Biological and therapeutic significance of targeting NLRP3 inflammasome in the brain and the current efforts to develop brain-penetrant inhibitors

Abstract

NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a pivotal regulator of the innate immune system, orchestrates inflammatory responses implicated in neurodegenerative and inflammatory diseases. Over the past 20 years, the exploration of NLRP3 activation pathways has advanced significantly. Upon NLRP3 activation, it initiates the formation of a cytosolic multiprotein complex known as the inflammasome. This complex activates caspase-1, which then processes proinflammatory cytokines IL-1β and IL-18 and leads to gasdermin-mediated cell death, pyroptosis. Structural insights into NLRP3 inflammasome assembly and caspase-1 activation have spurred development of novel small molecule inhibitors targeting this pathway, aiming to mitigate excessive inflammation without compromising immune surveillance. The initial NLRP3 inhibitor reported was glyburide, an FDA-approved antidiabetic drug of the sulfonylurea class, which was found to inhibit the release of IL-1β induced by stimuli in human monocytes and murine macrophages. Subsequently, MCC950 (also known as CRID3), a direct NLRP3 inhibitor, was discovered. While showing promising results in preclinical and clinical trials for treating diseases, higher doses of MCC950 led to elevated transaminase levels and hepatotoxicity concerns. Recent studies using MCC950 as a research tool have prompted the development of safer and more effective NLRP3 inhibitors, including a series of compounds currently undergoing clinical trials, highlighting the potential of NLRP3 inhibitors in attenuating disease progression and improving therapeutic outcomes. In this chapter, we delve into the latest progress in understanding the mechanism of NLRP3 inflammasome activation and its roles in the pathophysiology of neurological diseases. We also summarize recent development of small molecule NLRP3 inhibitors along with the associated obstacles and concerns.

1. Introduction

The NLRP3 inflammasome is a multiprotein signaling complex that plays a central role in the innate immune responses, primarily by regulating the activation of caspase-1 (Casp1) and subsequent maturation and secretion of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 (Gattorno et al., 2007; González-Benítez et al., 2008; Tschopp & Martinon, 2002). This protein complex is composed of a sensor molecule - NOD-like receptor family pyrin domain containing 3 (NLRP3), an adaptor protein - apoptosis-associated speck-like protein containing a CARD (ASC), and pro-Casp1. Upon activation by a diverse array of stimuli, including pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and environmental irritants, the NLRP3 inflammasome initiates an inflammatory cascade that is essential for host defense but can also contribute to pathological inflammation when dysregulated (Ramachandran, Manan, Kim, & Choi, 2024).

In the central nervous system (CNS), the NLRP3 inflammasome has emerged as a critical mediator of neuroinflammation, a common feature of many neurodegenerative and neuroinflammatory diseases (Anderson, Biggs, Rankin, & Havrda, 2023). Conditions such as Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and traumatic brain injury (TBI) are characterized by chronic inflammation, in which the NLRP3 inflammasome is often upregulated and hyperactive. The detrimental effects of chronic NLRP3 activation in the brain include neuronal damage, glial cell activation, and the disruption of normal brain function, highlighting its significance as a potential therapeutic target (Zhang, Xiao, Mao, & Xia, 2023).

The therapeutic potential of targeting the NLRP3 inflammasome in the brain is highlighted by a growing body of evidence linking its activation to the progression of neurological disorders. Consequently, there has been substantial interest in developing inhibitors that can modulate the NLRP3 activity in the CNS. However, one of the major challenges in this therapeutic strategy is the development of inhibitors that can effectively penetrate the blood-brain barrier (BBB), a selective permeability barrier that protects the brain but also limits the delivery of many therapeutic agents (Blevins, Xu, Biby, & Zhang, 2022).

This chapter aims to explore the biological and therapeutic significance of targeting the NLRP3 inflammasome in the brain, with a focus on the current efforts and challenges in developing NLRP3 inhibitors. We will discuss the role of NLRP3 in various CNS diseases, the current landscape of therapeutic strategies aimed at inhibiting the NLRP3 inflammasome, and the innovative approaches being employed to overcome the BBB penetration.

2. Structure, activation, and functions of the NLRP3 inflammasome

The immune response is initially mediated by host germline-encoded pattern recognition receptors (PRRs) that sense the endogenously derived damage-associated molecules and cellular activities induced by the virulence factors of the pathogen. Upon PRRs’ response, specific intracellular protein complexes are assembled and activated, leading to the activation of Casp1 which promotes the processing and release of inflammatory cytokines such as IL-1β and IL-18 and cleavage of pore-forming proteins gasdermins. Gasdermin D (GSDMD) controls the release of IL-1β and IL-18 via a lytic pyroptosis. Initially, Jürg Tschopp and associates termed inflammasome as these supramolecular protein complexes that assemble to mediate the activation of proinflammatory Casp1 and maturation and release of proinflammatory cytokines IL-1β and IL-18 in response to inflammatory stimuli (Martinon, Burns, & Tschopp, 2002; Schroder & Tschopp, 2010). An inflammasome typically consists of a sensor protein that recruits ASC and pro-Casp1 upon activation.

The known PRRs in innate immunity include RIG-I like receptors (RLRs), Toll like receptors (TLRs), NOD like receptors (NLRs), absent in melanoma 2 (AIM2) like receptors (ALRs), and cycle GMP-AMP synthase (cGAS)/STING. Among the NLRs family, which contains NLRP1, NLRP2, NLRP3, NLRP6, NLR family CARD domain-containing protein 4 (NLRC4), and NLRP12, the NLRP3 inflammasome is the most well-characterized upstream regulator of GSDMD-mediated pyroptosis. The NLRC4 inflammasome responds to conditions of bacterial infection and quite a few gram-negative bacteria can induce pyroptosis through the NLRC4-Casp1-dependent pathway. The direct recruitment of pro-Casp1 with the CARD domain of NLRC4 can facilitate the assembly of NLRC4 inflammasome and the Casp1-dependent NLRC4 activation can be triggered by delivering purified bacterial flagellin into the cytoplasm (Miao et al., 2006). AIM2 belongs to the interferon γ protein family and acts as a cytoplasmic DNA sensor recognizing double-stranded DNA. AIM2 oligomerizes to assemble with ASC and pro-Casp1 to form AIM2 inflammasome complex that can lead to PANoptosis, pyroptosis, apoptosis, and necroptosis, as a protection mechanism for the host (Lee et al., 2021).

2.1. Structure of NLRP3 protein

The most well-demonstrated NLR family of inflammasomes uses ATP-binding nucleotide-binding oligomerization domain (NACHT) containing NLRP3 and NLRC4 as sensor proteins, whereas the ALR family of inflammasomes uses sensor proteins such as AIM2 and Gamma-interferon-inducible protein (IFI16) that do not consist of a NACHT domain (Man & Kanneganti, 2015). In both types of inflammasomes, pro-Casp1 cleaves itself into an active enzyme and thereby mediates the maturation and production of the proinflammatory cytokines IL-1β and IL-18 (Black, Kronheim, Merriam, March, & Hopp, 1989; Giamarellos-Bourboulis et al., 2009; Joosten et al., 2010). In addition to the cytokines, Casp1 also cleaves GSDMD into its N-terminal fragment which forms a pore in the cytoplasmic membrane followed by swelling and cell lysis known as pyroptosis, a type of inflammatory cell death (Liu et al., 2016). Till now, >1000 proteins have been identified as substrates of Casp1 and the number continues to grow. Among the proteins, Casp7 is known to be associated with apoptosis by cleaving substrates such as the DNA damage sensor protein poly (ADP-ribose) polymerase 1 (PARP1). Activation of PARP1 by Casp7 can occur at the promoters of a subset of NF-κB target genes, providing a role of NLRP3 inflammasome activation on proinflammatory gene expression (Erener et al., 2012). The inflammatory sensor proteins recognize specific signals from pathogens. For example, the type III secretion system (T3SS) or bacterial flagellin activates NLRC4, cytoplasmic double-stranded DNAs are recognized by AIM2, whereas NLRP3 inflammasome is activated by structurally diverse signals from host cell molecules, such as mitochondrial reactive oxygen species (ROS), extracellular ATP, microbial components, such as nigericin, double- or single-stranded RNA, and muramyl dipeptide.

2.1.1. Major Components and Domains

The NLRP3 inflammasome is composed of three major components: NLRP3, ASC, and Casp1. NLRP3 is a protein of ~118 kDa, consists of an N-terminal pyrin domain (PYD), a centrally located adenosine triphosphatase (ATPase) domain known as NOD or NACHT, and a C-terminal leucine-rich repeat (LRR) domain which contains 12 repeats (Fig. 1A). The NACHT domain acts as an ATP-induced oligomerization domain of NLRP3 by binding to ADP/ATP. The recent cryo-EM structure revealed a binding pocket formed by the four subdomains of the NACHT domain and their spatial arrangement (Dekker et al., 2021). The LRR domain is evolutionarily conserved in proteins associated with innate immunity in vertebrates, invertebrates, and plants, and the LRR in NLRP3 is known to play a key role in ligand sensing and autoregulation of NLRP3 (Dick, Sborgi, Rühl, Hiller, & Broz, 2016). Recent studies revealed that the formation of oligomeric ring-like structures of inactive NLRP3 in cells is mediated by “back-to-back” and “face-to-face” interactions of LRRs. Additionally, the fish-specific NACHT-associated (FISNA) domain of NLRP3, between the PYD and NACHT domains, works as a conformational switch in NLRP3 activation and a transition of N-terminal LRR domain takes place during the structural change (Tapia-Abellán et al., 2021). Never in mitosis gene A–related kinase 7 (NEK7) is a mitotic kinase that binds to NLRP3 and facilitates its activation at the interphase stage of the cell cycle. Activated NLRP3 oligomerizes and recruits ASCs through homotypic binding between their PYDs. PYD, also termed as a death motif, is a conserved motif in proteins involved in lytic cell death such as pyroptosis and necroptosis. The PYD domains of NLRP3 and ASC mediate the binding between NLRP3 and ASC, and the NLRP3-ASC complex in turn recruits multiple pro-Casp1s through binding between their CARDs to initiate the activation of pro-Casp1, the proteolytic processing, and maturation of IL-1β and IL-18. Pro-Casp1 contains a CARD domain and a p20-p10 domain that is cleaved into p20 and p10 upon activation.

