Ion fluxes (K⁺, Ca²⁺, Cl⁻, and Na⁺) are suggested as one of the major mechanisms that trigger the activation of the NLRP3 inflammasome. Inflammasome activators such as extracellular ATP (via the non-selective cation channel receptor P2X7), particulate matter (aluminum hydroxide, silica, and calcium pyrophosphate crystals), and nigericin (ionophore) can induce K⁺ efflux, a necessary signal for NLRP3 activation (Muñoz-Planillo et al., 2013; Katsnelson et al., 2015). Monosodium urate (MSU) crystals cause cellular swelling from water influx resulting in an intracellular decrease of K⁺, leading to NLRP3 inflammasome activation (Schorn et al., 2011). Quinine, an inhibitor of two-pore domain K⁺ channels can prevent caspase-1 activity in a dose-dependent manner, highlighting the role of this family of ionic channels in inflammasome activation (Di et al., 2018). Conversely, K⁺ channel inhibitors Ba²⁺ (inhibitor of inward-rectifier K⁺ channels), tetraethylammonium (inhibitor of voltage-gated K⁺ channels), and iberiotoxin (inhibitor of large-conductance calcium-activated K⁺ channels) fail to prevent caspase-1 activation in macrophages primed with LPS (Di et al., 2018). Combined, these results leave questions and uncertainty about the role that potassium may have on inflammasome activation. The second messenger inositol 1,4,5-triphosphate (IP₃) appeared to promote inflammasome activation by inducing efflux of Ca²⁺ from the endoplasmic reticulum (ER) through a ligand-gated ion channel, subsequently increasing intracellular concentrations (Murakami et al., 2012). The same study found that either depletion of ER Ca²⁺ by incubation with an inhibitor of sarcoplasmic/ER Ca²⁺-ATPase pump or incubation with Ca²⁺ free media to prevent extracellular Ca²⁺ entry attenuated NLRP3 inflammasome activation in response to ATP, suggesting mobilization of both extracellular and ER Ca²⁺ is required for inflammasome activation. Moreover, blockers of volume-activated Cl⁻ channels that are inactive against K⁺ channels are able to inhibit inflammasome activation, demonstrating the role of Cl⁻ efflux in NLRP3 inflammasome assembly (Compan et al., 2012; Daniels et al., 2016). It’s suggested that Na⁺ influx also plays a regulatory role in NLRP3 inflammasome activation in conjunction with K⁺ efflux induced by different stimuli. Na⁺ influx caused by ionophores cannot activate the NLRP3 inflammasome, however, lysosomal delivery of MSU crystals causes an increase in intracellular Na⁺ and water influx, subsequently lowering intracellular K⁺ concentration and activating the NLRP3 inflammasome (Muñoz-Planillo et al., 2013). More recently, the use of amiloride, a common epithelial Na⁺ channel blocker, can reduce cytokine secretion and caspase-1 activity in primary monocytes (Scambler et al., 2019).

Mitochondrial reactive oxygen species (mtROS) is one of the first discovered activators of the NLRP3 inflammasome and is produced during mitochondrial damage, stress, or dysfunction. Early studies suggested the role of mtROS in NLRP3 inflammasome activation when they noted the inhibition of NADPH oxidase-dependent mtROS prevents ATP-induced caspase-1 activation and IL-1β production in macrophages (Cruz et al., 2007). In addition, downregulation of voltage-dependent anion channels (VDAC), which are responsible for regulating mtROS homeostasis, results in decreased mtROS, caspase-1 activation, and IL-1β release in THP-1 cells (Zhou et al., 2011). Palmitate, a fatty acid commonly found in palm oil, is shown to lead to NLRP3 inflammasome activation in a ROS-dependent manner since the use of the ROS inhibitor APDC blocks palmitate-dependent IL-1β production (Wen et al., 2011). The complete mechanism by which NLRP3 senses mtROS is not well understood. Some studies suggest NEK7 may detect mtROS since it is an NLRP3 modulator (Shi et al., 2016). Supporting this idea, imiquimod, a TLR7 agonist, can activate the NLRP3 pathway and induce IL-1β release in a ROS-dependent manner, but fails to induce IL-1β production in cells deficient in NEK7 (Groß et al., 2016). Other studies have suggested mtROS’s involvement in the priming step, not activation, since some ROS inhibitors are able to block NLRP3 expression (Bauernfeind et al., 2011). Furthermore, mtROS is also implicated to be involved in the deubiquitination of NLRP3, subsequently promoting its activation (Juliana et al., 2012).

