitors research. During the activation stage, P2X7 is activated by ATP and causes K⁺ efflux.²⁵ In addition, both Ca²⁺ release from the ER and influx of extracellular Ca²⁺ are essential for NLRP3 activation.²⁶ Taurodeoxycholate, is known as G-protein coupled receptor 19 (GPCR19) agonist, which was proved to downregulate the expression of P2X7 receptor (P2X7R) and inhibit Ca²⁺ influx, thereby suppressing NLRP3 signaling pathway.⁷⁶ Brilliant blue G (BBG), a P2X7R antagonist, enhanced the inhibition of NLRP3 inflammasome combined with OLT1177.⁷⁷

Acrylamide derivative INF39 targeted ATPase domain of NLRP3 and inhibited the interaction between NEK7 and NLRP3. This compound was proved to be safe and non-toxic, while its binding site has not been definitively clarified.^78,79 NEK7 serves as a binding protein of NLRP3, and Cl⁻ efflux can activate NLRP3 inflammasome as well as modulate NEK7-NLRP3 interaction.^80,81 Chloroquine compounds are capable of inhibiting inflammation. 7‑position phenolic hydroxyl group and nitrogen atom of 8‑hydroxyquinoline were important and methyl group at 2‑position enhanced activity. However, introducing strong electron-withdrawing groups at 2- or 4-position, or methylating 8-position or removing hydroxyl group, led to loss of activity. Compound 10 bears a para-methoxy substituent and exhibits potent inhibitory efficacy. It disrupted interactions between key proteins such as NEK7 and NLRP3.⁸²

In activation steps, phagocytosis of particulate leads to lysosomal damage, rupture, and leakage of contents. This process promotes CatB releasing into cytoplasm and facilitates K⁺ efflux.^30,83 These mechanisms highlight significance of blocking K⁺ efflux in the activation process. Aryl hydrocarbon receptor signaling promotes polyamine biosynthesis by enhancing transcription of ornithine decarboxylase 1. Supplementation with polyamines, particularly spermine, could inhibit K⁺ efflux, block NLRP3 inflammasome assembly.⁸⁴

Target NLRP3-Associated Proteins

Targeted regulation of upstream and downstream proteins related to NLRP3 provides a novel intervention strategy for specific inhibition. Nevertheless, compared to direct modulation, research on small molecule compounds targeting NLRP3-associated proteins remains exploratory. Roles of related proteins have been gradually elucidated, among which oxidative stress signaling pathway regulated by mitophagy is particularly pivotal. When mitophagy is inhibited, continuous accumulation of ROS causes dissociation of thioredoxin-interacting protein (TXNIP) from thioredoxin-1 (TRX). The C-terminus of TXNIP preferentially binds to the LRR and NACHT domains of NLRP3 inflammasome, thereby driving the activation.^85–87 A synthetic flavonoid, compound VI-16, enhanced antioxidant capacity in colonic macrophages, inhibiting dissociation of TXNIP from Trx-1 and its subsequent binding to NLRP3.⁸⁸ Representative structure of these compounds and their mechanism in targeting NLRP3 pathway are presented in Table 1.

Table 1 Preclinical Research in Synthesized Compounds Inhibit NLRP3 Inflammasome to Treat UC

