A Patent Review of NLRP3 Inhibitors to Treat Autoimmune Diseases

Abstract

Introduction:

NOD-like receptor family pyrin domain containing 3 (NLRP3) can sense a plethora of exogenous and endogenous dangers. Upon activation, a multimeric protein complex, the NLRP3 inflammasome, is formed to initiate the innate immune responses. Emerging studies have implicated the pathophysiological roles of this protein complex in human disorders, highlighting that it represents a druggable target for therapeutics development.

Areas covered:

The current review summarizes the functional facets of the NLRP3 inflammasome, its association with autoimmune diseases, and recent patents on the development of NLRP3 inhibitors. Literature search was conducted using SciFinder and Google Patents with the key word NLRP3 and NLRP3 inhibitors.

Expert opinion:

Although significant advances have been made in understanding the NLRP3 inflammasome, more studies are still needed to elucidate the molecular mechanisms underlying its roles in autoimmune diseases. A number of NLRP3 inhibitors have been patented, however, none of them have been approved for clinical use. Due to the complex nature of the NLRP3 inflammasome, novel screening assays along with target engagement methods could benefit the drug discovery and clinical translation. In addition, clinical trials on NLRP3 inhibitors are still in their early stages, and continuous investigations are needed to fully assess their safety and effectiveness.

1. Introduction

The innate immunity and adaptive immunity constitute the immune system and are responsible for surveilling the host from endogenous and exogenous dangers [1]. The innate immune system serves as the first line of defense and relies on pattern-recognition receptors (PRRs) to recognize pathogens and danger signaling through pathogen-associated molecular patterns (PAMPs), and/or damage-associated molecular patterns (DAMPs), and to initiate inflammatory responses [2–4]. PRRs can be classified into membrane-bound PRRs, e.g., Toll like receptors (TLRs) and C-type lectin receptors (CLRs), and cytoplasmic PRRs including NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) [5,6]. Several cytoplasmic PRRs are able to form a multimeric protein complex known as “inflammasome” to activate the proinflammatory caspases and lead to the production of inflammatory cytokines [7–10]. Among the known inflammasomes, the NLR family pyrin domain containing 1 (NLRP1), NLRP3, NLRP6, and NLR family card domain containing 4 (NLRC4) inflammasomes are subsets of the NLR family. Absent in melanoma 2 (AIM2) and interferon (IFN)-inducible protein 16 (IFI16) inflammasomes belong to the pyrin and HIN (hematopoietic expression, interferon-inducible nature, and nuclear localization) domain-containing (PYHIN) family [11–13]. Recent studies have highlighted the pathological roles of inflammasome dysregulation in certain human diseases, and the NLRP3 inflammasome has attracted much attention as a viable drug target for therapeutic interventions. Herein, we review the NLRP3 inflammasome activation process, its association with autoimmune diseases, and summarize the small molecule inhibitors developed by targeting the inflammasomes from patents filed recently and their potential application in autoimmune diseases.

2. NLPR3 inflammasome and its activation

Assembly of the NLRP3 inflammasome requires a sensor protein, NLRP3 (containing Leucine rich repeat-NACHT-PYD domains), an adaptor apoptosis-associated speck-like protein (ASC, containing PYD and CARD domains), and an effector pro-caspase 1 (containing the CARD domain) [14,15]. Under canonical conditions, two steps are typically needed to assemble and activate the NLRP3 inflammasome (Fig. 1). The priming is to upregulate the expression of NLRP3 protein upon the signaling of PAMPs or DAMPs, e.g., microbial products or endogenous cytokines such as tumor necrosis factor (TNF-α) or interleukin (IL)-1β through the activation of the NF-κB (nuclear factor kappa light chain enhancer of activated B cells) pathway [16–18]. Additionally, the priming step also induces posttranslational modifications (PTMs) of NLRP3, including ubiquitylation, phosphorylation, and sumoylation, which leads to the stabilization of inactive NLRP3 [19]. A plethora of molecular and cellular events have been implicated to trigger NLRP3 inflammasome activation, including K⁺ efflux [20–22], Ca²⁺ flux [23], Cl⁻ efflux [24,25], lysosomal disruption [26], mitochondrial dysfunction [27], metabolic changes [28,29], and trans-Golgi disassembly [30], among others. Upon activation, the NIMA-related kinase 7 (NEK7) binds to NLRP3, causing structural rearrangement that allows for NLRP3 oligomerization through its NACHT domain. This is followed by the recruitment of ASC via the PYD-PYD interactions between NLRP3 and ASC to form the “speck” [31]. Then, pro-caspase 1 is recruited to form the complex through CARD–CARD interactions with ASC [32,33]. Upon assembly of the NLRP3 inflammasome complex, self-cleavage converts pro-caspase 1 to its active form, caspase-1, followed by the proteolytic cleavage of pro-IL-1β and pro-IL-18 into proinflammatory cytokines, IL-1β and IL-18. This ultimately leads to pyroptosis, a highly regulated and programmed mechanism of cell death [34]. Another critical component involved in the pyroptotic process, gasdermin D (GSDMD), is also cleaved by the active caspase-1 to produce an N-terminal fragment (NT-GSDMD) by which a pore structure will be formed in the cell membranes to release IL-1β and IL-18 [35].