After the discovery of mtROS and its potential involvement in the inflammasome pathway, mitochondria in general have shown to play an intricate and important role in the activation of inflammasomes through various mechanisms. Mitophagy, the clearance of damaged mitochondria, is negatively correlated with NLRP3 activation, and deficiency causes the accumulation of dysfunctional mitochondria and the release of mtROS and mitochondrial DNA (mtDNA), both known activators of NLRP3 (Nakahira et al., 2011; Zhou et al., 2011). Further investigation revealed that other NLRP3 inflammasome activators, such as alum and nigericin, cause mitochondrial dysfunction and that oxidized mtDNA directly binds to NLRP3 (Shimada et al., 2012). Mitochondrial fission is also implicated in NLRP3 activation through LPS signaling; although the mechanism remains unclear, it’s suspected that fission facilitates NLRP3 localization and complex assembly (Park et al., 2015). The mitochondrial-specific lipid cardiolipin also plays a direct role in NLRP3 activation by binding to NLRP3 in a ROS-dependent and independent manner (Iyer et al., 2013). In addition, hexokinase, the first enzyme in glycolysis, dissociates from the mitochondrial membrane upon inhibition with bacterial peptidoglycans from Gram-positive bacterial cell walls and consequently activates NLRP3 (Wolf et al., 2016). Mitochondrial dysfunction and stress play a significant role in inflammation in vitro and in vivo (Gong et al., 2018; Mahalanobish et al., 2020; Ko et al., 2021); however, some studies dispute the requirement of mitochondrial dysfunction and the mtROS in NLRP3 activation (Muñoz-Planillo et al., 2013; Allam et al., 2014; Lawlor and Vince, 2014).

Lysosomes are responsible for degrading extracellular material by endocytosis or phagocytosis and degrading or recycling intracellular material via autophagy. Lysosomal damage is considered to play a significant role in NLRP3 activation, and direct evidence of this is shown when the lysosomal damage-inducing dipeptide, L-leucyl-L-leucine methyl ester (Leu-Leu-OMe), is able to activate the NLRP3 inflammasome (Hornung et al., 2008). In addition, NLRP3 activators such as particulate matter (e.g., cholesterol crystals, silica, aluminum salts, uric acid crystals, and asbestos) and misfolded proteins (e.g., amyloid-β) can induce lysosomal damage, indicating an interesting mechanism of NLRP3 activation (Emmerson et al., 1990; Martinon et al., 2006; Halle et al., 2008; Hornung et al., 2008; Duewell et al., 2010; Ito et al., 2020). There are several theories for how lysosomal damage activates NLRP3, one of the most popular being the release of cathepsin proteases, the proteins that mediate lysosomal degradation, into the cytosol where they can have apoptotic or apoptotic-like consequences (Wang F. et al., 2018) In support, genetic and pharmacological inhibition of cathepsin B impairs caspase-1 activation and IL-1β release in response to amyloid-β, Leu-Leu-OMe, and palmitate (Halle et al., 2008; Hornung et al., 2008; Weber and Schilling, 2014). However, no difference in caspase-1 cleavage or IL-1β secretion is observed with genetic or pharmacological inhibition of cathepsin B in response to MSU, hemozoin, alum, or ATP, suggesting a distinct mechanism for activators or cathepsin B (Halle et al., 2008; Dostert et al., 2009). In addition to this, cathepsins L, C, S, and X are also implicated to play a role in inflammasome activation (Orlowski et al., 2015). Other theories on how lysosomal damage induces NLRP3 activation include lysosomal acidification and influencing ion fluxes. Blocking the vacuolar H⁺-ATPase, a proton pump responsible for lysosomal acidification, prevents IL-1β release in response to silica, suggesting acidification is required (Hornung et al., 2008). In addition, lysosomal damage induced by Leu-Leu-OMe can affect K⁺ efflux and Ca²⁺ mobilization, subsequently activating the NLRP3 inflammasome (Murakami et al., 2012; Katsnelson et al., 2016). This also highlights the capability of NLRP3 activation pathways to work in conjunction.