Fig. 1.

Structures of NLRP3 inflammasome proteins. (A) Domain structure of the NLRP3 inflammasome and active NLRP3 inflammasome complex. (B) Crystal structure of human NACHT (PDB: 7ALV) (Dekker et al., 2021). (C) Crystal structure of human PYD (PDB: 3QF2) (Bae & Park, 2011). (D) Cryo-EM structure of human NLRP3 NACHT-LRR bound with NEK7 (PDB: 6NPY) (Sharif et al., 2019). (E) Cryo-EM structure of inactive full-length human NLRP3 (PDB: 7PZC) (Hochheiser et al., 2022).

2.1.2. Structures of Domains, the Full-Length Protein, and Oligomers of NLRP3 in inactive/active conformations

The NACHT domain consists of four subdomains –the nucleotide-binding domain (NBD), helical domain (HD) 1, winged helix domain (WHD), and HD2, respectively. Structurally, NACHT is composed of STAND elements, that is, a Sensor-1 motif (residues 346-LLITTR-351) followed by a Walker A motif or P-loop (residues 226- GAAGIGKT-233) and a Walker B motif (residues 296- RILFLMDGFDE-306). The motifs are spatially arranged which is believed to be involved in interactions of key residues to stabilize the inactive conformation of NLRP3 and control domain rearrangement upon activation (Dekker et al., 2021) (Fig. 1B). Also, in the absence of LRR and NEK7, the NACHT domain tends to adopt an inactive closed conformation. When ADP binds to the NBD pocket, the β-phosphate group of ADP forms extensive contact with Walker A by interacting with His522 of WHD which keeps NACHT in its closed conformation. Then in the presence of ADP, a salt bridge between the conserved Arg351 of sensor-I motif with Glu527 stabilizes the inactive closed conformation. When ATP binds, Arg351 is released from the salt interaction to coordinate the γ-phosphate of ATP. Arg262 adjacent to Walker B forms a salt bridge with Glu511 and contributes to the stabilization of the inactive conformation by providing additional interdomain interactions between NBD and WHD. The three-dimensional (3D) structure of NLRP3 obtained from X-ray crystallography (PDB: 3QF2) and Nuclear Magnetic Resonance Spectroscopy (NMR) show a high degree of similarity to each other with a backbone root mean square deviation of 1.66 Å (Fig. 1C). Overall, the PYD domain holds a canonical antiparallel six-helical bundle fold composed of six helices (a1–a6) and five connecting loops which resembles PYD domains of NLRP4 and NLRP10 proteins. During inflammasomal assembly, the activated NLRP3 binds to ASC via homotypic interactions between their PYDs forming electrostatic and hydrophobic bonds. A recent cryo-EM structure revealed a disulfide bond between the Cys8 residue in helix 1 and the Cys108 residue in the PYD loop that connects the PYD and NBD domains. The formation of this disulfide bond serves as a contributor in NLRP3 activation by relieving auto-inhibition of monomeric inactive NLRP3. Downstream of the K⁺ efflux events, NEK7 binds to NLRP3 to induce inflammasome activation during the interphase stage of cell cycle. In a recent cryo-EM study at a resolution of 3.8 Å (PDB: 6NPY), NLRP3-NEK7 interactions in inactive conformation of human NLRP3 confirmed the role of NEK7 in NLRP3 activation. This 3D structure revealed two interfaces of NLRP3-NEK7 interactions. The Interface I has interactions between the LRR of NLRP3 domain and the first half of NEK7 C-lobe whereas Interface II shows interactions between the C-lobe of the second half of NEK7 with NACHT domain of NLRP3. Modeling of hypothetical NLRP3-NEK7 active conformation based on the NLRC4 structure predicted an additional contact between an NLRP3-bound NEK7 and a neighboring NLRP3 suggesting that NEK7 might help in bridging NLRP3 subunits together (Sharif et al., 2019) (Fig. 1D).

Moreover, recent studies revealed that inactive NLRP3 exists in an equilibrium between monomeric and oligomeric proteins in cells with ADP bound and with or without NLRP3 inhibitor MCC950 (Hochheiser et al., 2022). The cryo-EM structure (PDB: 7PZC) shows that the ring-like structures differ from one another considerably regarding the number of monomers and configurations of rings, and are capable of associating with membrane structures such as Golgi apparatus in cells (Fig. 1E).

All the domains of NLRP3 stay flexibly linked together in individual NLRP3 subunits. Ten such subunits form a large flower-shaped circular disc with a diameter of about 32 nm, forming an interconnection between the LRR domains and NEK7 extending away from the disc’s center whereas FISNA-NACHT domains interact near the center of the disc (Xiao, Magupalli, & Wu, 2023). The recent cryo-EM structure revealed that the conformation of active NLRP3 differs from the previously resolved inactive NLRP3 in caged form and complex with NEK7 (Fig. 2A). Comparing the active and inactive states of NLRP3, the WHD-HD2-LRR module rotates by approximately 85.4° along an axis at the junction between HD1 and WHD to turn into the active state from the inactive state (Xiao et al., 2023) (Fig. 2B). However, the interaction between the NEK7 and LRR domains does not participate in disc assembly. Overall, the conformational change is a rigid body rotation where the WHD-HD2-LRR and FISNA-NBD-HD1 modules align well between the active and inactive states. The FISNA domain undergoes notable structural changes in these two states. For instance, during NLRP3 conformation changes, a disordered loop 1 (residues 151–163) in the middle of the FISNA domain becomes ordered and loop 2 (residues 212–217) undergoes conformational change in the active conformation. At the assembly of FISNA-NACHT region, the structural changes in FISNA and NACHT domains are accompanied by exchange of ADP in the inactive conformation to ATPγS (mimicking ATP) in the active conformation.

Fig. 2.

Cryo-EM structures of active, inactive, and MCC950-bound NLRP3 structures. (A) Cryo-EM structure of full-length NLRP3 structure (PDB: 8EJ4). (B) Structural change and rotation of NLRP3 during the switch from inactive to active conformation. Different domains are highlighted with color variations. (C) MCC950-bound full-length inactive NLRP3 (PDB: 7VTP). (D) Binding interactions of MCC950 with NLRP3 (inactive conformation), MCC950 is highlighted in magenta (Figures made by ‘The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC’).

In separate studies, the ligand-bound structure of NLRP3 also revealed differences between the inactive and active states. For example, the cryo-EM structure of full-length mouse NLRP3 (residues 1 to 1033) bound with ADP and MCC950 was obtained as a dodecamer ring at 3.6 Å resolution (Fig. 2C) (Ohto et al., 2022). Also, a human NLRP3 protein lacking PYD (NLRP3ΔP residues 130 to 1036) bound with ADP and MCC950 showed a spheric hexamer with a diameter of 160 Å. The structure of NLRP3ΔP presented an inactive and closed conformation like the NLRP3-NEK7 structure. The hexamer formation involves a head-to-tail interaction between LRR, NBD, and WHD of NACHT and forms a back-to-back interaction between LRR3 and LRR6 of two protomers (Ohto et al., 2022). MCC950 binds to the cavity distinct from the nucleotide-binding site in the NACHT domain and stabilizes the closed conformation of NLRP3. The oxygen atom of the urea moiety forms two strong hydrogen bonds or ionic interactions with ARG351 (NBD) and ARG578 (HD2) (distance 3.4 Å and 2.9 Å, respectively). The tricyclic sulfonyl amide moiety of MCC950 was lined up with residues GLY226, ALA227, and ALA228 from the ATP-binding Walker A motif of NBD. Also, MCC950 formed multiple hydrophobic contacts with the residues A228 (NBD), PHE410, ILE411, LEU413, THR439, TYR443, THR524 (WHD), PHE575, ARG578, TYR632 (HD2), and MET661 (LRR) (Fig. 2D).

2.2. Molecular Activation of NLRP3

In the CNS, NLRP3 is mainly expressed in microglia, the resident immune cells of the CNS, and astrocytes in response to stimuli (Heneka, McManus, & Latz, 2018). In addition, NLRP3 expression has been found in neurons (Zhang et al., 2016), peripheral (Navarro et al., 2016) as well as brain vascular pericytes (Stark et al., 2013; Weiskirchen & Tacke, 2014). NLRP3 is distributed in the hippocampus (Zhuang et al., 2017), midbrain (Zhou et al., 2016), white matter, striatum, and cortex region of brain (Chivero et al., 2017; Walsh, Reinke, et al., 2014). Outside of the brain, NLRP3 has a limited tissue distribution, with notable expression in the non-keratinizing epithelia of the oropharynx, food pipe, ectocervix, and the urothelial layer of the bladder. Within these tissues, NLRP3 is predominantly cytoplasmic, likely facilitating the rapid detection of pathogens and initiating an immune response (Kummer et al., 2007).