Figure 1.

Canonical activation of the NLRP3 Inflammasome (created with BioRender.com). Step 1 illustrates the priming step in the process of NLRP3 inflammasome activation. Upon the activation of the NF-κB pathway, the expression of NLRP3, pro-IL-1β, and pro-IL18 are increased. Step 2 shows the activation process of the NLRP3 inflammasome. The activation triggers, such as K⁺ efflux and lysosomal rupture, lead to the assembly of the inflammasome complex. This is followed by the proteolytic cleavage of pro-caspase-1 to active caspase-1, ultimately leading to the release of IL-1β and IL-18.

The non-canonical activation of the NLRP3 inflammasome involves the recognition of cytosolic lipopolysaccharide (LPS) by human caspase-4, caspase-5, or mouse caspase-11 during gram-negative bacterial infection (Fig. 2) [36]. The activation of these caspases results in the proteolytic cleavage of GSDMD and pyroptosis without IL-1β and IL-18 release [37,38]. Notably, pannexin-1 (a membrane channel for adenosine triphosphate (ATP)) can be cleaved by the activated caspase-11 to induce an efflux event of ATP and K⁺, which in turn activates the NLRP3 inflammasome and leads to IL-1β and IL-18 secretion [39]. This reflects the interconnection between the non-canonical and canonical pathways.

Figure 2.

The noncanonical and alternative activations of the NLRP3 inflammasome (created with BioRender.com). Under the non-canonical activation pathway, caspase-4/5 or caspase-11 is activated to cleave GSDMD and lead to pyroptosis. Under the alternative activation pathway, caspase-8 is activated and this subsequently leads to the activation of the NLRP3 inflammasome.

The activation of the NLRP3 inflammasome via the alternative pathway is observed in human and porcine monocytes but not in murine monocytes (Fig. 2) [40]. Upon stimulation with LPS, TLR4 activates myeloid differentiation primary response 88 (MyD88) and Toll/IL-1R (TIR)-domain-containing adapter-inducing interferon-β (TRIF), leading to the activation of the NF-κB pathway, and the formation of a complex comprising receptor-interacting serine/threonine protein kinase 1 (RIPK1) and FAS-associated death domain protein (FADD). This complex then triggers the activation of caspase-8, ultimately leading to the activation of the NLRP3 inflammasome. In the TRIF-RIPK1-FADD-caspase 8 cascade, the events observed under canonical NLRP3 inflammasome activation, such as ASC speck formation, K⁺ efflux, GSDMD cleavage, and pyroptosis, are typically dispensable [40].

3. Dysregulation of the NLRP3 inflammasome in autoimmune diseases

As a crucial component of the innate immune system, the NLRP3 inflammasome is widely expressed in a variety of immune cells, including monocytes, macrophages [41], osteoblasts [42], epithelial cells [43], fibroblasts [44], T cells, and B cells [45]. The T-helper (Th)17 cells, a subpopulation of the CD4⁺ T cells, have been implicated to play roles in autoimmune and inflammatory disorders, e.g., multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis [46]. Regulatory Th17 cells (Treg17) modulate the inflammatory responses via Th17/Th1 signaling [47,48]. Dysregulation of Th17/Treg has been shown to have pathophysiological roles in autoimmune diseases [49–52]. Studies have shown that IL-1β prolongs the survival of T cells and induces their differentiation and polarization, especially towards the Th17 population. In addition, IL-1β stimulates the proliferation of B cells leading to enhanced antibody production [53]. Furthermore, studies demonstrated that IL-18 enhances the proliferation of Th1 cells and leads to the production of IFN-γ [53–55]. Collectively, the NLRP3/IL-1 axis plays an essential role in promoting the differentiation and amplification of Th17 and Th1 cells, highlighting the importance of the NLRP3 inflammasome in autoimmune diseases.