Glyburide is an FDA-approved, anti-diabetic drug that falls into the sulfonylurea class of medications. Glyburide treats diabetes by blocking ATP sensitive potassium (K_ATP) channels in pancreatic β cells (Ashcroft, 2005). Studies have shown that glyburide inhibits NLRP3 activation and IL-1β secretion induced by LPS/ATP in bone marrow-derived macrophages (BMDMs) with an IC₅₀ of 10–20 μM (Lamkanfi et al., 2009; Hill et al., 2017). Lamkanfi et al. (2009) demonstrated that glyburide inhibits caspase-1 activation and IL-1β secretion independently of K_ATP channels and later concluded that glyburide likely targets downstream of the purinergic 2X7 receptor (P2X7R) and upstream of inflammasome formation. Pilot structure-function studies revealed that the cyclohexylurea group is responsible for insulin secretion, whereas the benzamide and sulfonyl moiety are critical for NLRP3 inflammasome inhibition (Hill et al., 2017). Unfortunately, the concentration that glyburide is able to elicit its anti-inflammatory properties can induce hypoglycemia which limits its further development as an NLRP3 inflammasome inhibitor (Coll et al., 2015).

MCC950 is a sulfonylurea compound initially discovered in 2001 under the name CP-424, 174 (Perregaux et al., 2001). The term CRID3 is also used synonymously with MCC950. Recent studies have demonstrated that MCC950 is a potent and selective NLRP3 inflammasome inhibitor that can inhibit IL-1β release in BMDM cells with an IC₅₀ of 7.5 nM (Coll et al., 2015; O’Neill et al., 2016). However, it should be noted that in a 2011 article by Coll et al. (2011), a different compound was incorrectly termed CRID3 and was described to be nonselective and inhibiting both NLRP3 and AIM2 inflammasomes. Mechanistic studies revealed that MCC950 is a direct NLRP3 inhibitor that binds to the Walker B motif of the ATPase binding pocket in the central NACHT domain subsequently inhibiting ATPase activity, a critical step in inflammasome activation (Coll et al., 2019). Further docking studies supported this binding site (Tapia-Abellan et al., 2019; Andreeva et al., 2021; Hochheiser et al., 2021). MCC950 fails to stimulate insulin secretion in vitro; however, a later study found it’s able to increase insulin sensitivity in an in vivo diabetic mouse model, although this was attributed to MCC950’s effects at NLRP3 (Hill et al., 2017; Zhai et al., 2018). In 2017, Hill et al. (2017) synthesized a series of MCC950, sulfonylurea derivatives that can inhibit NLRP3 inflammasome with nanomolar potencies and also inhibit insulin secretion. Since the discovery of MCC950 as an NLRP3 inhibitor, it’s been employed as a chemical tool to understand the pathological roles of the NLRP3 inflammasome in a variety of disease models including AD, atherosclerosis, asthma, allergic airway inflammation, inflammatory bowel disease (IBD), and many more (Dempsey et al., 2017; van der Heijden et al., 2017; van Hout et al., 2017; Ismael et al., 2018; Perera et al., 2018; Robinson et al., 2018; Xu et al., 2018; Zhai et al., 2018; Theofani et al., 2019; Fu et al., 2020; Ren et al., 2020). The commonly used dose in these studies was 10 mg/kg, relatively high compared to its low nanomolar potency. Recent studies of radio-labeled MCC950 PET tracer demonstrate its low CNS penetration, which may limit its development and use as a CNS agent (Hill et al., 2020).

Formerly known as 16673-34-0, JC121 was developed as a sulfonamide derivative of glyburide. JC121 is able to inhibit ASC aggregation, caspase-1 activity, IL-1β secretion, and inflammatory cell death in cardiomyocytes in response to ATP and nigericin (Marchetti et al., 2014). Furthermore, JC121 inhibits the activity of constitutively active NLRP3 in BMDMs from genetically modified mice, suggesting that this compound inhibits downstream inflammasome activation rather than upstream targets (Marchetti et al., 2015). Notably, JC121 does not show hypoglycemia effects in vivo, a concern for its scaffold predecessor glyburide, even at doses as high as 500 mg/kg. This compound exhibits in vivo protective activity in mouse acute myocardial infarction models and preserves cardiac function after ischemic injury (Marchetti et al., 2014, 2015). To overcome the observed solubility issues of JC121, another analog, JC124, was designed that incorporates a methylated sulfonamide into the structure. The results demonstrated that, like JC121, JC124 is also a selective inhibitor of the NLRP3 inflammasome that inhibits IL-1β release with an IC₅₀ of 3.25 μM without significant inhibition on NLRC4 or AIM2 inflammasomes (Fulp et al., 2018). Mechanistic studies employing a photo-affinity probe suggested that JC124 directly interacts with the NLRP3 protein; however, unlike MCC950, JC124 can bind to NLRP3 without affecting the ATPase activity, suggesting a distinct mode of binding for these sulfonamide derivatives (Kuwar et al., 2019). In vivo, JC124 can actively reduce disease pathology and improve functional performance in animal models of TBI, AD, and acute myocardial infarction by engaging the NLRP3 inflammasome (Fulp et al., 2018; Yin et al., 2018; Kuwar et al., 2019). Structure-activity relationship (SAR) studies of JC124 revealed that the sulfonamide moiety can be modified to improve inhibitory potency.