Research on Clinical Drugs

On the basis of fundamental research, NLRP3 inflammasome inhibitors have been used in clinical trials. Cytokine release inhibitory drugs (CRIDs) were created, targeting IL-1β. CRID3, also known as MCC950 or CP-456773, binds to NLRP3 via the Walker B motif within NACHT.^89–91 MCC950 is the first specific NLRP3 inflammasome inhibitor entering clinical trials and has been widely used in studies of diseases related with NLRP3 inflammasome. However, due to hepatotoxicity observed in a Phase II clinical trial for rheumatoid arthritis, the clinical development of MCC950 was forced to terminate.⁹² Selnoflast (formerly somalix/RG6418/IZD334) is a derivative modified from MCC950. Compared to MCC950, the sulfonyl side chain of selnoflast contains N-ethylpiperidine, replacing the furan ring and tertiary alcohol of MCC950. This modification improved water solubility and enhanced reversibility of target binding. Clinical trials have verified that selnoflast exhibits favorable safety and tolerability (No. NCT04086602). Administration of 450 mg selnoflast daily to patients with moderate-to-severe UC could maintain inhibition on IL-1β above 90% inhibitory concentration (IC₉₀) during dosing interval, and the concentration of selnoflast in sigmoid colon tissues was higher than IC₉₀. Blood tests revealed that IL-1β release was inhibited within 30 minutes after dosing and maintained for at least 10 hours (mean [± SD] >95% [± 4.16%]). However, no significant differences in Geboes score, Nancy index and caspase-1 expression were observed before and after treatment (data not shown). Although expression of inflammatory cytokine-related genes in colon tissue showed a slight decrease, there was no significant difference. These outcomes halted clinical development of selnoflast for UC, terminating its progress at the Phase Ib clinical trial.⁹³

ROS is also indispensable in NLRP3 activation. NAC, a ROS scavenger, stemmed LPS-induced NLRP3 from activation.⁹⁴ In the acetic acid (AA)-induced colitis model, intracolonic administration of NAC alleviated severity of colonic injury, accompanied by a reduction in myeloperoxidase.⁹⁵ In a randomized, double-blind controlled clinical trial (No. IRCT20190713044185N1), UC patients in the steroid-tapering phase who took NAC demonstrated significantly lower relapse rates (P = 0.007) compared with placebo group. Taking NAC also showed lower mean fecal calprotectin and serum erythrocyte sedimentation rate (p < 0.05). However, the incidence of nausea was higher in the NAC group (19.51%) than in the placebo (3.48%) (P = 0.001).⁹⁶ Although NAC has demonstrated promising efficacy in the clinical trial, further investigation is required to determine whether it specifically targets NLRP3 inflammasome in human. Based on these two clinical trials, no clinical studies have reported adverse effects or risks of targeting NLRP3 inflammasome for UC treatment. MCC950 was halted due to hepatotoxicity. Another MLRP3 inflammasome inhibitor GDC-2394, based on MCC950 structure, effectively inhibited IL-1β and IL-18 release, but exhibited potential hepatotoxicity in Phase I trial.⁹⁷ This suggests that future drug development should also focus on screening for reactive metabolites in the early stages of drug discovery and rigorously monitoring liver function indicators in animal studies.

MCC950 serves as a critical structural scaffold for research and development of NLRP3 inflammasome inhibitors. While its furan ring moiety is likely to induce drug-induced liver injury. Therefore, structural optimization of MCC950, especially replacing its furan ring fragment, is particularly essential. Based on MCC950, the furan ring was replaced with a phenyl group and a dimethylaminomethyl group was introduced at the 4-position. This yielded 4-(2-(dimethylamino) ethyl)-N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl) carbamoyl) benzenesulfonamide (N14). N14 had an oral bioavailability of 85.2%. Compared with MCC950, it exhibited no significant hepatocyte toxicity at 500 μM and more effectively alleviated symptoms in UC mice.⁹⁸

Expanding Therapeutic Application of NLRP3 Inflammasome Inhibitors for UC Treatment

Other drugs currently in clinical trials or already marketed also exhibit capacity to inhibit NLRP3 inflammasome.