3.1. Role of the NLRP3 inflammasome in Systemic Lupus Erythematosus (SLE)

SLE is a chronic autoimmune illness that damages several organs as a result of impaired innate and adaptive immunity, which leads to the generation and buildup of nuclear autoantibody complexes in response to inadequate auto-antigen recognition. Although the exact cause of SLE is undetermined, excessive activation of the NLRP3 inflammasome is thought to be a major factor in the disease’s pathogenesis. Nearly 70% of SLE patients suffer from lupus nephritis (LN) [56,57]. Studies have shown that patients with SLE, especially those suffering with LN, more significantly expressed a gain-of-function mutation on the NLRP3 gene than other groups, resulting in increased activity of the NLRP3 inflammasome in patients with SLE [58]. Additionally, increased levels of NLRP3 gene expression, as well as other genes in the NLRP3 inflammasome pathway, have been shown in both cells isolated from SLE patients and in lupus-like mouse models, such as MLR/lpr mice [59–61]. Moreover, over activation of the NLRP3 inflammasome was observed in samples derived from SLE patients [59,60]. The accumulation of DAMPs, such as reactive oxygen species (ROS), intracellular ATP, nucleic acids, and others, as a result of an elevated rate of apoptosis, decreased clearance of neutrophil extracellular traps (NETs), formed by activated low density granulocytes (LDGs), and abnormal autophagy are thought to be major factors influencing the hyperactivation of NLRP3, which results in the aberrant release of pro-inflammatory cytokines that ultimately cause tissue damage. Inhibition of the NLRP3 inflammasome has exhibited marked improvement of the disease state both in bone marrow-derived mesenchymal stem cells from SLE patients (SLE-BMSCs) and MRL/lpr mice, showing decreased inflammation, significant improvement of renal pathology, reduced autoantibody production, and reduced incidence of mortality compared to untreated mice [59,62]. Furthermore, assessment of wild-type and caspase-1^−/− Balb/c and C57BL/6 mice treated with pristane to induce lupus showed the importance of the NLRP3 inflammasome in autoantibody production and the development of LN [63]. These studies suggested the importance of the NLRP3 inflammasome in the progression of SLE and highlight the need for NLRP3 inhibitors as potential treatments for SLE. However, there are also reports that the decreased level of NLRP3 results in the progression of disease in SLE patients as the NLRP3 pathway is associated with programmed cell death, which aids in the maintenance of homeostasis by removal of overactivated lymphocytes [64]. These studies also indicated decreased levels of NLRP3 and its inflammasome components in patients with SLE, which is contradictory to the information published by others [64,65].

3.2. Role of the NLRP3 inflammasome in Systemic Sclerosis (SSc)

Characterized by autoimmunity, vascular injury, and aggressive tissue fibrosis, SSc is an autoimmune disease mainly powered by genetic and environmental factors including exposure to viruses, silica dust, organic solvents, and chemicals. Immune dysregulation and uncontrolled inflammation play a pivotal role in the synthesis of extracellular matrix components (EMCs), e.g., collagen, whose over-accumulation leads to hardening, scarring, and deformation of tissue which ultimately leads to organ damage [66]. The overproduction of collagen in SSc has been linked to inflammasome activation, and studies have shown the increased level of NLRP3 and its downstream cytokines in SSc patients. Comparing the multiple skin layers of healthy people versus SSc patients, increased level of NLRP3 can be observed in SSc patients [67]. Additionally, studies have shown a ~7-fold increase of both NLRP3 and IL-1β gene expression levels in SSc patient-derived versus normal dermal fibroblast cell lines. Furthermore, studies have shown that pharmacological suppression of the NLRP3 inflammasome by caspase-1 inhibitor Z-YVAD(OMe)-FMK results in a decrease in the amount of collagen and stress fibers [68]. Regulated by the NLRP3-IL1 signaling, the skewing of Th1/Th2 towards Th2 plays an important role in the pathogenesis of SSc due to an increased production of pro-fibrotic cytokines in comparison to anti-fibrotic IFN-γ by Th1 cells [69]. NLRP3-mediated activation of B cells results in fibrosis and production of autoantibodies and pro-fibrotic cytokines [70,71]. This causal relationship between NLRP3 activation and the pathogenesis of SSc has also been demonstrated in mouse models treated with bleomycin to induce dermal fibrosis associated with SSc. The skin thickness in the NLRP3^−/− and ASC^−/− mice was significantly less than the wild-type controls and is comparable to their untreated knockout counterparts [68].

3.3. Role of the NLRP3 inflammasome in Irritable Bowel Disease (IBD)

Resulting from chronic and recurring inflammation of the gastrointestinal (GI) tract, Crohn’s disease (CD) and ulcerative colitis are the two main clinical manifestations of IBD. Development of IBD might be due to the dysfunction of the mucosal immune system of the GI tract caused by the disruption of the intestinal mucosa that keeps gut micro-organisms separated from the immune cells [72]. The permeability of the tight junctions of the mucosal membrane has been shown to be impaired under increased levels of proinflammatory cytokines, such as IL-1β and TNF-α [73], and these increased levels are attributed to the severity of inflammation in IBD [74–76]. Caspase-1 activation and maturation of IL-1β and IL-18 are thought to occur as a result of abnormal NLRP3 activation caused by NF-κB in IBD [77]. Genetic analysis of CD patients associated a single nucleotide polymorphism of the NLRP3 gene to a higher susceptibility of developing the disease [78]. However, the exact role of NLRP3 in IBD pathogenesis is not completely understood, both NLRP3’s protective and pathogenic functions have been proposed. In a study employing a dextran sodium sulfate (DSS)-induced colitis mouse model, the NLRP3^−/− mice exhibited far lower clinical and histological scores associated with colitis as well as lower proinflammatory cytokine levels in the colon. Additionally, these lowered levels and scores were comparable to those of the NLRP3^+/+ mice treated with a caspase-1 inhibitor, suggesting that the NLRP3 inflammasome plays a critical role in the pathology of IBD [79].