To increase the space for structural optimization, a new chemical scaffold was created based on the structure of JC124 and further medicinal chemistry campaign led to the design of YQ-II-128 (YQ128) as a novel NLRP3 inhibitor (Jiang et al., 2019). YQ128 significantly inhibits NLRP3 mediated IL-1β production in vitro with an IC₅₀ of 0.30 μM in mouse macrophages. Further in vitro studies demonstrated YQ128 as a selective inhibitor of the NLRP3 inflammasome, since it did not inhibit IL-1β production mediated by NLRC4 or AIM2 inflammasomes. In vivo studies also confirmed its selective inhibition on IL-1β production upon LPS challenge in mice. A YQ128 PET tracer showed rapid BBB penetrance with moderate brain uptake; however, preliminary pharmacokinetic studies revealed that YQ128 displays poor oral bioavailability (Jiang et al., 2019; Xu et al., 2021). Ongoing SAR studies are currently underway to improve the potency and pharmacokinetics of these compounds.

Flavonoids are a group of phytonutrients found in a variety of fruits, vegetables, grains, and plants and are well recognized for their neuroprotective, anti-inflammatory, antimutagenic, and antioxidant properties.

X-11-5-27 is a synthetic derivative of daidzein, a natural isoflavonoid found in soybeans, with antioxidant and anti-inflammatory properties. Daidzein has been shown to suppress the secretion of pro-inflammatory cytokines in vitro and it’s suspected that these effects are the result of a variety of targets in the MAPK pathway (Hämäläinen et al., 2007; Liu et al., 2009; Sakamoto et al., 2016). X-11-5-27 inhibits NLRP3 expression and activation, ASC speck formation, and subsequent caspase-1 activation and IL-1β secretion in BMDMs and THP-1 cells (Zhou et al., 2017). It was also observed that X-11-5-27 inhibits NLRP3 through the induction of autophagy, a process that is negatively correlated with NLRP3. Further studies suggested that the inhibitory activity of X-11-5-27 in these studies may be through antioxidant effects by reducing ROS production and protecting mitochondrial function (Zhou et al., 2017).

In 2014, a series of α, β-unsaturated warheads from an electrophilic fragment-based library were reported to irreversibly inhibit the NLRP3 inflammasome (Cocco et al., 2014). Further characterization of one of the compounds, INF4E (compound 9), demonstrated its irreversible inhibition of the NLRP3 protein via its Michael acceptor to form a covalent bond with NLRP3, which subsequently inhibits IL-1β secretion in ATP-induced THP-1 cells. INF4E also inhibits NLRP3’s ATPase and caspase-1 activity, suggesting those as targets. Furthermore, INF4E prevents caspase-1 dependent pyroptosis and attenuates ATP- and nigericin-induced cell death (Cocco et al., 2014). To decrease toxicity associated with these types of compounds, further structural derivatization led to a series of acrylamide analogs using the ligand-merging strategy to combine the Michael acceptor moiety and a sulfonamide moiety. One of the analogs, INF58 (compound 14), irreversibly inhibits NLRP3 ATPase with improved potency (Cocco et al., 2016). Further optimization led to the identification of an ethyl acrylate derivative, INF39 (compound 11), that possesses decreased cytotoxicity and reactivity compared to both INF4E and INF58. Mechanistic studies suggest its direct interaction with the NLRP3 ATPase domain and can inhibit IL-1β production with an IC₅₀ of 10 μM (Cocco et al., 2017). The same study found that INF39 can attenuate NEK7-NLRP3 interactions in HEK293 cells expressing NLRP3 and NEK7, confirming that INF39 blocks NLRP3 inflammasome assembly. INF39 continues to be investigated in the inflammasome pathway, a recent study has further verified INF39’s selective interaction with NLRP3, as it does not suppress AIM2 or NLRC4 inflammasomes, and showed the inhibition of ASC speck formation and the cleavage of caspase-1, IL-1β, and GSDMD proteins in THP-1 cells (Shi et al., 2021). Other studies have continued to derivatize INF39 and investigate the mechanism of binding (Gastaldi et al., 2021).