2.2.1. Priming and Activation of NLRP3

The activation of the NLRP3 inflammasome involves many signals and follows a two-step process that requires a prior priming step. Under unstimulated conditions, a low level of NLRP3 protein exists in its latent form and its translation is increased during priming stage which maintains NLRP3 in an activation-competent state (Fig. 3). Proinflammatory cytokines such as Tumor Necrosis Factor-α (TNF-α) and IL-1β activate their respective receptors and contribute to the induction that occurs at the transcription level via the cytoplasmic NOD2 or TLRs pathways. The activation and function of NLRP3 are mediated by the post-translational modifications of the NLRP3 protein such as phosphorylation, ubiquitylation, and sumoylation. For instance, TNF receptor-associated factor 6 mediates the non-transcriptional priming of NLRP3 through TLR and IL-1R signaling in an E3 ligase-dependent manner (Xing et al., 2017). Also, protein-protein interactions can mediate priming such as the NLRP3-NEK7 binding licenses the activation of NLRP3 at the interphase of cell cycle (He, Zeng, Yang, Motro, & Núñez, 2016).

Fig. 3.

Diagram of activation of the NLRP3 inflammasome. Priming is induced by signal 1, such as LPS and TNF-α and subsequent activation of their membrane-bound pattern recognition receptors. Canonical activation of the NLRP3 inflammasome involves docking of NLRP3 at mitochondrial outer membrane, is elicited by signal 2 including PAMPs, such as nigericin, mtDNA, and mtROS, viral RNA, and MDP, and DAMPs, such as extracellular ATP, and particles and crystals. Activation of NLRP3 involves multiple signaling events including K⁺ efflux, Ca²⁺ flux, Cl⁻ efflux, lysosomal disruption, mtROS production, and release of oxidized mtDNA. Noncanonical activation of the NLRP3 inflammasome is induced by gram-negative bacteria. Release of LPS from engulfed bacteria into the cytoplasm activates human Casp4/5 or mouse Casp11. The alternative pathway is mediated by LPS-induced TLR4 which activates the TLR4-TRIF-RIPK1-FADD-Casp8 signaling (Created with BioRender.com).

2.2.1.1. Canonical Activation of NLRP3

Full activation of NLRP3 follows a multistep process known as canonical pathway starting with the release of NLRP3 autoinhibition, oligomerization of NACHT domain, ASC specks formation, recruitment and auto-activation of Caspase-1, and cleavage and maturation of IL-1β and IL-18 (Fig. 3). Several cellular and molecular events have been recognized as critical steps in the canonical activation of NLRP3, however, elucidation of the mechanism by which primed cells recognize NLRP3 activating signals has been a subject of considerable debate. Nevertheless, these steps are not mutually exclusive but may occur in sequence or in parallel in a context-dependent manner to activate NLRP3. There is no single-consensus model for NLRP3 inflammasome activation by all activators of NLRP3 to this date.

Efflux of intracellular K⁺ is a key step of NLRP3 activation by activators such as extracellular ATP, bacterial toxins, and various particulates. A reduction in intracellular K⁺ concentration is sufficient to induce the activation of NLRP3. Among microbial toxins, nigericin as a potassium ionophore induces IL-1β maturation through pannexin-1-dependent K⁺ efflux. Then, the extracellular ATP activates P2X purinoceptor 7 (P2X7), which coordinates with TWIK2 (two-pore domain potassium (K2P) channel) to promote K⁺ efflux and influx of Ca²⁺ and Na⁺, thereby leading to NLRP3 activation (Di et al., 2018). Also, the activation of NLRP3 is enhanced by the release of intracellular ATP via C3a activation by LPS alongside priming. Furthermore, particulates, e.g., calcium pyrophosphate, alum, dihydrate crystals, and silica, have been shown to promote nigericin-induced NLRP3 activation by inducing K⁺ efflux and Ca²⁺ mobilization (Asgari et al., 2013). Besides K⁺ efflux and Ca²⁺ mobilization, the efflux of Cl⁻ has also been implicated in the activation of NLRP3. Reduced levels of extracellular Cl⁻ can enhance, whereas elevated levels Cl⁻ can hinder the activation of NLRP3. The Efflux of Cl⁻ is speculated to be downstream of K⁺ efflux and affects ASC polymerization, whereas K⁺ efflux promotes NLRP3 oligomerization (Tang et al., 2017).

Apart from the flux of ions, there are other pathways of NLRP3 activation such as via ROS, lysosomal degradation, metabolic changes, mitochondrial dysregulation, and trans-Golgi disassembly (Heid et al., 2013). ROS is generated during the synthesis of ATPs by mitochondria due to one-electron transfer to O₂ in the respiratory chain at complex 1 and complex III. Although the direct mechanism of NLRP3 activation by ROS is unclear, TLR7 ligand imiquimod was shown to inhibit mitochondrial complex I to result in a burst of ROS production from mitochondria and, consequently, NLRP3 activation (Lee et al., 2020). Mitochondria also serve as a docking site for inflammasome formation by providing ROS, DNA, and DAMPs. Under mitochondrial stress, the inner membrane of mitochondria releases cardiolipin to the outer membrane and binds Casp1 and NLRP3, promoting inflammasome activation.

2.2.1.2. Non-canonical and alternative activation of NLRP3

The non-canonical activation of NLRP3 can consequently result from the oligomerization of the caspases and their auto-cleavage. Caspases, for example, casp4 and casp5 in humans and casp11 in mice, can be stimulated by cytoplasmic LPS. The endogenous ligand of casp11, oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine) along with LPS activate casp11 by binding to different domains of Casp11 and trigger NLRP3 inflammasome activation followed by Casp1-dependent IL-1β maturation (Fig. 3). Active caspases cleave GSDMD to induce pyroptosis which releases ATP by activating pannexin-1 and induces K⁺ efflux, all of which in turn drive the activation and oligomerization of NLRP3, formation of ASC specks, and Casp1-dependent maturation and release of IL-1β and IL-18 (Ma, 2023).

In alternative pathways, activating NLRP3 doesn’t necessarily lead to Casp1-dependent IL-1β maturation. Under this circumstance, Casp8, an apoptosis-initiating caspase works to cleave IL-1β and IL-18 and thereby activates NLRP3 inflammasome via the TLR4-TRIF-RIPK1- FADD-CASP8 pathway independently of K⁺ efflux (Gaidt et al., 2016). Casp8 along with FADD are known mediators of apoptosis and were found to mediate NF-κB-dependent priming and post-translational activation of NLRP3 in murine macrophages (Zhong et al., 2016).

2.3. Functions of the NLRP3 Inflammasome

Inflammasomes in various types of cells act as a bridge between innate and adaptive immunity and react to a wide range of physiological and pathogenic stimuli. The major function of inflammasome activation lies in the regulation of the production of pro-inflammatory cytokines, ultimately triggering inflammatory responses and maintaining immune homeostasis. NLRP3 protein serves as a sensor to diverse substances including microbial, environmental, and host-derived factors. A number of viral pathogens such as vesicular stomatitis virus (Rajan, Rodriguez, Miao, & Aderem, 2011) and Influenza A virus (Thomas et al., 2009), pathogenic bacteria such as E.coli and S. aureus (Rathinam et al., 2012), parasite Dermatophagoides pteronyssinus (Dai et al., 2011), and fungal pathogens such as Aspergillus fumigatus (Saïd-Sadier, Padilla, Langsley, & Ojcius, 2010) and Candida albicans (Gross et al., 2009) has been shown to activate the NLRP3 inflammasome. Consequently, the NLRP3 inflammasome mediates to secret cytokines, including IL-1, IL-1β, IL-18, IL-17A and IFN-γ to limit the systemic spread of the pathogens (Ceballos-Olvera, Sahoo, Miller, Del Barrio, & Re, 2011; Liu et al., 2012). NLRP3 also acts as a sensor to host-derived danger signals and can detect changes in mitochondrial function, membrane lipids, ion efflux, and ROS production (Heid et al., 2013; Wei, Yang, Zheng, Tang, & Li, 2019; Zhou, Yazdi, Menu, & Tschopp, 2011). The activation of the NLRP3 inflammasome is a double edge sword, on one side to maintain system homeostasis by secreting proinflammatory cytokines against pathogen infection and on the other side to initiate cell pyroptosis to cause tissue damage. It is worth noting that NLRP3 inflammasome-mediated cell death not only depends on pyroptosis but also on PANoptosis, a more complex integration of pyroptosis, apoptosis, and necroptosis (Kuriakose et al., 2016). Necroptosis is usually known to be mediated by the activation of mixed-lineage kinase domain-like protein (MLKL). The MLKL-RIPK3 signaling is involved in the activation of NLRP3 and IL-1β release during necroptotic cell death in a cell-intrinsic manner (Conos et al., 2017).

The NLRP3 inflammasome has been implicated roles in B cell development. NLRP3-mediated IL-1β release has been shown to regulate energy homeostasis, triglyceride metabolism, and the promotion of insulin signaling in adipocytes, which helps the development of B cells in the peritoneal cavity as it requires a metabolic program including glycolysis, lipid uptake, and predominant fatty acid synthesis (Clarke, Riffelmacher, Braas, Cornall, & Simon, 2018). Furthermore, studies demonstrated that NLRP3 regulates B cell development by playing a role in the transcriptional network. NLRP3 has been found to promote the activation of gene promoters via interacting with Interferon Regulatory Factor 4 (IRF4) in the nucleus of lymphocytes (Bruchard et al., 2015). Moreover, in B cells, NLRP3 stimulates the expression of chemokine receptors CXCR4 and CCR7 and Immunoglobulin M (IgM) production to initiate protective immunity and immune responses (Honda, Ku, & Anders, 2023). NLRP3 can directly bind to interleukin receptor gene promoter regions and acts as a gene transcription factor in T cells and epithelial cells to regulate the expression of IL-33, a nuclear cytokine that is crucial to induce type 2 immune response (Honda et al., 2023).