3.4. Role of the NLRP3 inflammasome in RA

Driven by pro-inflammatory cytokines, RA is a systemic polygenic autoimmune disease that causes synovial inflammation of joints resulting in cartilage and bone destruction. NLRP3, as the major regulator of the maturation of IL-1β and IL-18, has been implicated an active role in the pathogenesis of RA [80,81]. NF-κB, a critical transcriptional modulator of NLRP3 and proinflammatory cytokine expression, is constitutively activated in the synovial tissue of RA patients [82,83]. Consequently, gene expression of NLRP3 in PBMCs of RA patients, as well as other inflammasome components, has been shown to be significantly higher than in the healthy control population [84,85]. Additionally, single nucleotide polymorphisms associated with NLRP3 have been shown to modulate the susceptibility of an individual to develop RA and response to therapy [84]. Furthermore, it was determined that not only do RA patients exhibit increased NLRP3 inflammasome activity, but that upon exogenous stimulation, caspase-1 contributes most significantly to IL-1β release compared to other caspases [85]. Studies have also highlighted the role of IL-1β following pyroptosis in stimulating the priming step of the NLRP3 inflammasome formation in RA tissues, thus perpetuating the inflammatory cycle [83]. Through IL-1β, NLRP3 in synovial tissue promotes Th17 cell differentiation in CD4⁺ T cells which further aggravates RA by the production of TNF-α and IL-17a. Through NLRP3 activation, anti-citrullinated protein antibodies (ACPA) increase the production of IL-1 in RA patients. Additionally, genetic knock out studies in mice also suggested the crucial roles of the NLRP3 inflammasome in the pathology of arthritis [86].

4. Small molecule inhibitors of the NLRP3 inflammasome

Given the growing evidence of the NLRP3 inflammasome’s involvement in multiple pathological disorders, efforts have been dedicated to the development of small molecule inhibitors of this protein platform to achieve disease interventions. Recently, patents have been filed from both industry and academia to cover a variety of chemical scaffolds and their use. In this review, we summarized the compounds that are reported as NLRP3 inhibitors from patents published from 2016 to present. We conducted a thorough search using SciFinder and Google Patents. NLRP3 and NLRP3 inhibitors were used as the key words for this search. The list of references identified was then checked individually for patent publications. In total, forty-four patents were identified. The institutions and their related patents numbers are shown in Table 1. Additionally, the summarized NLRP3 inhibitors in preclinical or early clinical stages reported from recently filed patents and articles are illustrated in Table 2.

Table 1.

Summary of the institutions and their related patents numbers.

Company/Institution Name	Numbers of patents
Inflazome Ltd.	11
IFM Therapeutics, Inc.	6
Janssen Pharmaceuticals	5
NodThera Ltd.	4
Novartis AG	4
The University Of Queensland	3
AC Immune SA	1
Cadila Healthcare Ltd.	1
Galderma Research and Development SNC.	1
Icahn School of Medicine at Mount Sinai.	1
Innate Tumor Immunity, Inc.	1
Jecure Therapeutics, Inc.	1
Pfizer Inc.	1
The University Of Manchester	1
University of Turin	1
Ventus Therapeutics, Inc.	1
Virginia Commonwealth University	1

Table 2.

Representative NLRP3 inhibitors are in preclinical or early clinical stages reported from recently filed patents and articles.

Inhibitor	MOAs	IC50 (IL-1β release)#	Preclinical disease models*	Development Stage
CY-09	Binds to Walker A site in NACHT domain Inhibits ATPase activity	6 μM	T2DM, diabetic retinopathy, diabetic liver injury, NAFLD, peritonitis, Muckle-Wells syndrome, epilepsy	Preclinical
Tranilast	Disrupts NLRP3-NLRP3 oligomerization through NACHT domain binding	<25 μM	T2DM, NASH, colitis, peritonitis, Muckle-Wells syndrome, atherosclerosis	Phase II clinical trials to treat cryopin associated periodic syndrome (CAPS) (NCT03923140) Phase IV trial for percutaneous coronary intervention (NCT05130892)
Oridonin	Covalently binds to Cys279 of NACHT domain and reduces NLRP3-NEK7 interaction	<0.5 μM	T2DM, liver fibrosis, IBD, peritonitis, hearing loss, pleurisy	Phase IV trial for percutaneous coronary intervention (NCT05130892)
RRx-001	Covalently binds to Cys409 of NACHT domain and reduces NLRP3-NEK7 interaction	117 nM	EAE, DSS colitis	Phase III clinical trials adjuvant treatment of small cell lung cancer with platinum (NCT03699956)
Bay-11-7082	Inhibits ATPase activity Unknown binding site	~3 μM	T2DM, IBD, psoriasis, lupus nephritis	Preclinical
BOT-4-one	Inhibits ATPase activity Unknown binding site	1.28 μM (LPS/ATP) 0.67 μM (LPS/nigericin)	Peritonitis, dermatitis, psoriasis, arthritis	Preclinical
OLT1177 (Dapansutrile)	Inhibits ATPase activity Unknown binding site	ND	AD, EAE, colitis, arthritis	Phase II clinical trials to treat acute gouty arthritis (EudraCT 2016-000943-14)
INF39	Covalent inhibitor of ATPase activity Unknown binding site	~10 μM	IBD, colitis, pancreatitis	Preclinical
JC-171	Reduces NLRP3-ASC interaction Unknown binding site	8.45 μM	EAE	Preclinical
VTX2735	Peripherally restricted NLRP3 inhibitor	4 nM	N/A	Phase II clinical trial to treat cryopyrin associated periodic syndrome (NTC05812781)
Emlenoflast (Inzomelid; MCC7840; IZD174)	NLRP3 inflammasome inhibitor Unknown binding site	4.7 nM	N/A	Phase I clinical trials for safety and tolerability (NCT04015076) Phase IIb clinical trials to treat cryopyrin associated periodic syndrome (CAPS) (EudraCT2020-000489-40)
Selnoflast (RG6418; RO7486967)	NLRP3 inflammasome inhibitor Unknown binding site	≤ 1 μM (THP-1 cells)	N/A	Phase Ib clinical trials for safety and tolerability in ulcerative colitis patients (BP43099) Phase Ib clinical trials for safety and tolerability in patients with early idiopathic Parkinson’s disease (BP43176, CPMS 50823, IRAS 307220)