2-aminoethyldiphenyl borate (2APB), a boron containing compound, is known to inhibit IP₃-mediated calcium release, store-operated calcium entry, and disrupt other calcium-dependent pathways in HeLa cells and cardiac myocytes, which may affect the ion flux balance required to activate NLRP3 (Peppiatt et al., 2003). Further studies demonstrated that 2APB can inhibit nigericin- and ATP-induced NLRP3 inflammasome signaling cascade independently of its effect on calcium flux (Katsnelson et al., 2015). However, 2APB’s effects on calcium flux limits its drug development. Further optimization of 2APB to weaken its effects at calcium flux while strengthening the effects on NLRP3 resulted in compound NBC6 with significantly enhanced potency (IC₅₀ = 574 nM) (Baldwin et al., 2017). NBC6 can inhibit ASC oligomerization and was found to be a potent inhibitor of NLRP3; however, it’s also able to inhibit NLRC4 and AIM2 pathways at higher concentrations. Interestingly and unlike 2APB, NBC6 retained activity after washing away free drug, suggesting it may act as a covalent inhibitor. NBC13, a similar analog to NBC6 with better solubility, inhibited IL-1β release in vivo (Baldwin et al., 2017). NBC compounds have yet to be investigated in disease states.

BAY 11-7082 (BAY) is a phenyl vinyl sulfone and is known for its inhibition of TNF-α induced IKK phosphorylation which subsequently inhibits activation of NF-κB and expression of NLRP3 (García et al., 2005). However, due to its non-specific modification of cysteine residues, BAY shows polypharmacological properties with multiple mechanisms suggested (Lee et al., 2012). In 2010, a study found that BAY reduces NLRP3’s ATPase activity and inhibits ASC oligomerization (Juliana et al., 2010). Upon further investigation, this study found that BAY inhibits NLRP3 activity by irreversibly alkylating cysteine residues in the ATPase region of NLRP3 through a Michael addition mechanism and SAR studies of BAY revealed the importance of the vinyl sulfone in the observed inhibitory activity. BAY can inhibit IL-1β production in BMDM’s with an IC₅₀ of ~5 μM (Jiang H. et al., 2017). Increased ubiquitination of NLRP3 by BAY was also observed which may contribute to its inhibition of the NLRP3 protein by interfering with NLRP3-ASC interactions (Shim et al., 2017). A recent study demonstrated that BAY inhibits pore-formation of GSDMD by covalently modifying the critical cysteine residue C191 which is involved in GSDMD pore-formation (Hu et al., 2018). This study also found that BAY can inhibit canonical and non-canonical caspases. BAY has been investigated in a number of disease states and injuries including psoriasis, diabetic nephropathy, spinal cord injury, and extensively in cancer for its effects on the NF-κB pathway (White and Burchill, 2008; Kolati et al., 2015; Irrera et al., 2017; Jiang W. et al., 2017).

3,4-Methylenedioxy-β-nitrostyrene (MNS) is a well-known Syk and Src tyrosine-kinase inhibitor; however, it is also shown to be a direct, selective inhibitor of the NLRP3 inflammasome (He et al., 2014). MNS can block IL-1β production with an IC₅₀ of 2 μM in BMDMs. Pull-down studies suggest that MNS binds to the LRR and NACHT domains of the NLRP3 protein, and further investigation showed that the MNS inhibits NLRP3 ATPase activity. MNS has no inhibitory activity towards AIM2 or NLRC4 inflammasomes. Moreover, SAR studies of MNS indicated that the nitrovinyl group is essential for its inhibition of NLRP3, whereas the dioxole is dispensable. It’s suspected that the vinyl group may interact with the cysteine side chains in NLRP3, possibly in the ATPase region, making it an irreversible inhibitor (He et al., 2014). A recent study found that MNS induced ubiquitination of NLRP3, which may contribute to its inhibitory mechanism (Shim et al., 2017). MNS has been studied in wound healing and in osteosarcoma tumors (Messerschmitt et al., 2012; Xiao et al., 2016).

Tranilast has historically been approved to treat bronchial asthma in Japan, China, and South Korea. Tranilast was initially found to inhibit NF-κB by interfering with NF-κB and CREB-binding protein (CBP) association, but recent studies demonstrated that tranilast is a selective inhibitor that directly binds to the NLRP3 protein and inhibits IL-1β production with an IC₅₀ of 25–50 μM (Spiecker et al., 2002; Huang et al., 2018). Mechanistically, tranilast binds to the NACHT domain of NLRP3, consequently inhibiting NLRP3-NLRP3 and NLRP3-ASC interactions, but not NLRP3-NEK7, inhibiting inflammasome formation and activation of caspase-1. Unlike other direct NLRP3 inhibitors, tranilast does not inhibit ATPase activity (Huang et al., 2018). Recently, tranilast has been investigated for the treatment of neuropathic pain, gouty arthritis, CAPS, and cancer, and has been suggested to be a potential therapy for vitiligo (Huang et al., 2018; Saito et al., 2018; Moore et al., 2019; Zhuang et al., 2020).

