Pharmacological Analysis of NLRP3 Inflammasome Inhibitor Sodium [(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide in Cellular and Mouse Models of Inflammation Provides a Translational Framework

John R Doedens; Pamela Smolak; MyTrang Nguyen; Heather Wescott; Christine Diamond; Ken Schooley; Andy Billinton; David Harrison; Beverly H Koller; Alan P Watt; Christopher A Gabel

doi:10.1021/acsptsci.4c00061

. 2024 Apr 18;7(5):1438–1456. doi: 10.1021/acsptsci.4c00061

Pharmacological Analysis of NLRP3 Inflammasome Inhibitor Sodium [(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide in Cellular and Mouse Models of Inflammation Provides a Translational Framework

John R Doedens ^†, Pamela Smolak ^†, MyTrang Nguyen ^‡, Heather Wescott ^†, Christine Diamond ^†, Ken Schooley ^†, Andy Billinton ^§, David Harrison ^§, Beverly H Koller ^‡, Alan P Watt ^§, Christopher A Gabel ^†,^*

PMCID: PMC11091978 PMID: 38751618

Abstract

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Interleukin (IL)-1β is an apex proinflammatory cytokine produced in response to tissue injury and infection. The output of IL-1β from monocytes and macrophages is regulated not only by transcription and translation but also post-translationally. Release of the active cytokine requires activation of inflammasomes, which couple IL-1β post-translational proteolysis with pyroptosis. Among inflammasome platforms, NOD-like receptor pyrin domain-containing protein 3 (NLRP3) is implicated in the pathogenesis of numerous human disorders in which disease-specific danger-associated molecular patterns (DAMPS) are positioned to drive its activation. As a promising therapeutic target, numerous candidate NLRP3-targeting therapeutics have been described and demonstrated to provide benefits in the context of animal disease models. While showing benefits, published preclinical studies have not explored dose–response relationships within the context of the models. Here, the preclinical pharmacology of a new chemical entity, [(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide (NT-0249), is detailed, establishing its potency and selectivity as an NLRP3 inhibitor. NT-0249 also is evaluated in two acute in vivo mouse challenge models where pharmacodynamic/pharmacokinetic relationships align well with in vitro blood potency assessments. The therapeutic utility of NT-0249 is established in a mouse model of cryopyrin-associated periodic syndrome (CAPS). In this model, mice express a human gain-of-function NLRP3 allele and develop chronic and progressive IL-1β-dependent autoinflammatory disease. NT-0249 dose-dependently reduced multiple inflammatory biomarkers in this model. Significantly, NT-0249 decreased mature IL-1β levels in tissue homogenates, confirming in vivo target engagement. Our findings highlight not only the pharmacological attributes of NT-0249 but also provide insight into the extent of target suppression that will be required to achieve clinical benefit.

Keywords: NT-0249, NLRP3, IL-1β, inflammation, pharmacological inhibition, inflammasome, pyroptosis

Monocytes and macrophages serve key roles in host defense and are equipped with numerous pattern recognition receptors (PRRs) to facilitate the recognition of pathogen-associated molecular patterns (PAMPs). Following the binding of PAMPs to PRRs, monocytes/macrophages rapidly engage inflammatory response pathways, which are essential to innate immunity.^1,2 Once activated, these pathways may lead to the production of proinflammatory cytokines, which can signal neighboring cells of impending danger, and chemokines, which can promote the recruitment of other immune and inflammatory cells to combat the pathogen.³ In some cases, death of the responding cell also may occur.^4,5 Monocytes and macrophages are armed both with intracellular and surface-exposed PRRs. In the former category, Nod-like receptor protein 3 (NLRP3) is activated in response to a number of pathogens, including Toxoplasma gondii, influenza virus, and Candida albicans.⁶⁻⁸ From a therapeutic perspective, however, NLRP3 has garnered interest not only for its role in pathogen defense but also for its ability to engage in response to numerous endogenous danger-associated molecular patterns (DAMPs) that are encountered in human disease states. Examples of sterile DAMPs include monosodium urate crystals, as found in gout; cholesterol crystals, as found in cardiovascular diseases; saturated lipids, as found in metabolic disorders; and aggregates of α-synuclein, as found in Parkinson’s disease.⁹⁻¹²In vitro, LPS-activated monocytes/macrophages treated with sterile DAMPs assemble NLRP3 inflammasome complexes and activate caspase-1. Activated caspase-1, in turn, cleaves pro-interleukin (IL)-1β and pro-IL-18 to generate signaling-competent cytokine species; the corresponding biologically inactive procytokine species accumulate intracellularly as a result of the absence of leader sequences required for entry into the standard secretory apparatus.¹³ Caspase-1 also cleaves gasdermin D, yielding an N-terminal polypeptide, which subsequently transitions from a soluble to a membrane-bound state to form nonselective pore structures.^14,15 The resulting loss of ionic homeostasis leads to the death of the cell (pyroptosis) and the release of mature IL-1β and IL-18 to the medium.^16,17 The multistep nature of this atypical secretory process suggests that the maturation and release of IL-1β and IL-18 are carefully controlled to limit proinflammatory outcomes attendant to these cytokines.