NLRP3 inflammasome activation has been found to play a crucial role in maintaining CNS homeostasis via necessary inflammatory response and programmed cell death in various CNS cells including dendritic cells and neurons (Hanslik & Ulland, 2020; Jha et al., 2010). In response to peripheral injury, production of the NLRP3 inflammasome-mediated proinflammatory cytokines helps microglia switch to effector cells from DAMP-sensing sentinel cells to induce an immune response in the brain. Thereby, the glial cells undergo proliferation, process motility, morphological changes, and upregulate the release of inflammatory factors such as interleukins, NO, and TNF-α. The inflammatory factors then promote network-wide adaptive response by engaging neurons and glial cells (Tejera et al., 2019).

3. Association of the NLRP3 Inflammasome with Diseases

3.1. Alzheimer’s Disease (AD)

AD is a form of dementia that results in progressive cognitive decline and can range in severity as the disease progresses (Gaugler et al., 2024). As of May 2024, it is estimated that 6.9 million Americans aged 65 and older suffer from AD, and this number is expected to double by 2060. Additionally, AD is currently ranked as the 7th leading cause of death in the United States, 5th among the 65 and older population (Gaugler et al., 2024). The pathological hallmarks of AD are the presence of amyloid plaques and tau tangles. The buildup of these plaques and tangles in brain regions associated with memory formation and storage, such as the cerebral cortex and hippocampus, is thought to be a key contributor of the cognitive decline seen in AD (Glenner & Wong, 1984; Kidd, 1963; Selkoe, Ihara, & Salazar, 1982; Tagliavini, Giaccone, Frangione, & Bugiani, 1988; Terry, 1963; Yamaguchi, Hirai, Morimatsu, Shoji, & Harigaya, 1988).

The role that the NLRP3 inflammasome plays in the pathology of AD has been extensively studied over the years (He et al. 2016; Kayagaki et al., 2011; Latz, Xiao, & Stutz, 2013; Schmid-Burgk et al., 2016; Schroder & Tschopp, 2010; Shi et al., 2016; Tan, Yu, Jiang, Zhu, & Tan, 2013; Walsh, Muruve, & Power, 2014). Studies have highlighted an increased level of proteins of the NLRP3 inflammasome pathway, such as Casp1, ASC, and IL-1β, and increased inflammasome activity in mouse models and patients with AD, as well as frontotemporal dementia models (Couturier et al., 2016; Halle et al., 2008; Heneka et al., 2013; Ising et al., 2019; Saresella et al., 2016; Schindowski et al., 2006; Zhu et al., 1999). Studies have also shown that the NLRP3 inflammasome can be activated by Aβ fibrils through damage to the lysosomes of microglia, triggering subsequent inflammatory signal cascade (Halle et al., 2008; Lučiūnaitė et al., 2020). The activation of the NLRP3 inflammasome by Aβ can also be seen in astrocytes of 5xFAD mice (Couturier et al., 2016). To support the roles of NLRP3 in AD development, studies have shown that downregulation of the NLRP3 inflammasome leads to increased phagocytosis and decreased Aβ pathology in AD mouse astrocytes (Couturier et al., 2016). Additionally, the downstream secretion of IL-1β has been shown to drive tau pathology by inducing tau phosphorylation and promoting the formation of neurofibrillary tangles. Knocking out NLRP3 and ASC in Tau22 mice has been found to reduce tau pathology (Griffin, Liu, Li, Mrak, & Barger, 2006; Ising et al., 2019; Sheng et al., 2001). Furthermore, transgenic APP/PS1 mice that lacked NLRP3 inflammasome-related proteins showed significant protection from AD pathology and associated cognitive impairment (Heneka et al., 2013). The evidence linking NLRP3 inflammasome to the progression of AD makes it a viable drug target to develop effective treatment for this debilitating disease. Indeed, several NLRP3 inhibitors have been shown to alleviate or even reverse cognitive decline, as well as reduce insoluble Aβ levels, lower neuroinflammation, and even promote neurogenesis (Haseeb et al., 2022; He et al., 2020; Kuwar et al., 2021; Yin et al., 2018).

3.2. Parkinson’s Disease (PD)

PD is a progressive and neurodegenerative disorder that primarily impacts motor skills, resulting in symptoms such as tremor and bradykinesia (Mayo Clinic Staff, 2024a). Approximately 1 million people in the United States and 10 million people worldwide are diagnosed with PD, and that number is expected to keep growing. The prevalence of the disease increases with age, making it the second most-common neurodegenerative disorder, right behind AD. A prominent pathology of PD is the aggregation of α-synuclein (α-syn) proteins, forming clusters known as Lewy bodies (LB) (Polymeropoulos et al., 1997; Spillantini et al., 1997). This, in combination with the loss of the nigrostriatal dopaminergic neurons, culminates in the manifestation of the motor and cognitive symptoms associated with PD, including bradykinesia, cognitive impairment, and hypomimia (Alexander, 2004; Calabresi et al., 2023; Dauer & Przedborski, 2003; Mayo Clinic Staff, 2024a).

Studies have highlighted the relationship between the dysregulation of the NLRP3 inflammasome and the development of PD. Recent evaluations of PD patients have shown that levels of NLRP3 and IL-1β are significantly higher than those of healthy patients (Anderson et al., 2021; Chatterjee et al., 2020; Havrda, 2016; von Herrmann et al., 2018). Also found were elevated NLRP3 inflammasome activation markers such as ASC speck formation in mesencephalic tissues of early-stage and even pre-diagnosis PD patients (Anderson et al., 2021). There also appears to be a feedback loop between the NLRP3 inflammasome, α-syn, and dopamine. A positive correlation has been seen between the level of NLRP3 and the level of α-syn in PD patient serum samples (Chatterjee et al., 2020). Studies also show that inflammatory proteins Casp1 and IL-1β are responsible for the formation of LBs and the decreased level of dopamine in the brain, whereas other studies show that dopamine actually possesses anti-inflammatory properties through inhibition of the NLRP3 inflammasome (Gordon et al., 2018; Griffin et al., 2006; Qiao et al., 2017; Wang et al., 2016; Yan et al., 2015). Additionally, α-syn has been shown to upregulate the expression of NLRP3 inflammasome components through the Atg5 protein in astrocytes of PD patients (Wang, Chi et al. 2020). Furthermore, α-syn has been shown to activate the NLRP3 inflammasome in microglia, resulting in the secretion of IL-1β (Pike et al., 2021).

3.3. Multiple Sclerosis (MS)

MS is a debilitating neurodegenerative disorder in which the immune response damages the myelin sheath around neurons. The shedding of this protective covering causes a wide variety of symptoms, including pain, numbness, and vision problems (Tobin, 2022). As of 2019, it was estimated that 1 million Americans and 2.3 million people worldwide suffer from MS (Empowering people affected by MS to live their best lives, n.d.; Nelson et al., 2019). Unlike AD and PD, MS onset typically occurs in young adulthood, between the ages of 20 and 40, with symptoms progressing over time (“Multiple Sclerosis - Symptoms and Causes,” 2022; Nelson et al., 2019). The pathology of MS is thought to be caused by the abnormal activation of immune cells, such as T-helper (Th) cells and CD8⁺ cytotoxic T lymphocytes (CTLs), followed by their infiltration to the CNS, leading to the increased production of inflammatory cytokines and ROS by astrocytes and microglia. The combination of these events leads to the demyelination of neurons and the subsequent clinical manifestations associated with MS (Brinkmann et al., 2010).

Studies have shown the critical role of the NLRP3 inflammasome in the pathogenesis of MS. In experimental autoimmune encephalomyelitis (EAE) mice, an MS preclinical mouse model, studies found that expression of NLRP3 was significantly higher at the height of the disease state compared to prior to disease progression (Gris et al., 2010). Additionally, they found that depletion of NLRP3 was associated with delayed disease progression and preservation of the myelin sheath around the spinal cord (Gris et al., 2010). Furthermore, they highlighted that NLRP3 is crucial for the pathology of EAE through proliferation and differentiation of Th1 (IFN-γ-secreting CD4⁺ T cells) and Th17 (IL-17-secreting CD4⁺ T cells) cells, and that depletion of NLRP3 resulted in reduced IL-18, IFN-γ, and IL-17 levels, as well as delayed disease course in EAE mice (Gris et al., 2010; Inoue, Williams, Gunn, & Shinohara, 2012). Studies have also highlighted the role of the NLRP3 inflammasome in demyelination. In fact, NLRP3^−/− EAE mice were shown to have significantly delayed demyelination in the corpus callosum and spinal cord, likely due to the increased levels of mature myelinating oligodendrocytes present compared to the wild-type EAE mice (Inoue et al., 2012; Jha et al., 2010). A decreased infiltration rate of microglia and astrocytes to the corpus callosum was also seen in these knockout mice, due to the NLRP3 inflammasome involvement in this immune response, particularly through downstream effector proteins Casp1 and IL-18 (Jha et al., 2010). Additionally, the NLRP3 inflammasome was shown to be crucial for the migration of other immune cells, such as Th17 cells, to the CNS as well, and that injection of these cells into NLRP3 knockout mice resulted in a much less aggressive case of EAE (Inoue et al., 2012). To echo these observations, pharmacological inhibition with NLRP3 inhibitors have shown significant relief of symptoms and delayed disease progression in EAE mice (Coll et al., 2015; Guo et al., 2017).