OLT1177 is a β-sulfonyl nitrile compound that selectively blocks the NLRP3 inflammasome by inhibiting its ATPase activity, preventing NLRP3-ASC interactions and IL-1β release in J774A.1 macrophages with an IC₅₀ of ~1 nM. OLT1177 has demonstrated to be a highly potent and selective inhibitor of NLRP3 since it failed to inhibit IL-1β secretion upon activation of NLRC4 or AIM2 inflammasomes (Marchetti et al., 2018a). OLT1177 has shown both in vitro and in vivo success and has recently been developed into an oral tablet and topical gel, Dapansutrile, that has proven to be safe in humans and completed phase II clinical trial for osteoarthritis of the knee by Olatec Therapeutics LLC (Marchetti et al., 2018a). Aside from clinical trials, OLT1177 has recently been tested as a potential treatment for numerous disease states including AD, autoimmune encephalomyelitis, myocardial ischemia, arthritis, and more (Marchetti et al., 2018b; Sánchez-Fernández et al., 2019; Toldo et al., 2019; Lonnemann et al., 2020).

CY-09 has recently been reported to selectively and directly bind to the ATPase domain of NLRP3 and inhibit IL-1β release in BMDM cells with an IC₅₀ of ~6 μM (Jiang H. et al., 2017; El-Sharkawy et al., 2020). Mutating the Walker A motif of NLRP3’s ATPase pocket abolishes the activity of CY-09, whereas mutating the Walker B motif shows no effect, suggesting the Walker A motif of the ATPase pocket as the binding site of CY-09 where it interferes with ATP binding. This is contrary to MCC950 which binds to the Walker B motif of NLRP3. ATP also competes with CY-09 to bind to NLRP3 which further confirms this interaction. Using microscale thermophoresis (MST), CY-09 binds to recombinant NLRP3 with a K_D of 500 nM (Jiang H. et al., 2017). CY-09 has shown to be effective in ex vivo and in vivo models for CAPS, type 2 diabetes, gout, non-alcoholic fatty liver disease (NAFLD), pain, and more (Jiang H. et al., 2017; Fan et al., 2021; Wang X. et al., 2021).

BOT-4-one is a benzoxathiole derivative that exhibits anti-inflammatory activities through several different mechanisms of action due to its ability to irreversibly alkylate its targets. A 2016 study found that BOT-4-one inhibits the NF-κB pathway by alkylating IKKβ, subsequently inhibiting NLRP3 expression (Lee H. G. et al., 2016). Recent studies demonstrated that BOT-4-one inhibited NLRP3 ATPase activity through alkylation (Shim et al., 2017). Furthermore, alkylation of NLRP3 by BOT-4-one can increase ubiquitination of NLRP3, which could contribute to its inhibitory mechanism. While it’s not clear whether alkylation induces ubiquitination or inhibits deubiquitination, other NLRP3 alkylators BAY and MNS also increase NLRP3 ubiquitination. BOT-4-one is able to inhibit IL-1β secretion induced by ATP with an IC₅₀ of 1.28 μM and by nigericin with an IC₅₀ of 0.67 μM. This compound has been investigated in arthritis, pathogenic skin inflammation, and cancer, particularly Hodgkin’s lymphoma, in animal models (Kim et al., 2011, 2016; Lee H. G. et al., 2016).

Methylene blue has a long history of use in medicine, from treatment for methemoglobinemia and septic shock to being used as a dye in surgeries for tissue labeling. Methylene blue is known to have anti-inflammatory, antioxidant, and neuroprotective effects through a variety of mechanisms including its non-selective inhibition of canonical and non-canonical NLRP3, NLRC4, and AIM2 inflammasomes (Ahn et al., 2017). Studies have shown that methylene blue decreases NLRP3, pro-IL-1β, and iNOS expression by interrupting transcription factors NF-κB and STAT1. Methylene blue exhibits no effects on the nuclear transportation of NF-κB and STAT1 but markedly decreases their binding to DNA, thus suggesting methylene blue’s potential interference at binding (Huang et al., 2015). The attenuation of IL-1β and caspase-1 secretion as well as ASC oligomerization when methylene blue is added after LPS suggests that methylene blue inhibits both the priming and activation step of the NLRP3 pathway. In addition, methylene blue also inhibits the activity of recombinant capase-1 (Ahn et al., 2017). Methylene blue has been investigated in inflammation in spinal cord injury, cognitive impairment from neuroinflammation, diabetic retinopathy, AD, and more (Lin et al., 2017; Hao et al., 2019; Soeda et al., 2019; Zhou et al., 2019).