^#IC₅₀ values for IL-1β release inhibition in murine macrophage cells.

^*Abbreviations: AD: Alzheimer’s disease, PD: Parkinson’s disease, EAE: Experimental autoimmune encephalomyelitis, EAT: Experimental autoimmune thyroiditis, T2DM: Type 2 diabetes mellitus, NAFLD: Non-alcoholic fatty liver disease, NASH: Non-alcoholic steatohepatitis, IBD: Irritable bowel disease, DSS: Dextran sulfate sodium. ND: not determined.

4.1. CRID3/MCC950-based sulfonylurea, sulfonamide, and urea derivatives

Early studies from Perregaux et al. reported the inhibitory effects of glyburide, a sulfonylurea type of FDA approved antidiabetic agent, and a series of biaryl sulfonylurea compounds on the release of IL-1β induced by stimuli in human monocytes and murine macrophages [87]. These biaryl sulfonylurea compounds were termed as cytokine release inhibitory drugs (CRIDs). Studies from Genentech demonstrated inhibition of the NLRP3 inflammasome as the mechanism of action (MOA) for glyburide’s anti-inflammatory effects [88]. However, further studies of the CRIDs initially suggested glutathione S-transferase omega-1 (GSTO1) as the molecular target for these compounds. CRID3 (also known as CP-456773 and MCC950) was used as a competitor for the affinity chromatography experiments in this study from which the GSTO1 was eluted. Although further studies by Coll et al. suggested that CRID3 targets the NLRP3 and AIM2 inflammasomes, later studies from the same group demonstrated CRID3 as a potent NLRP3 inhibitor and this compound was re-termed as MCC950 [89–90]. To credit the original inventor and to reflect the current term use in most of the literature, we use CRID3/MCC950 for this compound in the rest of the manuscript. Recently, mechanistic studies from two groups demonstrated that CRID3/MCC950 functions as a direct NLRP3 inhibitor by binding to the NACHT domain of NLRP3 [91–93]. Recent structural biology studies supported this conclusion by revealing that CRID3/MCC950 interacts with multiple sites within the NACHT domain, including the nucleotide binding domain (NBD), helical domain 1 (HD1), winged-helix domain (WHD), and helical domain 2 (HD2). The unique binding by CRID3/MCC950 locks NLRP3 in an inactive state, therefore preventing the assembly of the inflammasome [94]. Since the discovery of CRID3/MCC950 as a potent NLRP3 inhibitor, this compound has been extensively tested in multiple disease models including autoimmune diseases. CRID3/MCC950 showed good skin penetration and tolerance for the treatment and/or prevention of an inflammatory skin disorder, such as atopic dermatitis and psoriasis [95]. In EAE (experimental autoimmune encephalomyelitis) mice, an experimental mouse model of MS, treatment with CRID3/MCC950 alleviated cognitive deficits, hippocampal pathology and synaptic loss [96]. CRID3/MCC950 has also been shown to significantly reduce infiltrating T cells and proinflammatory cytokines, e.g., IL-17a, in experimental autoimmune thyroiditis (EAT) mice [97]. Furthermore, pharmacological suppression of the NLRP3 inflammasome by CRID3/MCC950 led to significant improvement in cognitive function and insulin sensitivity in the diabetic encephalopathy mice [98]. Given the promising results obtained from studies using CRID3/MCC950 as a tool compound, several patents based on the CRID3/MCC950 scaffold have been reported to cover a series of analogs. The structure of CRID3/MCC950 consists of a central sulfonylurea moiety flanked with a dimethyl-3-furanmethanol moiety and a 1,2,3,5,6,7-hexahydro-s-indacene moiety (Fig. 3). The derivatives of CRID3/MCC950 covered in these patents are compounds with modifications on these three moieties of CRID3/MCC950.