3.4. Amyotrophic Lateral Sclerosis (ALS)

ALS is a degenerative disease of the CNS that results in a loss of muscle control manifested as muscle weakness, trouble swallowing, and slurred speech (Mayo Clinic Staff, 2024b; Ravits et al., 2013; Rowland & Shneider, 2001). The current estimated survival for ALS usually ranges from 3–5 years, with progressive decline in motor and cognitive skills over time (Caplliure-Llopis et al., 2020; Rothstein, 2009; Traynor, Alexander, Corr, Frost, & Hardiman, 2003). As of 2018, approximately 30,000 people in the United States were reported to suffer from ALS and 80 % of those die within 2–5 years (Mayo Clinic Staff, 2024b; Mehta et al., 2023; National ALS Registry Dashboard, Amyotrophic Lateral Sclerosis ALS, & CDC, 2024). One of the ALS pathology is the aggregation of superoxide dismutase 1 (SOD1) proteins, leading to the degeneration of motor neurons and muscles, causing paralysis eventually (Clénet et al., 2023; Johann et al., 2015; Rothstein, 2009; Rowland & Shneider, 2001; Zhang et al., 2022).

The pathophysiological role of the NLRP3 inflammasome in ALS has been demonstrated by recent studies, especially its involvement in motor neuron degeneration, similar to the neuronal degeneration in MS (Banerjee et al., 2022; Cihankaya et al., 2024; Clénet et al., 2023; Deora et al., 2020; Moreno-García et al., 2021; Zhang et al., 2022). Studies have shown increased levels of NLRP3 inflammasome proteins in diseased mice compared to wild-type controls, with early-stage localization to the ventral horn neurons and late-stage localization to astrocytes and microglia (Banerjee et al., 2022; Johann et al., 2015; Kadhim, Deltenre, Martin, & Sébire, 2016; Zhang et al., 2022). Studies of human ALS brain tissues further confirmed the upregulation of these proteins compared to normal and healthy control brain samples (Johann et al., 2015; Kadhim et al., 2016). Genetic studies have identified 50 risk genes for ALS, among which were genes associated with the NLRP3 inflammasome (Banerjee et al., 2022). NLRP3 inflammasome gene alterations were also seen in the skeletal muscle of SOD1G93A mice, an ALS animal model (Moreno-García et al., 2021). Another study found that upregulation of Casp1 and GSDMD in the spinal cords of wobbler mice, another ALS animal model, corresponded to increased microglia and astrocyte infiltration to the spinal cord tissue and subsequent motor neuron degeneration and neuroinflammation (Cihankaya et al., 2024). Additionally, it has been shown that ALS-associated proteins, such as SOD1G93A and TDP-43, can activate the NLRP3 inflammasome and promote the release of pro-inflammatory cytokines (Deora et al., 2020).

3.5. Traumatic Brain Injury (TBI)

TBI is caused by an external force, such as a blow to the head, leading to concussion in mild cases and behavioral changes or even coma in severe cases (Facts About TBI, 2024; Mayo Clinic Staff, 2021). Severity of the injury is typically determined using the Glasgow Coma Scale (GCS), a clinical evaluation of motor responsiveness, verbal performance, and eye opening (Teasdale & Jennett, 1974). In 2021, there were approximately 69,000 TBI-related deaths and more than 210,000 hospitalizations in the United States, approximately one-third of which were people aged 75 or older, and that is in addition to the 1 million Americans annually treated and discharged from emergency rooms due to TBI (TBI Data, 2024; Brain Injury Facts, n.d.). Additionally, it is estimated that 5.3 million people in the United States are currently suffering with long-term disabilities related to TBI (Brain Injury Facts, n.d.).

Neuroinflammation has long been known as a secondary injury post-TBI contributing to poor prognosis (Coughlin et al., 2015; Lin et al., 2017; Liu et al., 2013; Perez-Polo et al., 2013; Wallisch et al., 2017). In fact, over the years, molecules including ROS, ATP, and well known DAMPs that target the NLRP3 inflammasome (Fig. 3) have been shown to play a major role in the severity of TBI and the long-term complications associated with it (Faroqi et al., 2021; Marklund, Clausen, Lewander, & Hillered, 2001; Marklund, Salci, Ronquist, & Hillered, 2006; Moro, Ghavim, & Sutton, 2021; Tavazzi et al., 2005). Studies have also shown that in human and rat brain and cerebrospinal fluid (CSF) samples, levels of proteins associated with the NLRP3 inflammasome, such as ASC and Casp1, are significantly elevated (Ismael, Nasoohi, & Ishrat, 2018; Lin et al., 2017; Liu et al., 2013; Wallisch et al., 2017; Xu et al., 2018). Consequently, the levels of pro-inflammatory cytokines like IL-1β are increased post-TBI as well (Lin et al., 2017; Liu et al., 2013; Perez-Polo et al., 2013). Additionally, studies with NLRP3 knockout mice have shown decreased inflammatory response in hippocampal brain samples when compared to wild-type mice post-TBI (Irrera et al., 2017). This inflammatory response is abnormally regulated through microglial activation and can even lead to Aβ and tau pathologies (Cherry et al., 2016; Collins-Praino & Corrigan, 2017; Ising et al., 2019). Furthermore, the neuroinflammation caused by severe and repeated mild TBI can have long lasting effects, including mood disorders, cognitive decline, and motor skill impairment (Cherry et al., 2016; Collins-Praino & Corrigan, 2017; Coughlin et al., 2015). Pharmacological inhibition of the NLRP3 inflammasome has shown to alleviate the symptoms, such as reduction of cerebral edema and neuronal cell death, and prevent long-term outcomes associated with TBI, such as improving motor and cognitive functions (Ismael, Nasoohi, et al., 2018; Kuwar et al., 2019; Lin et al., 2017; Xu et al., 2018; Yan et al., 2020).

3.6. Stroke

Stroke is an acute and severe cerebrovascular disorder that occurs when there is either a clot preventing blood flow to the brain (ischemic stroke) or excessive bleeding in the brain (hemorrhagic stroke) (Stroke - What Is a Stroke?, NHLBI, NIH, 2023). Either of these cases leads to damage of the brain cells, subsequently cognitive deficits, disability, and even brain death if not treated immediately. Signs of stroke progression can include slurred speech, muscle weakness on one side of the body, and severe headache (Stroke—Symptoms and causes, n.d.). More than 795,000 people in the United States and 15 million people worldwide have a stroke every year, about 87% of which are ischemic strokes (Stroke Facts & Statistics,” 2023; WHO EMRO, 2024). Stroke is the 5th leading cause of death in the United States and known risk factors include increased age, high blood pressure, obesity, and tobacco use (Stroke Overview, National Institute of Neurological Disorders and Stroke n.d.).

The involvement of the NLRP3 inflammasome in stroke progression has been documented over the years (Abulafia et al., 2009; Bellut et al., 2023; Ismael, Zhao, Nasoohi, & Ishrat, 2018; Lemarchand et al., 2019). Increased levels of NLRP3 and downstream effector proteins were seen in post-mortem brain samples of stroke patients, as well as in ischemic stroke rat models (Chen, Wang, Yao, & Lin, 2022; Li, Liu et al., 2021; Yang-Wei Fann et al., 2013). Additionally, this elevation was also seen in transient middle cerebral artery occlusion (tMCAO) mice within 24 h post-ischemic injury (Franke et al., 2021). Elevated level of NLRP3 in ischemic stroke patients was found to correlate with cases of malignant brain edema, a massive swelling of the brain that results in rapid degeneration and death (Wang et al., 2021; Wu et al., 2018). Furthermore, studies with NLRP3 knockout mice have demonstrated decreased brain swelling and improved neurological functions within 72 h in a intracerebral hemorrhage (ICH) mouse model (Ma et al., 2014). Deletion of downstream effectors IL-1α and IL-1β has also been shown to reduce ischemic injury and edema compared to wild-type tMCAO mice (Boutin et al., 2001). Pharmacological inhibition of the NLRP3 inflammasome also led to beneficial effects in several stroke animal models (Bellut et al., 2023; Franke et al., 2021; Ismael, Zhao, et al., 2018; Ito et al., 2015; Wang, Yao et al., 2020).

3.7. The NLRP3 inflammasome and gut microbiota

Studies have provided evidence to support a central role of the NLRP3 inflammasome to the microbiota–gut–brain axis, affecting the bidirectional communication between the gut and brain. Research has shown that imbalance in gut microbiota by acute-on-chronic alcohol consumption in mice causes gut dysbiosis, leading to NLRP3 inflammasome activation, neuroinflammation, and increased inflammatory markers, e.g., IL-18 and IL-1β, in both the gut and brain (Lowe et al., 2018). HIV-induced NLRP3 activation through gut microbiota imbalance has been identified as a factor worsening ischemic stroke outcomes and impairing post-stroke recovery (Torices et al., 2023). Additionally, dysregulation of gut microbiota and NLRP3 upregulation have been linked to postoperative cognitive dysfunction (POCD) due to sevoflurane anesthesia, with similar mechanisms contributing to neuroinflammation and cognitive decline (Han et al., 2024). Chronic ethanol exposure-induced dysbiosis and NLRP3 upregulation are associated with neuroinflammation and depressive-like behavior, suggesting new treatment avenues for CEE-related psychiatric disorders (Huang et al., 2023). Furthermore, treating early-stage 3xTg AD mice with SLAB51 probiotics improved gut microbiota, reduced inflammatory cytokines, and restored key neuronal pathways, leading to decreased cognitive decline, less brain damage, and lower Aβ accumulation, underscoring microbiota modulation as a promising strategy to slow AD progression (Yang, Wang, Chen, Guo, & Dong, 2023).