Disulfiram is an FDA-approved drug to treat chronic alcohol dependence through an irreversible cysteine residue modification on aldehyde dehydrogenase. However, it’s recently been discovered that disulfiram contains anti-inflammatory properties. Deng and colleagues found that disulfiram provides lysosomal protection and regulates ROS production that is independent of mitochondria, inhibiting NLRP3-dependent IL-1β secretion with an IC₅₀ of ~5 μM under LPS/ATP conditions in mouse macrophages (Deng et al., 2020). In addition, disulfiram can directly inhibit canonical and non-canonical caspases (Hu et al., 2018). Recent studies demonstrated that disulfiram inhibits GSDMD pore formation by modifying Cys191 of GSDMD and, as a result, suppressing IL-1β secretion and the subsequent pyroptosis (Hu et al., 2020). Others have investigated the potential to repurpose disulfiram to treat several diseases including cancer and bacterial infections and recently has been a suggested as a potential therapy for COVID-19 infection (Viola-Rhenals et al., 2018; Frazier et al., 2019; Fillmore et al., 2021).

Fenamic acid derivatives are known non-steroidal anti-inflammatory (NSAID) agents. While the primary role of NSAIDs is inhibiting prostaglandin synthesis through cyclooxygenase (COX) enzymes, recent studies have shown that fenamic acid derivatives also exhibit anti-inflammatory effects by inhibiting the NLRP3 inflammasome pathway via interference with the Cl⁻ volume-regulated anion channel (VRAC), a regulator of NLRP3 (Daniels et al., 2016). While flufenamic, meclofenamic, and mefenamic acids were able to inhibit IL-1β release in iBMDMs under challenging for the NLRP3 pathway, flufenamic, and mefenamic acid were unable to inhibit IL-1β under NLRC4 and AIM2 challenge, suggesting these acid derivatives are specific to the NLRP3 pathway. In addition, other COX enzyme inhibitors such as Ibuprofen and celecoxib did not affect IL-1β release (Daniels et al., 2016). These fenamic acid derivatives have been studied in a variety of disease models including AD, prostate cancer, and more (Delgado-Enciso et al., 2015; Mangan et al., 2018).

Fluoxetine, also known as Prozac, is an FDA-approved drug used to treat clinical depression. Recently, fluoxetine was found to directly inhibit the NLRP3 protein, stopping inflammasome formation and subsequent IL-1β release in retinal pigmented epithelium (RPE) and macrophage cells (Ambati et al., 2021). In addition, the study demonstrated the physical interaction of fluoxetine with the NLRP3 protein by pull-down. Further binding studies found that a CY-09 probe could also pull-down NLRP3, and that excess fluoxetine was able to compete with the binding of CY-09, an NLRP3 ATPase inhibitor, suggesting these compounds share a similar mode of binding. Both CY-09 and fluoxetine contain a (trifluoromethyl)phenyl moiety and SAR studies suggest that this moiety may assist in the interaction with NLRP3’s ATPase domain. Unlike other antidepressants, fluoxetine reduced RPE degeneration in vivo caused by activating the NLRP3 inflammasome (Ambati et al., 2021). This may open new avenues for the use of fluoxetine; however, more research is needed to prove its potential in different disease states.

β-Hydroxybutyrate (BHB) is an endogenous ligand produced in the liver and functions as an energy source during nutrient deprivation and low-carb diets. During long fasting periods or ketogenic diets, circulating concentrations of BHB can increase which is shown to be correlated with decreased immune response. Youm and colleagues showed that BHB can inhibit the NLRP3 pathway by blocking potassium efflux, an activator of NLRP3 (Youm et al., 2015). The concentrations of BHB that produced these anti-inflammatory effects in vitro were similar to its endogenous level after strenuous exercise or 2 days of fasting. Unfortunately, in vivo administration of BHB alone resulted in rapid clearance; however, when complexed with nanolipogels, BHB retained its anti-inflammatory activity, reducing NLRP3-driven neutrophil infiltration and decreasing IL-1β release at the injection site of MSU crystals (Youm et al., 2015). In LPS-primed BMDMs, BHB can inhibit NLRP3-mediated IL-1β with an IC₅₀ of ~5 μM (Jiang H. et al., 2017). BHB has been studied in stress-related mood disorders, hypertension, seizures, and gout (Goldberg et al., 2017; Yamanashi et al., 2017; Chakraborty et al., 2018; Rho et al., 2019).