OLT1177, a β-sulfonyl nitrile compound, is a selective NLRP3 inhibitor. It has completed Phase I (No. NCT02134964) and Phase II clinical trials for osteoarthritis (No. NCT02104050) and is currently undergoing Phase III clinical research for gout (No. NCT05658575).^99,100 OLT1177 was well tolerated in humans who continuously administrated OLT1177 for 8 days at a maximum dose of 1000 mg. OLT1177 blocked interactions between NLRP3 and ASC, as well as between NLRP3 and caspase-1, showing significant efficacy in the early stage of DSS-induced UC mice.^101,102 Nevertheless, a target engagement assay revealed that although OLT1177 could ameliorate inflammation, it failed to suppress NLRP3 inflammasome assembly.⁹⁹ Current clinical studies have confirmed its safety. However, its ability to specifically target and inhibit NLRP3 inflammasome remains to be verified.

Transilast is an analog of tryptophan metabolites used for treating allergic diseases such as bronchial asthma.¹⁰³ Its anti-allergic mechanism involves stabilizing cell membranes of mast cells and basophils, preventing their degranulation, and blocking inflammatory responses.¹⁰³ Since excessive inflammation drives UC progression, exploring impacts of anti-allergic drugs on NLRP3 represents a new direction for UC treatment. Tranilast has been proved that it can covalently bind to NACHT.¹⁰⁴ Enema administration was shown tranilast ameliorated inflammatory manifestations in UC mice.¹⁰⁵ However, there is still a long way to go before tranilast can be truly applied to curing UC, as its pharmacological properties pose the main bottleneck, restricting its translational application. In human, the plasma concentration of tranilast peaks at 2 h after a single oral dose, with a half-life of 5 h. Oral administration of conventional therapeutic doses (600 mg/day) yields plasma concentrations ranging from 30 to 300 μM.¹⁰³ Multiple hydrophobic groups (eg, cinnamoyl group) in the tranilast molecule result in extremely low water solubility (14.5 μg/mL), particularly under acidic conditions. In addition, the highly flexible acrylamide moiety in tranilast increases entropy loss and reduces binding affinity.¹⁰⁶ Tranilast possesses reliable safety profiles, and structural optimization is required to develop its application in UC therapy.

RRx-001 (1-bromoacetyl-3,3-dinitroazetidine) is an agent for small cell lung cancer, colorectal cancer and brain metastatic tumors and has completed Phase III clinical trials for small cell lung cancer (No. NCT03699956).^107,108 Its bromoacetyl group can covalently bind to cysteine (Cys) 409 of human NLRP3 and Cys405 of mouse NLRP3, alleviating clinical symptoms in UC mice.¹⁰⁹ However, RRx-001 contains high-energy nitro functional groups, which may produce highly toxic oxides during production and processing. Compound 149–01, an RRx-001 analogue devoid of high-energy nitro groups, blocks the NLRP3–NEK7 interaction, though its efficacy has not yet been validated in UC mice.¹¹⁰

Chloroquinaldol, an 8-hydroxyquinoline derivative, is a topical antimicrobial agent used for skin infections. It has been proved to improve psoriasiform dermatitis by suppressing NLRP3 inflammasome activation.^111,112 Chlorquinaldol blocked NLRP3-ASC interaction, and hydroxyl group on its benzene ring contributed to inhibitory effect. It could inhibit caspase-1 cleavage and IL-1β maturation and was demonstrated therapeutic effects in DSS-induced UC mice.¹¹³ These findings provide a theoretical basis for inhibition of NLRP3 inflammasome by 8-hydroxyquinoline and support the development of its derivatives.

While these drugs have not been clinically tested for UC, they have shown capacity to target and inhibit NLRP3 inflammasome. Their safety has also been confirmed through previous clinical studies. Despite remaining challenges such as unclear target specificity and suboptimal druggability, these agents, with established clinical safety evidence and confirmed NLRP3 inhibitory activity, represent one of the most promising research directions for NLRP3-targeted therapy of UC.