4. Therapeutic Significance of NLRP3

Owing to the complex signaling pathway of NLRP3, multiple strategies such as suppressing upstream/downstream events, preventing inflammasome complex formation, inhibiting caspase-1, P2X7 receptor or GSDMD cleavage can be employed to treat NLRP3-mediated diseases. Although IL-1β is a potent pro-inflammatory cytokine released from the activation of the NLRP3 inflammasome, inhibiting it alone may not be an ideal approach because this will only block one pro-inflammatory cytokine while failing to address other inflammatory factors and processes involved in NLRP3-driven diseases. Targeting downstream caspase-1 can lead to a broader disruption of immune regulations, as caspase-1 is crucial for the function of multiple inflammasomes such as NLRC4 and AIM2. This could increase the risk of infections. While caspase-1 inhibitors, e.g., VX-740 and VX-765, showed efficacy in preclinical animal models and early clinical trials, they were discontinued due to concerns about hepatotoxicity despite not showing major side effects initially (Dhani, Zhao, & Zhivotovsky, 2021). Consequently, efforts have shifted towards targeting the NLRP3 inflammasome specifically. Over the past decade, the growing understanding of the pathophysiological roles of the NLRP3 inflammasome has led to the development of several chemical entities with promising potential for clinical translation. In our previous review (Xu, Biby, Kaur, & Zhang, 2023), we thoroughly examined the development of NLRP3 inhibitors. Here, we build upon that foundation by exploring the latest advancements and ongoing clinical trials.

4.1. Small Molecule NLRP3 Inhibitors in Clinical Trials

The anti-diabetic drug glyburide, a sulfonylurea compound, was identified to selectively inhibit NLRP3-mediated IL-1β secretion and pyroptosis, while sparing other inflammasomes like NLRP1b and NLRC4 (Juliana et al., 2010). Subsequently, MCC950 or CP-456773, emerged as a potent inhibitor of the NLRP3 inflammasome (Primiano et al., 2016). Its selectivity for NLRP3 has positioned MCC950 as a crucial tool for studying and treating various NLRP3-mediated inflammatory diseases (Bakhshi & Shamsi, 2022). Mechanistically, MCC950 binds reversibly to the NACHT domain of NLRP3, preventing inflammasome activation and reducing pro-inflammatory cytokine secretion (Coll et al., 2019). However, MCC950 was found to be less effective against cryopyrin-associated periodic syndrome (CAPS) driven by NLRP3 gain-of-function mutations, requiring higher doses to suppress IL-1β and IL-18 (Vande Walle et al., 2019). MCC950 has been extensively tested and showed efficacy in multiple disease models including atopic dermatitis, psoriasis, AD, EAE, MS, among others. Notably, MCC950 had previously undergone Phase II clinical trials for rheumatoid arthritis (RA); however, at high doses (1200 mg total daily dose, >100 μM plasma C_max), it caused elevation of transaminases and hepatotoxicity (Could an NLRP3 inhibitor be the one drug to conquer common diseases?, 2020). Given the promising results obtained from studies using MCC950 as a tool compound, a series of analogs have been developed as safer and more efficacious NLRP3 inflammasome inhibitors, some of which have successfully entered clinical studies (Table 1).

Table 1.

NLRP3 inhibitors in clinical trials.

Inhibitor	Mechanism of action	Conditions	Clinical stage	Brain- penetrant
MCC950 based NLRP3 inhibitors
	Directly NLRP3 inhibitor NACHT domain	Atopic dermatitis, psoriasis, EAE, MS	Rheumatoid arthritis Phase II clinical trials	N/A
	NLRP3 inflammasome inhibitor Unknown binding site	UC, Parkinson’s disease, CAPS, Pulmonary disease	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)	N/A
	Unknown binding site	CAPS	Phase 2 clinical study, CAPS (NCT05186051)	N/A
	Directly NLRP3 inhibitor NACHT domain	Acute peritonitis, Gouty arthritis,	unknown	N/A
	Unknown binding site	CAPS, PD	Phase I clinical trials for safety and tolerability (NCT04015076) Phase IIb clinical trials to treat CAPS (EudraCT2020-000489-40)	Y
	Unknown binding site	Neuroinflammatory diseases	Phase I trials for brain penetration and PK/PD profiles (NCT06129409) Phase Ib/IIa study in Parkinson’s disease.	Y
Non MCC950 based NLRP3 inhibitors
	Inhibits ATPase activity Unknown binding site	AD, EAE, colitis, arthritis	Phase II clinical trials to treat acute gouty arthritis (EudraCT 2016-000943-14)	N/A
	Disrupts NLRP3-NLRP3 oligomerization through NACHT domain binding	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)	N/A
	Covalently binds to Cys279 of NACHT domain and reduces NLRP3-NEK7 interaction	T2DM, liver fibrosis, IBD, peritonitis, hearing loss, pleurisy	Phase IV trial for percutaneous coronary intervention (NCT05130892)	N/A
	Covalently binds to Cys409 of NACHT domain and reduces NLRP3-NEK7 interaction	EAE, DSS colitis	Phase III clinical trials adjuvant treatment of small cell lung cancer with platinum (NCT03699956)	N/A
Undisclosed NLRP3 inhibitors
VTX2735	Unknown binding site	Systemic inflammation	Phase II clinical trials to treat cryopyrin associated periodic syndrome (NTC05812781)	N/A
VTX3232	Unknown binding site	PD, MS, AD and ALS	Phase II trials	Y
Somalix	Unknown binding site	Inflammatory diseases	Phase I clinical trials of inflammatory diseases	N/A
DFV-890	Unknown binding site	FCAS, Symptomatic knee osteoarthritis, Cardiovascular diseases, COVID-19, CARDS	Phase II clinical trials for the treatment of FCAS (NCT04868968) symptomatic knee osteoarthritis (NCT04886258) and cardiovascular diseases (NCT06031844, NCT06097663), COVID-19, (NCT04382053) CARDS	N/A
VTX3232	Unknown binding site	PD, MS, AD and ALS	Phase II trials	Y
VENT-01	Unknown binding site	Nonalcoholic steatohepatitis, chronic kidney disease, and other cardiometabolic conditions	Phase I trials for PK/PD and safety	Y
VENT-02	Unknown binding site	Neuroinflammation, Neurodegeneration	Phase I trials for PK/PD, safety, and tolerability	Y
NT-0249	Unknown binding site	Inflammatory disorders	Phase I clinical trials for safety and tolerability	N/A

Selnoflast, initially developed by Inflazome, is an orally available NLRP3 inhibitor and has completed a Phase I clinical trial for safety and tolerability in patients with CAPS that affects both CNS and peripheral organs. In a Phase Ib study in 19 ulcerative colitis patients, despite showing effective NLRP3 inhibition and reduced IL-1β production, selnoflast did not significantly impact plasma IL-18 levels or IL-1 based colon inflammation. It was deemed safe but unlikely to improve ulcerative colitis (Klughammer et al., 2023). Another trial is ongoing with 72 early PD patients focusing on adverse events, suicidality, pharmacokinetics, and brain neuroinflammation, and is expected to conclude in January 2025 (Selnoflast, ALZFORUM, n.d.). ZYIL1, another orally available NLRP3 inhibitor, was developed by Zydus and has achieved a positive proof-of-concept in its Phase 2 clinical study in patients with CAPS without abnormalities observed in liver and kidney function (NCT05186051) (Hissaria et al., 2024). Emlenoflast, previously known as IZD174 or Inzomelid, has been acquired by Roche from Inflazome UK Ltd., and is an orally available and brain penetrant NLRP3 inhibitor. Phase 1 interventional studies assessing the safety, efficacy, pharmacodynamics, and pharmacokinetics (PK) of emlenoflast in patients with CAPS were completed in 2020. The results revealed that emlenoflast is safe and well tolerated. One CAPS patient demonstrated rapid improvement in clinical parameters after taking emlenoflast, though the specifics were not detailed. In April 2020, a phase 1 dose-escalation study for Parkinson’s disease was registered. However, it was withdrawn due to the strategic decision by the sponsor (NCT04338997). With the concept of prodrug, NodThera released NT-0796 as a brain penetrant and highly potent NLRP3 inhibitor, in which the isopropyl ester facilitated NT-0796 to cross BBB, and then undergoes intracellular conversion to the parent compound. NT-0796 has completed its Phase 1 study confirming brain penetration with excellent PK properties (NCT06129409) in 2022 (NodThera Announces Positive Phase 1 Study Readouts for the NLRP3 Inflammasome Inhibitors NT-0796 and NT-0249, 2022). Recently, NodThera reported positive Phase Ib/IIa trial results confirming that NT-0796 reverses neuroinflammation by reducing IL-1β, IL-6, CCL2, CXCL1 and CXCL8 (key biomarkers) in PD patients (NodThera’s NLRP3 Inhibitor NT-0796 Reverses Neuroinflammation in Parkinson’s Disease Phase Ib/IIa Trial, 2022).