MicroRNA are small, non-coding single-stranded RNA molecules that regulate gene expression post-transcriptionally by RNA silencing. These molecules represent an attractive strategy for drug discovery due to their high specificity. In addition, miRNAs have multiple binding sites, also known as seeding regions, which allows them to be involved in multiple pathways. While there are several miRNAs that influence different targets in the NLRP3 inflammasome pathway, there are several miRNAs that target the NLRP3 protein (Boxberger et al., 2019). The first miRNA discovered to target NLRP3 is miRNA (miR)-223-3p which was found to negatively regulate the NLRP3 protein (Bauernfeind et al., 2012; Haneklaus et al., 2012). It’s been proposed that miR-223-3p is important for mediating protein expression in different cell types since differentiation of monocytes to macrophages resulted in a decreased expression of miR-223-3p and increased levels of NLRP3 protein. This could potentially explain why some cell lines have low responses to inflammatory NLRP3 stimuli. MiR-7-5p and miR-30e-5p were also found to negatively regulate NLRP3 protein expression in vitro and in vivo (Zhou et al., 2016; Li et al., 2018). In addition, delivery of miR-7-5p and miR-30-5p mimics were found to protect dopaminergic neurons from degeneration and decrease inflammatory cytokines through inflammasome suppression in an MPTP-induced PD mouse model. Other miRNA’s that negatively regulate NLRP3 protein expression are miR-22-3p, miR-133b-3p, miR-186-5p, and miR-495-3p which have been studied in a variety of disease states including asthma, AD, allergic inflammation, cardiac microvascular endothelial cell injury (CMEC) and more (Xiao et al., 2017; Chen M. L. et al., 2018; Zhou et al., 2018; Han et al., 2020; Guo et al., 2021). There are currently extensive efforts underway to explore the therapeutic potentials of miRNA. However, the major limitation of these molecules is their delivery and membrane permeability. Nanoparticles show promising results in delivering peptides and could provide hope for the future of miRNAs as a therapy to treat numerous diseases (Wang et al., 2015; Sezlev Bilecen et al., 2017).

There are currently three FDA-approved biologics that target the NLRP3 pathway. Anakinra was the first to be developed and is a modified IL-1Ra that blocks the binding of IL-1β and IL-1α, inhibiting their signaling pathways and further inflammation. Anakinra was approved for the treatment of RA in 2001 and was later approved for the treatment of CAPS in 2013 (Calabrese, 2002; Jesus and Goldbach-Mansky, 2014). The short plasma half-life of anakinra (4–6 h) is a pitfall among patients due to required daily injections (Granowitz et al., 1992). Rilonacept is a dimeric fusion protein with decoy receptors containing extracellular residues of the two IL-1R subunits, IL-1R1 and IL-1R accessory protein (IL-1RAcP), which can bind to and neutralize IL-1β and IL-1α (Jesus and Goldbach-Mansky, 2014). With an improved half-life (7 days) compared to anakinra, rilonacept was approved for the treatment of CAPS in 2008 and was recently approved for recurrent pericarditis in 2021 (Hoffman, 2009; Fava et al., 2022). Canakinumab is an anti-IL-1β monoclonal antibody that selectively binds to and neutralizes IL-1β. With a half-life of 26 days, canakinumab is a preferred treatment option and has been approved for CAPS, juvenile idiopathic arthritis, rare periodic fever syndromes, and adult-onset Still’s disease (AOSD) (Curran, 2012; Orrock and Ilowite, 2016; Malcova et al., 2020; Sfriso et al., 2020). In addition, a number of clinical trials have recently investigated anakinra and canakinumab as a therapeutic treatment for patients with COVID-19 (Kooistra et al., 2020; Landi et al., 2020; Kyriazopoulou et al., 2021; Kharazmi et al., 2022). These therapies have proven to be safe and effective in treating inflammation-driven diseases, however, some issues remain. All three biologics require administration by injection, a route that is not preferred by patients and is particularly problematic for anakinra due to its short half-life. In addition, due to the nature of biologics and protein-based therapies, it’s likely that these therapeutics have poor BBB penetrance, potentially limiting their applications to diseases outside of the CNS. Furthermore, increased infection is a concern for therapies blocking all IL-1β signaling regardless of the source, an issue that was observed with canakinumab (Dinarello et al., 2012; Mangan et al., 2018). These problems emphasize the necessity for small-molecule inhibitors of NLRP3 to overcome pharmacokinetic and selectivity issues.