Figure 3.

Chemical structures of CRID3/MCC950 and sulfonylurea derivatives.

4.1.1. Sulfonylurea derivatives

Similar to CRID3/MCC950, compound 1 showed potent inhibition on IL-1β release with an IC₅₀ of 11 nM in human monocyte-derived macrophages (HMDM) [99]. A structurally similar compound, emlenoflast (also known as MCC7840, IZD174, or inzomelid) (Table 2), was reported in the same patent with an IC₅₀ of 13 nM in HMDM cells [99] and an IC₅₀ of 4.7 nM in murine microglia under LPS/ATP stimulation conditions [100]. Emlenoflast was originally developed by the University of Queensland and licensed to Inflamzome Ltd.. This compound has entered Phase IIb clinical trials to treat cryopyrin associated periodic syndrome (EudraCT2020-000489-40) [101–103]. Inflazome Ltd., merged with F. Hoffman-La Roche Ltd. in 2020 [104], also developed selnoflast (also known as RG6418 or RO7486967) with an IC₅₀ of ≤ 1 μM for IL-1β inhibition in THP-1 cells [105], and has since entered Phase Ib clinical trials to treat patients with ulcerative colitis [106], as well as patients with early idiopathic Parkinson’s disease [107]. IFM Therapeutics Inc. designed a series compounds as exemplified by 2-5 showing an IC₅₀ < 1 μM on inhibition of IL-1β release in THP-1 cells [108,109]. Pyroptosis assays using human THP-1 cells demonstrated the protective effects of compounds 6-9 with IC₅₀ < 1 μM [110]. Structural extension delivered compounds 10-14 that exhibited similar potency with IC₅₀ < 1 μM to inhibit the production of IL-1β in BMDM cells under the stimulation of LPS/ATP [111]. Jecure Therapeutics Inc., acquired by F. Hoffman-La Roche Ltd. in 2018 [112], reported a series of pyrazolo-oxazine compounds (15 and 16, Fig. 3) and showed inhibitory effects by these compounds on the production of IL-1β and pyroptosis in human Kupffer cells (KC). In a C57BL/6 mouse model, a single oral dose of compound 15 at 1.5 mg/kg, and compound 16 at 1 mg/kg reduced the release of IL-1β induced by LPS/ATP by > 98% [113]. Replacement of the furan moiety and the 1,2,3,5,6,7-hexahydro-s-indacene in CRID3/MCC950 with heterocycles yielded compounds 17-19 that retained the inhibitory effects on IL-1β production [114,115]. Inflazome Ltd. also developed pyrazole-containing compounds, exemplified by compounds 20 and 21, to show inhibitory effects on the NLRP3 inflammasome with improved oral bioavailability [116]. Removal of the furan moiety and replacement of the 1,2,3,5,6,7-hexahydro-s-indacene moiety with a bi-aryl ring system in CRID3/MCC950 led to compounds 22 and 23 that showed potent inhibition of the NLRP3 inflammasome [117]. The change of the tricyclic indacene moiety to a bi-aryl ring system might be intended to explore the chemical space within NLRP3 where CRID3/MCC950 binds to.

4.1.2. Acyl sulfonamide and Acyl sulfoximine compounds

Novartis reported a series of compounds that contain an acyl sulfonamide moiety, among which compounds 24-26 showed potent inhibition on IL-1β release with an IC₅₀ < 1 μM upon stimulation by gramicidin in PMA-differentiated THP-1 cells (Fig. 4) [118]. IFM Therapeutics, Inc., acquired by Bristol-Myers Squibb in 2017 [119], developed compounds 27-30 with comparable inhibitory potency under the same assay conditions [120]. Further modifications led to compounds 31-33, and the biological results suggested that the aryl sulfonamide moiety is dispensable to retain the activities as NLRP3 inhibitors [121]. However, as evidenced by compounds 34 and 35 in a following study, the aryl sulfonamide moiety might be important to provide favorable pharmacokinetic (PK) properties [122]. Change of the sulfonylurea to a sulfoximine led to improved inhibitory potency on the NLRP3 inflammasome as reflected by 36-39 [123]. Similarly, compounds 40-43 also exhibited single digit nanomolar potency to inhibit the NLRP3 inflammasome [124]. Combination of urea and sulfoximine into a sulfonimidamide moiety also led to active compounds as NLRP3 inflammasome inhibitors (44-49) [125]. A potential covalent alkenyl sulfonylurea derivative, 50, developed by Zydus Lifesciences Ltd. (originally known as Candila Healthcare Ltd.) [126], demonstrated promising potency both in vitro and in vivo [127]. Interestingly, Inflazome Ltd. designed and reported macrocyclic 1,3,4-triazole and 3-pyrrazole sulfonamide, sulfonylurea and acyl sulfonamide analogs as evidenced by 51-59 as NLRP3 inhibitors. The compounds seem to be weaker inhibitors with sub-micromolar potency (Fig. 5) [128–130]. Although the rationale behind such design was not disclosed, it might be intended to explore the effects of conformational constrain on the biological activities of these scaffolds.