OLT-1177, which features a β-sulfonyl nitrile skeleton, was tested to treat gout flare in phase IIa trials. The results indicated its satisfactory safety profile and efficacy in the reduction of joint pain (EudraCT Number, 2016–000943-14—Clinical trial results—EU Clinical Trials Register, n.d.). Notably, OLT1177 reaches effective plasma levels in vivo greater than 100-fold of those needed to inhibit the NLRP3 inflammasome in vitro (Marchetti et al., 2018). Currently, it is under multi-center Phase II/III randomized trial to evaluate the efficacy/safety using tablets in patients with acute gout flare (NCT05658575). Tranilast, a clinical anti-allergic drug, was repositioned as a direct NLRP3 inhibitor that acts on the NACHT domain and inhibits the assembly of NLRP3 inflammasome with the α,β-unsaturated amide moiety as Michael addition receptor (Huang et al., 2018). This compound is in Phase II clinical trials, currently under recruiting phase to investigate its effects on treating CAPS (NCT03923140). Tranilast has completed Phase IV trials for the treatment of high-sensitivity C-reactive protein (hsCRP), percutaneous coronary intervention (PCI) (NCT05130892). The natural product, Oridonin, also features a Michael addition receptor that reacts with Cys279 of the NACHT domain to interfere the interaction between NLRP3 and NEK7, ultimately inhibiting NLRP3 inflammasomes activation (H. He et al., 2018). It has completed the Phase IV trial for percutaneous coronary intervention (NCT05130892). Irreversible inhibitor RRx-001 that binds to Cys409 of NLRP3 and blocks NLRP3-NEK7 interaction has been shown therapeutic efficacy in a range of mouse models, including DSS-induced colitis, LPS-induced systemic inflammation, and EAE (Y. Chen et al., 2021).

Somalix, a peripherally restricted inhibitor, was found safe and well-tolerated in Phase I clinical trials supporting the progression to Phase II. (Inflazome’s Somalix Demonstrates Positive Safety, Tolerability and Pharmacodynamic Profile in its Phase I Study, 2020). However, no recent updates have been reported on this candidate. DFV-890 is an orally administered NLRP3 inhibitor that was acquired by Novartis from IFM Tre. The Phase II clinical trial for familial cold autoinflammatory syndrome (FCAS) (NCT04868968) has been completed. The trial for symptomatic knee osteoarthritis (NCT04886258) is currently recruiting, and studies on inflammatory marker reduction in adults with coronary heart disease and elevated hsCRP (NCT06031844) and clonal hematopoiesis of indeterminate potential (CHIP) (NCT06097663) are both actively recruiting participants. It has also completed a clinical trial (NCT04382053) for COVID-19 patients with pneumonia and respiratory impairment (ClinicalTrials.gov, n.d.). VTX2735 was developed by Ventyx Biosciences for systemic inflammation. A Phase II trial of VTX2735 was completed in patients with CAPS (NCT05812781), and the trial observed reductions in inflammatory biomarkers, which aligned with improvements in disease activity (Ventyx Biosciences Reports Clinical Data for its NLRP3 Inhibitor Portfolio and Provides Pipeline Updates at Virtual Investor Event, 2024). VTX 3232, also developed by Ventyx Bioscience, is an orally available and brain penetrant NLRP3 inhibitor, and has shown efficacy in a range of animal models of neurodegenerative diseases, including PD, MS, AD and ALS (Initiation of Dosing Begins in Phase 1 Trial of VTX3232, an Innovative CNS-penetrant NLRP3 Inhibitor, by Ventyx Biosciences, 2023). The Phase I trial of VTX3232, which included single and multiple ascending doses, was completed in healthy adult volunteers to evaluate its safety, PK, and pharmacodynamics. The company announced the initiation of dosing for phase II trials in 2023, but results are not reported. VENT-01 and VENT-02 were discovered by Ventus, from which VENT-01 was developed as a peripherally restricted NLRP3 inhibitor for the treatment of nonalcoholic steatohepatitis, chronic kidney disease, and other cardiometabolic conditions, while VENT-02 was developed as a brain-penetrant NLRP3 inhibitor (Pipeline—Ventus Therapeutics, 2023). NodThera’s second clinical compound, NT-0249, a peripherally restricted NLRP3 inhibitor, completed its safety and tolerability study in Phase I single ascending dose clinical study. The multiple-ascending dose (MAD) cohorts of NT-0249 demonstrate a potentially best-in-class PK/pharmacodynamic profile, suitable for once-daily dosing, thereby confirming a low clinical dose for efficacy. In healthy volunteers, reductions in key inflammatory biomarkers, C-reactive protein (CRP) and fibrinogen, were maintained throughout treatment with NT-0249.

4.2. Small Molecule NLRP3 Inhibitors under preclinical investigation

4.2.1. MCC950 derivatives

As shown in Fig. 4, numerous NLRP3 inhibitors have been developed based on the structure of MCC950, which can be divided into three key parts: the tricyclic moiety, the sulfonylurea group, and the 4-(1-hydroxy-1-methylethyl)furan-2-yl motif. Structural modifications have been extensively investigated in these three regions to understand the chemical space and to develop effective inhibitors. Overall, although the modifications led to active compounds, they are in general less potent than MCC950.

Fig. 4.

Derivatives of MCC950.

The hexahydroindacene moiety of MCC950 contains two cyclopentane rings that are susceptible to metabolic processes, producing hydroxylated metabolites (Salla et al., 2016). Therefore, a wide range of modifications have been explored on the tricyclic moiety. Varying the size of one of the cyclopentyl rings to cyclobutyl ring formed compound 2 with an IC₅₀ in the range between 0.04 to 0.2 μM in PMA differentiated THP-1 cells (Roush et al., 2020). Replacement of the hexahydroindacene ring with a substituted imidazole ring led to an active compound 3 with IC₅₀ ≤ 0.25 μM (Macleod, Shannon, Thom, Arregui, & Alanine, 2020). Studies have revealed that the aryl ring within the hexahydroindacene moiety is crucial for its binding in the hydrophobic pocket of the NLRP3 protein. To support this notion, replacing the hexahydroindacene moiety with a 3,5-dimethylbenzene led to an active compound (4) showing 98.3% inhibition on IL-1β release at 1 μM concentration. Further studies revealed an IC₅₀ of 0.23 nM in LPS primed BMDMs cells and was found to exhibit significant anti-inflammatory effects in vivo (Narros-Fernández et al., 2022). However, removal of one methyl group as shown in compound 5 made it inactive compound, suggesting importance of both methyl groups.

The search for NLRP3 inhibitors by removing the furan toxicophore and incorporating different aryl and heteroaryl moieties led to the discovery of compounds 6–18. These findings suggest that structural modifications on the furan ring are well-tolerated, resulting in potent inhibitors (O’Neill, Coll, Cooper, Robertson, & Schroder, 2016; O’Neill et al., 2017; Cooper et al., 2019; Mohan, Nuss, Harris, & Yuan, 2021; Qian et al. 2021). Guided by lipophilic ligand efficiency (LLE), compound 17 and GDC-2394 (18) with comparable potency but lower lipophilicity compared to MCC950 were discovered (Fig. 4). Compound 17 showed renal toxicity in monkeys, prompting solubility improvements with a basic amine, resulting in GDC-2394 (18), which showed minimal renal or hepatic toxicity at high doses (McBride et al., 2022). Despite favorable PK and target engagement, phase I studies of 18 revealed severe drug-induced liver injury in two participants, thus leading to the halt of the trial and precluding its further development (Tang et al., 2023).

The sulfonylurea moiety in MCC950 is a crucial pharmacophore for binding to NLRP3 which is facilitated through hydrogen bonding interactions. Keeping sulfonylurea as a core, extensive modifications were reported by changing the hexahydroindacene ring and the furan moiety of MCC950. Replacement of one of the cyclopentyl rings with cyclopentanone (20) as well as substituted pyridine (21) did not improve the activity in comparison to 19 (Figs. 4-5). Introduction of halogen on the hexahydroindacene moiety (22-23) as well as replacement of one of the cyclopentane rings with an isopropyl group (24) or dihydrofuran (25) resulted in compounds with IC₅₀ ≤ 0.5 μM.

Fig. 5.

Derivatives of MCC950 with modifications of the hexadroindacene and furan moieties.

The results of 26–28 suggested the importance of the 2,6-diisopropylphenyl moiety in maintaining the activity as NLRP3 inhibitors of this scaffold (Cooper et al. 2022). Furthermore, replacement of the hexahydroindacene ring with other heterocyclic rings such as furan (29–30) or thiophene (31–32) led to compounds with an IC₅₀ < 1 μM (Bock, Harrison, & Scanlon, 2020) (Figs. 4 and 5). Replacement of the furan moiety of MCC950 with a 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine ring led to a compound (34) with comparable potency as MCC950. It has been also demonstrated that the hydrophobic pocket can tolerate large substituents on fluorenyl in compound 35 without compromising activity. Replacement of the hexahydroindacene ring with naphthalene in 36 led to a 61.2% inhibition of caspase-1 whereas replacement with an indole ring in compound 37completely lost the inhibitory activity (Kim, Im, Han, & Cho, 2024) (Fig. 4).

Bioisosteric replacement strategies were used to create diverse mimics of the sulfonylurea moiety while maintaining crucial hydrogen bond patterns (Fig. 6). To investigate the importance of the acidic center of sulfonylurea, Nodthera reported various compounds with a hexahydroindacene ring connected, via an amide linkage, to a carboxylate (38), ester (39), or sulfonamide group (40-41) (Harrison et al., 2020) (Fig. 6). None of these modifications resulted in active compounds. In contrast, compounds with a hexahydroindacene moiety linked to a carboxylic ester via a urea linkage as evidenced in 42, showed an IC₅₀ value of 6.8 μM in PBMC assay under LPS/ATP condition. The inhibitory activity can be maintained with an ethyl ester (43) or isopropyl ester (44) but was diminished with a tert-butyl group (45). Change of the ester moiety to an amide (46-47) or oxadiazole rings (48-49) led to loss of activity. These observations led to the hypothesis that the ester group may help in the permeability of compounds across the cell membrane which subsequently get converted into active carboxylic acid containing compounds via intracellular carboxylesterase enzymes. Substitution of compound 42 at the alpha-position of the ester led to compounds with comparable potency (50-56). Notably, a substitution with pyrimidine or pyrazine gave compounds with significantly improved potency (57 and 58). N-cyano sulfoximine urea analogue 59 was found to be potent NLRP3 inhibitor (IC₅₀ = 7 nM) along with good PK profile (Agarwal et al., 2020). Compound 60 with 4-isopropylbenzamide moiety showed an IC₅₀ of 30 nM in BMDMs cells under LPS/ATP stimulation (Li, Cao et al., 2021). NDT-30805 (61) with a triazolopyrimidine moiety was also found to be a potent NLRP3 inhibitor with good selectivity and solubility profile (Harrison et al., 2022).