Drugs Dependent on NLRP3 Inflammasome

The Nlrp3^−/− model enables the definitive identification of whether anti-inflammatory effects are exerted via the NLRP3 inflammasome. For example, 3-(2-Oxo-2-phenylethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (compound 1) interfered with NLRP3 priming step to prevent activation, yet compound 1 failed to ameliorate intestinal inflammation in Nlrp3^−/− mice.¹¹⁴

Despite suppressing the assembly and activation of NLRP3 inflammasome, promoting its degradation also represents a promising therapeutic strategy for UC. For instance, colonic clock gene Rev-erbα directly repressed Nlrp3 transcription by specifically binding to its promoter region, thereby alleviating UC symptoms in mice. However, this effect was lost in Nlrp3^−/− mice.¹¹⁵ Therefore, concentrating on Rev-erbα may become an innovative drug target for UC. Van Gogh-like 2 (Vangl2), a planar cell polarity-related protein, was demonstrated to interact with NACHT and LRR of NLRP3 via its Prickle/cadherin-binding domain (PKBD), and recruits the E3 ubiquitin ligase MARCH8 to induce K27-linked ubiquitination of NLRP3 at lysine site K823, leading to autophagic degradation. VANGL2 is downregulated in IBD patients and DSS-induced murine colitis, which highlights its promising application of inhibiting IBD progress.¹¹⁶ The novel sorbicillinoid compound JNUTS013, inducing proteasomal degradation of NLRP3, and autophagy-tethering compound MC-ND-18, recruiting NLRP3 and LC3B protein, could facilitate NLRP3 degradation.^117,118

Although the above compounds have been validated in Nlrp3^−/− mice, to reveal their specificity for NLRP3 inflammasome, further research is required to precisely identify binding sites and underlying mechanisms of various compounds with NLRP3 inflammasome, together with its upstream and downstream signaling pathways. In addition, transcriptional regulation involving Rev-erbα and the VANGL2-mediated ubiquitination degradation pathway provides a solid foundation for NLRP3 inflammasome-specific therapeutic agents.

Others

A growing collection of miscellaneous NLRP3 inflammasome inhibition measures do not fall neatly into the mechanistic categories discussed above. Recently, these agents have emerged as an excellent therapeutic strategy.

Libertellenone M is a secondary metabolite of the endophytic fungus Phomopsis sp. Immunofluorescence assays showed that S12 blocked NLRP3 inflammasome assembly.¹¹⁹ Lichen metabolite, atranorin combined with ASC and suppressed secretion of cytokines.¹²⁰ Beyond these, probiotics also offer a novel approach for NLRP3-targeted therapy. Roseburia intestinalis (R.I) is significantly reduced in patients with IBD. In vitro studies indicated that R.I. flagellin inhibited microRNA (miR)‑223‑3p, thereby modulating NLRP3 activation. Hence, R. I. flagellin will be a unique and promising probiotic product for UC.¹²¹

To achieve more precise regulation of the NLRP3 inflammasome, NLRP3-targeted delivery systems have been developed to improve therapeutic efficacy. Although bicalutamide promoted NLRP3 degradation, it had limitations in treating UC. Hence, a delivery system targeting NLRP3 in macrophages was invented, mediating NLRP3 protein degradation via autophagic pathway.¹²² Meanwhile, siNLRP3-loaded nanoparticles based on cationic liposomes were constructed, effectively smothering NLRP3 inflammasome activation.¹²³ A novel nanosystem carrying an NLRP3 detection probe, which was cleavable by caspase-1, together with MCC950 carried out early real-time monitoring of NLRP3 activation while exerting anti-inflammatory effects on UC mice.¹²⁴ Delivery systems overcome limitations of small molecule drugs and can precisely target NLRP3 inflammasome, holding promise for improving specificity and safety of NLRP3 modulation.