Figure 4.

Chemical structures of sulfonamide, sulfoximine, sulfonimidamide, and alkenyl sulfonylurea compounds.

Figure 5.

Chemical structures of macrocyclic sulfonyl triazole and sulfonamide compounds.

4.1.3. Replacement of sulfonylurea moiety with a heterocyclic ring

NodThera Ltd. reported small molecule NLRP3 inhibitors based on a thiazole scaffold (Fig. 6). Among them, 60 was found to be active with an IC₅₀ < 1 μM for the inhibition of IL-1β release in PBMC cells under stimulation by LPS/ATP or LPS/nigericin [131]. Replacement of the thiazole moiety with a 1,3,4-triazole, as observed in the patent by Inflazome Ltd., led to compounds 61 and 62 with IC₅₀ ≤ 2 μM under stimulation by LPS/nigericin in THP-1 cells [132]. Among the inhibitors reported by AC Immune SA, compounds 63-65 are the most potent inhibitors with an IC₅₀ < 1 μM. These compounds were also demonstrated with in vivo efficacy to suppress IL-1β by 99%, 73%, >99%, respectively, in an LPS/ATP induced peritonitis mouse model [133].

Figure 6.

Chemical structures of thiazole, triazole, and dihydroxazole compounds.

5. Non-MCC950-based NLRP3 inflammasome inhibitors

Janssen Pharmaceuticals reported SAR studies based on a pyridazine scaffold and compounds 66-73 were identified as active NLRP3 inhibitors (Fig. 7). While maintaining potent inhibition on the IL-1β release (IC₅₀ < 0.03 μM) induced by LPS, they also demonstrated good selectivity with no significant inhibition on TNF-α production (IC₅₀ > 10 μM) [134–138]. Novartis AG disclosed novel tricyclic thienopyrrolotriazinacetamide compounds 74 and 75 with an IC₅₀ of 0.4 nM and 0.7 nM, respectively, against IL-1β release induced by nigericin in THP-1 cells. Notably, no inhibition on TNF-α was observed even at much higher concentrations (> 100 μM) [139].

Figure 7.

Chemical structures of pyridazine and thienopyrrolotriazinacetamide compounds.

Compounds based on an imidazo-quinoline scaffold were disclosed by IFM Therapeutics, among those compounds, 76-85 showed activity as hNLRP3 agonists with an EC₅₀ ≤ 1 μM in PMA-differentiated THP-1 cells. Notably, these compounds showed no activity on the activation of TLR7 and TLR8 with an EC₅₀ > 100 μM [140,141]. Interestingly, compounds 86 and 87 showed potent activation of the NLRP3 inflammasome with an EC₅₀ of 0.19 μM and 0.11 μM, respectively [142]. (Fig. 8) This clearly suggests the versatility of this chemical scaffold to provide novel NLRP3 agonists.

Figure 8.

Chemical structures of NLRP3 inflammasome agonists with imidazo-quinoline scaffold.

Novartis AG disclosed novel pyridazin-3-yl phenol compounds as NLRP3 inhibitors (Fig. 9). Among the reported compounds, 88 and 89 exhibited potent inhibition on the production of IL-1β in PMA-differentiated THP-1 cells with an IC₅₀ of 4 nM and 6 nM, respectively, while no inhibition on TNF-α secretion was observed (IC₅₀ > 100 μM) [143]. Carbamate containing compounds, exemplified by compound 90, were also disclosed by NodThera Ltd. as active NLRP3 inhibitors [144]. Ventus Therapeutics recently discovered bicyclic pyridazine analogs as NLRP3 inhibitors, and among them, compounds 91 and 92 showed moderate inhibitory potency [145]. Sulfide containing compounds developed by the University of Turin were also reported. Compound 93 was demonstrated to show in vivo efficacy in a DSS-induced colitis animal model to reduce intestinal inflammation [146]. Mount Sinai reported benzoxazolone-containing compounds, and 94 is the most potent within this series and notably exhibited dual inhibition on the NLRP3 and NLRC4 inflammasomes. Mechanistically, this compound may compete with ATP for the ATP binding site within the NLRP3 and block ASC oligomerization. Compound 94 was also demonstrated with in vivo activity to reduce IL-1β in the brains of LPS-treated mice [147]. Our lab recently developed sulfonamide analogs from the structure of glyburide and demonstrated their activity as NLRP3 inhibitors. Among the compounds reported, compound 95 dose dependently inhibited IL-1β release with an IC₅₀ of 8.45 μM in J774A.1 cells under the stimulation by LPS/ATP. Notably, this compound selectively engaged the inflammasome in vivo and exhibited comparable efficacy as CRID3/MCC950 in an EAE mouse model to improve the clinical score and to suppress the pathology [148,149]. A series of cyclic diarylboron derivatives were disclosed by the University of Manchester, among which compound 96 was identified as the most potent one with an IC₅₀ of 0.54 μM to inhibit the production of IL-1β under stimulation conditions by LPS/nigericin in THP-1 cells [150].