Fig. 6.

Derivatives of MCC950 with bioisosteric modifications of the sulfonylurea.

4.2.2. Glyburide based NLRP3 inhibitors

Based on the structure of glyburide (62), JC-121 (63) (Fig. 7), was discovered with inhibitory activity on the NLRP3 inflammasome while no impact on glucose metabolism (Marchetti et al., 2014). To improve solubility of JC-121, JC-171 (64) with a hydroxamic acid on the sulfonamide moiety was developed with comparable activities (Guo et al., 2017). This compound was also demonstrated in vivo efficacy in an EAE mouse model to decrease IL-1β production and inhibit the pathogenic Th17 response. JC124 (65), a methylated analogue of JC-121 was developed as another NLRP3 inhibitor and has been shown in vivo efficacy in animal models of TBI (Kuwar et al., 2019), acute myocardial infarction (Fulp et al., 2018) and AD (Yin et al., 2018). Structure-activity relationship (SAR) studies of JC124 led to the discovery of 66 and 67, with improved potency (Fulp et al., 2018). Structural hopping of JC124 resulted in a new scaffold exemplified by HL-16 (68) with comparable potency on the NLRP3 inflammasome. Further modifications of this new scaffold led to the discovery of YQ128 (69) and MS-II-124 (70) with significantly improved potency and binding affinity (Yiming Xu, Biby, et al., 2024). The most recent modification of JC124 led to another series of analogues with 1,2,3-triazole scaffold exemplified with YM8 (71) with promising potency and binding affinity (Yiming Xu, Xu, et al., 2024).

Fig. 7.

Glyburide derivatives.

4.2.3. PET radiotracer

To help gain a deeper understanding of the mechanistic pathway of NLRP3 and its pathological roles in CNS disorders, positron emission tomography (PET) radiotracers based on the reported NLRP3 inhibitors have been attempted. Initially, [¹¹C]MCC950 (72) was developed as the first PET radiotracer to analyze brain uptake and investigate the target engagement of MCC950 (Hill et al., 2020) (Fig. 8). Unfortunately, this PET radiotracer showed poor brain uptake in mice, rats, and rhesus monkeys, with rapid washout occurring shortly after administration. Recently, [¹¹C]YQ128 (73) was developed and studies in mice demonstrated rapid but moderate brain uptake with a maximum percentage of injected dose per cubic centimeter (ID/cc) ~1.7 (Yulong Xu et al., 2021). A F-18 labeled radiotracer [¹⁸F]MS124 (74) showed improved brain uptake (ID/cc: 2.5%) in mice but without substantial washout over a scan period of 60 min (Yiming Xu et al., 2022). [¹¹C]MNS (75), synthesized from piperonal by Ogata et al., has been reported recently (Ogata et al., 2023). But the observed low brain penetration and rapid washout, coupled with the lack of specific binding to NLRP3 in the brain, limited its further development. Most recently, a radiotracer based on the triazole scaffold derived from YM8 (71), [¹¹C]YM8 (76), demonstrated significantly improved brain uptake with a %ID/cc of 2.7 in mice and specific binding to the NLRP3 as demonstrated by autoradiography studies using wild-type and NLRP3 knock out mouse brain tissues as well as competition studies by the parent compound YM8 and MCC950. Notably, this radiotracer has been shown promising brain uptake and kinetic properties in rhesus macaques with a standardized uptake value (SUV) > 3 by PET/MR studies, encouraging further development of this radiotracer in preclinical and clinical studies (Yiming Xu, Xu, et al., 2024).

Fig. 8.

NLRP3 PET radiotracers.

5. Challenges in the development of NLRP3 inhibitors

Targeting NLRP3 in the brain for treating neurological disorders presents a formidable hurdle due to the BBB’s inherent impermeability. Despite the discovery of potent NLRP3 inhibitors, their CNS efficacy is hampered by poor penetration through this barrier, rapid clearance, and concerns such as liver toxicity. These challenges complicate the clinical development of NLRP3 inhibitors as effective treatments for neurological conditions. Novel approaches are therefore needed to facilitate the transport of these inhibitors across the BBB, enabling them to reach the brain and exert CNS therapeutic effects. Nanotechnology has emerged as a promising avenue for delivering NLRP3 inhibitors across the BBB (Ahmad et al., 2024; Chaturvedi, Naseem, El-Khamisy, & Wahajuddin, 2022; Manna et al., 2023; Nandi et al., 2024). However, the use of engineered nanoparticles like carbon nanotubes (CNTs), graphene oxide (GO), rare earth nanoparticles (REOs), cellulose nanocrystals, and V₂O₅ has demonstrated unintended activation of NLRP3 due to their physical properties such as composition, charge, thickness, length, shape, crystallinity, suspension state, and surface characteristics (Liao et al., 2022). Incorporating antioxidants into polymeric nanoparticles, which are more stable than metallic ones, can provide controlled release and selective targeting but tend to aggregate and show neurotoxicity (Alqudah, Aljabali, Gammoh, & Tambuwala, 2024). Efforts to manage their size, charge, and morphology, along with PEG coating and have been attempted to design specific ligands that aim to enhance selective targeting of NLRP3 while avoiding cellular interactions in the brain (Zhang, Mehta, Tong, Esser, & Voelcker, 2021). Therefore, it is crucial to carefully consider factors like lipophilicity, molecular weight, charge, BBB permeability, and partition coefficient when designing NLRP3-targeting drugs. Given that NLRP3 belongs to the broader family of NOD-like receptors, developing inhibitors with high specificity for NLRP3 while sparing other related proteins is essential to avoid off-target effects. Since NLRP3 also plays a role in immune responses, inhibiting its function can potentially lead to immunosuppression, heightening susceptibility to infections and other immune-related complications. Hence, it is imperative to ensure that NLRP3 inhibitors selectively block its pathological activation without compromising its physiological roles in host defense mechanisms. Moreover, NLRP3 is implicated in various diseases, each with distinct pathological mechanisms. Developing inhibitors that are effective across multiple disorders or tailoring specific inhibitors for different conditions introduces additional complexity. Identifying reliable biomarkers to assess the efficacy of NLRP3 inhibitors in patients is critical for guiding clinical trials and eventual clinical applications. Selecting appropriate patient populations for trials poses a challenge due to the variability in disease presentation and progression among individuals.

6. Conclusion

Since the discovery of NLRP3 over two decades ago, our understanding of the NLRP3 inflammasome pathways and their diverse disease implications have advanced rapidly, catalyzing new avenues in research and propelling translational efforts. Results from structural biology studies of the NLRP3 inflammasome have provided valuable insights to the activation mechanisms and small molecule inhibitor binding interactions, all instrumental to facilitate drug discovery efforts. Recent endeavors in developing brain penetrant NLRP3 inhibitors have led to compounds into clinical studies for CNS indications. Therefore, it is expected that the drug development pipelines for devastating neurodegenerative disorders will be expanded by novel NLRP3 inhibitors. Moreover, the exploration of new drug delivery systems, such as safe nanoparticle delivery, are being actively attempted to optimize PK properties and minimize potential side effects. Rigorous clinical trials are imperative to affirm the therapeutic potential of NLRP3 inhibitors in neurodegenerative diseases, underscoring the critical need for biomarkers to confirm target engagement and drug efficacy. To this end, NLRP3 PET radiotracers will be valuable imaging probes to help both diagnostic and therapeutic agents’ development. Furthermore, the PET radiotracer discovery will be greatly accelerated by the growing pool of small molecule NLRP3 inhibitors given that different PK/PD properties are needed for CNS PET tracers.

Abbreviations

AD

Alzheimer’s disease.

AIM2

absent in melanoma 2.

ALRs

AIM2 like receptors.

ASC

Apoptosis-associated speck-like proteins containing CARD.

BBB

blood brain barrier.

BMDMs

bone marrow-derived macrophages.

CAPS

cryopyrin-associated periodic syndrome.

CASP1

Caspase-1.

cGAS

cycle GMP-AMP synthase.

CNS

central nervous system.

CARD

Caspase-recruitment and activation domain.

Cryo-EM

cryogenic electron microscopy.

GSDMD

Gasdermin D.

GSDME

Gasdermin E.

HD

Helical domain.

IFNs

type I interferons.

NBD

Nucleotide-binding domain.

NEK7

Never in mitosis gene A–related kinase 7.

NLRC4

NLR family caspase activation and recruitment domain-containing 4.

NLRs

NOD like receptors.

NLRP3

NOD-like receptor family pyrin-domain-containing 3.

oxPAPC

oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine.

PAMPs

pathogen-associated molecular patterns.

PARP1

poly (ADP-ribose) polymerase 1.

PBMC

human peripheral blood mononuclear cell.

PD

Parkinson’s diseases.

PRRs

Pattern recognition receptors.

RLRs

RIG-I like receptors.

TLRs

Toll like receptors.

TWIK2

two-pore domain K+ channel (K2P).

T3SS

type III secretion system.

WHD

winged helix domain.