Conclusion and Prospects

NLRP3 inflammasome has emerged as a central regulator of intestinal inflammation, driving mucosal damage and inflammatory infiltration through inflammatory cytokines release such as, IL-1β and IL-18. Its aberrant activation is linked to the pathogenesis and progression of UC, positioning NLRP3 inflammasome as a dominant feature in the inflammatory cascade and as a revolutionary therapeutic target. Structurally, NLRP3 inflammasome consists of multiple effector proteins, providing advantages of potential targeting sites. The treatment of NLRP3 inflammasome- related diseases mainly focuses on inhibiting IL-1β at present. However, IL-1β is not merely a product of NLRP3 inflammasome activation, nor is the sole output of such activation. Therefore, direct NLRP3 inhibitors may offer advantages in terms of safety and efficacy. Nevertheless, regulatory mechanisms of NLRP3 inflammasome in different stages and microenvironments of chronic inflammation remain unclear, and relevant inhibitory mechanisms or precise targets have not been fully elucidated.⁶⁰ Moreover, NLRP3 shares structural similarities with other inflammasomes, such as NOD-like receptors family CARD domain-containing protein 4 (NLRC4), absent-in-melanoma 2 (AIM2). Some inhibitors may interfere with other inflammatory pathways, potentially causing off-target effects that impairing immune defense. Furthermore, the activation mechanism of NLRP3 inflammasome is complex and involves multiple signaling pathways, which cannot be accurately recapitulated in animal models. This limits clarification of regulatory impacts of inhibition NLRP3 inflammasome on other pathways.¹²⁵ Additionally, long-term safety of drugs targeting NLRP3 inflammasome remains unknown.

To date, no small molecule drug directly targeting NLRP3 inflammasome has been approved for marketing. Although selnoflast did not show significant differences in Geboes score or Nancy index after treatment (data not disclosed), leading to termination of its clinical trial in UC, the exploration of selnoflast has advanced clinical translation of NLRP3-targeting strategies. In a randomized controlled trial for UC, ROS scavenger NAC significantly reduced recurrence rates (P = 0.007), with decreases in both fecal calprotectin and serum erythrocyte sedimentation rate. But its effects on NLRP3 inflammasome in intestinal tissue of UC patients remain unclear. As a highly promising candidate, NAC is expected to proceed to further clinical trials for UC.

Drugs already on the market or in clinical trials for other diseases that have been proved to specifically inhibit NLRP3 inflammasome warrant further development due to their well-defined safety. Drugs including tranilast, RRx-001, and antibacterial agent chlorquinaldol also merit structural modification to enhance efficacy against UC. Aberrant immune responses mediate UC progression. Anti-inflammatory and anti-allergic agents are also capable of suppressing inflammatory reactions. Therefore, future research may focus on repurposing marketed anti-allergic or anti-inflammatory drugs for UC, exploring their capacity to specifically modulate NLRP3 inflammasome, and modify their structure to optimize their therapeutic effects in UC. Acrylamide moiety of tranilast contributes to inhibiting NLRP3 inflammasome, yet this structure also reduces its binding affinity to the target protein. Compound Z48, derived from structural modification of this moiety, exhibited satisfactory therapeutic efficacy in animal models, with an oral bioavailability of 16.7% in mice, warranting further investigation.

Newly synthesized small molecule compounds have achieved high affinity and specific inhibition of NLRP3 inflammasome through structural modification and optimization. Quinoline, which can bind to NACHT and ASC domains, and sulfonamide compounds, which are based on MCC950 structure, hold the greatest development promise among various compounds. Although MCC950 was halted due to hepatotoxicity, derivatives generated by modifying its furan ring structure can still be exploited for novel NLRP3 inflammasome inhibitors.

Inhibiting NLRP3 inflammasome remains a highly promising therapeutic strategy for UC that warrants deep exploration. Artificial intelligence and advanced computational technologies offer unprecedented opportunities to accelerate screening of active compounds and to guide structural optimization of next-generation NLRP3 inhibitors. Combination therapies that pair NLRP3 inhibitors with NLRP3-targeted delivery systems may enhance therapeutic efficacy and overcome drug resistance mechanisms. Inhibition of NLRP3 offers new hope for patients with UC and other inflammatory disorders related to NLRP3.