Figure 9.

Chemical structures of pyridazine-3-yl phenol, benzoxazolone, and other associated compounds.

Conclusion

Dysregulation of the NLRP3 inflammasome has been linked to the development of inflammatory and autoimmune disorders, such as SSc, SLE, IBD, and RA. As a result, small molecule inhibitors to target the NLRP3 inflammasome have been actively pursued to achieve disease interventions. Since the discovery of CRID3/MCC950 as a direct NLRP3 inhibitor and the demonstrated efficacy of this compound in a variety of preclinical disease models, many NLRP3 inflammasome inhibitors have been developed and patented recently.

Overall, the majority of the reported compounds are based on the sulfonylurea skeleton found in CRID3/MCC950. Notably, most of the derivatives are able to inhibit the release of IL-1β upon activation of the NLRP3 inflammasome under various experimental conditions. Interestingly, structural modifications of the sulfonylurea moiety led to compounds with a wide range of potency from high micromolar to single digit nanomolar potency, highlighting the importance of the sulfonylurea moiety in interacting with the NLRP3 protein, consistent with the structural biology study results.

New chemical scaffolds that are completely free of the sulfonylurea moiety, evidenced by the pyridazine and carbamate analogs, were also reported as NLRP3 inflammasome inhibitors with significantly improved potency. It is worth noting that most of the patented compounds exhibit selectivity for inhibiting the production of IL-1β, without affecting the levels of TNF-α.

Expert opinion

Small molecule inhibitors of the NLRP3 inflammasome have gained growing attention as potential therapeutics. Meanwhile, accumulating evidence indicates the vital role of NLRP3 inflammasome in the pathogenesis and development of several autoimmune diseases, such as SSc, SLE, IBD, and RA. A review of the patent landscape in this area reveals a notable increase in the number of patents filed as NLRP3 inhibitors in recent years. Some of these compounds are currently in clinical trials. CRID3/MCC950, a widely used tool compound, has shown efficacy in a variety of preclinical models of autoimmune diseases. Treatment with CRID3/MCC950 has been shown to reduce the severity of the disease in animal models of gout and MS. However, CRID3/MCC950 displayed significant hepatotoxicity in phase II clinical trials as RA treatment [151,152]. Over the past few years, numerous compounds based on the CRID3/MCC950 scaffold have been developed and tested as NLRP3 inhibitors. In most of the cases, inhibition of activation and subsequent release of downstream cytokines, e.g., IL-1β, was reported. Combination therapy using NLRP3 inhibitors together with TNF-α inhibitors or IL-1 inhibitors has been the subject of several patent applications.

With the availability of structural biology information for the inactive and active states of NLRP3, as well as the cryo-EM structure of NLRP3 in complex with small molecule inhibitors, the development of novel NLRP3 inhibitors will continue to evolve. It is also hoped that, in the near future, the availability of small molecule NLRP3 inhibitors with different MOAs will complement the molecular and genetic studies to help elucidate the pathological roles of the NLRP3 inflammasome in human diseases including the autoimmune diseases. In addition, the recent interests in developing inhibitors of ASC, one of the components of the NLRP3 inflammasome, will be expanding the avenue to facilitate drug discovery efforts by targeting the inflammasome.

Despite the promising preclinical results, clinical trials of small molecule NLRP3 inhibitors for autoimmune diseases are still in their early stages, and further investigations are needed to fully assess their safety and validate their efficacy in human beings. There are several challenges that need to be considered in the future development of NLRP3 inhibitors. Firstly, possible immune suppression by the NLRP3 inhibitors may increase the chance of infections. Although the clinical success of IL-1β based treatments may alleviate the concerns, clinical evidence is still needed to prove the safety of such inhibitors. Secondly, the complexity of this protein platform and its activation under a plethora of conditions warrant biomarker and target engagement confirmation to facilitate clinical validation of NLRP3 inhibitors. In this regard, it is promising to see the recent development of imaging tools based on some of the NLRP3 inhibitors. Lastly, it is possible that not all patients with autoimmune disorders may benefit from NLRP3 suppression by the inhibitors due to the complex interplay between the innate and adaptive immune responses. Studies in understanding the cellular and molecular mechanisms underlying the association of the NLRP3 inflammasome with autoimmune diseases are still needed to help the drug discovery efforts. Although challenges exist down the road in developing NLRP3 inhibitors as effective treatments for autoimmune diseases, the results and data from preclinical and clinical studies of NLRP3 inhibitors are encouraging, and we may see breakthroughs in the near future to validate NLRP3 as a druggable target and successful trials with expected outcomes.

Article highlights.

• The review provided a comprehensive examination of the NLRP3 inflammasome, including its architecture, activation, and role in autoimmune diseases.

• The review provided a coverage of the patents on NLRP3 inflammasome inhibitors from 2016-present. Their efficiencies in preclinical disease models are also discussed.

• We comprehensively reviewed the inhibitors based on different chemical scaffolds and their SARs from published patents.

• The potential applications and mechanism of action of MCC950 in treating autoimmune diseases are discussed in detail.