From a clinical perspective, NLRP3′s therapeutic promise was established by the discovery of gain-of-function (GoF) mutations, which are responsible for a rare autoinflammatory disorder termed cryopyrin-associated periodic syndrome (CAPS).¹⁸ Depending on the specific mutation, disease manifestations range from a less severe form, termed familial cold autoinflammatory syndrome, to a more severe form, termed Muckle-Wells syndrome, and the most severe form, neonatal-onset multisystem inflammatory disease.¹⁹ Most NLRP3 GoF mutations leading to CAPS are located within the NACHT domain of the protein. However, it is currently unclear why specific GoF mutations cause more or less severe disease manifestations.²⁰ Importantly, CAPS patients are effectively treated with anti-IL-1β biologicals, supporting the importance of NLRP3 in the generation of mature IL-1β.²¹

The search for small molecule therapeutics to harness NLRP3 activation began in earnest following the realization that a previously identified inhibitor of IL-1β post-translational processing (CP-456,773) is a selective inhibitor of NLRP3 activation.^22,23 CP-456,773 originally was advanced into clinical trials, but its development was halted due to liver safety concerns.²⁴ Recent biochemical, cryo-EM, and X-ray crystallography studies identified CP-456,773 and structural analogues as binding directly to the NACHT domain of NLRP3.²⁵⁻²⁸ A new wave of directed medicinal chemistry efforts have led to the discovery of novel chemotypes (recently reveiwed^29,30), including a series of carboxylate esters, which are highly effective inhibitors of human monocyte NLRP3-induced IL-1β output.³¹ Clinical trials are now underway to evaluate the pharmacokinetics and pharmacodynamics of this new generation of small molecule inhibitors of NLRP3 activation.³²⁻³⁵ One such candidate, GDC-2394, contained a sulfonylurea core as found in CP-456,773 and was also found to carry liver safety concerns when administered at a high dose to healthy volunteers.³² However, other sulfonylurea-containing NLRP3 activation inhibitors have not reported similar liver safety issues in clinical trials,³³⁻³⁵ and a mechanistic understanding of the root cause for the liver toxicity is unknown. Given this uncertainty, a better understanding of the degree of target suppression required to achieve biological benefit is needed to mitigate possible side effects resulting from excessive pathway inhibition and/or compound overexposure. Disease models conducted to date with NLRP3 inhibitors have not explored dose–response relationships aimed at defining the extent and duration of NLRP3 inhibition required to achieve therapeutic benefit.

In this study, we present the preclinical pharmacological profile of a new sulfonylurea-based inhibitor of NLRP3 activation, NT-0249. In addition to establishing selective activity in a panel of cell-based assays, NT-0249 is shown to be an effective inhibitor of NLRP3-mediated IL-1β output when administered orally to wild-type mice subjected to acute peritonitis and lung challenge models. To demonstrate therapeutic utility, NT-0249 is profiled in a syntenic mouse model of CAPS. In this model, genetically altered mice that constitutively express human D305N GoF NLRP3 spontaneously develop inflammatory disease manifestations mimicking those observed in CAPS patients.³⁶ Biochemical analyses conducted on tissues derived from NT-0249-treated D305N mice demonstrated widespread anti-inflammatory impact. Importantly, within the context of the D305N model, NT-0249 inhibited the formation of mature IL-1β, establishing target engagement. The extent of mature IL-1β inhibition and associated reduction of anti-inflammatory outcome measures were dependent on the dose and frequency of NT-0249 administration. Findings from the D305N model of chronic inflammation provide important insight for designing future clinical trials that seek to demonstrate the therapeutic benefit of NT-0249.

Results

NT-0249 Selectively Inhibits IL-1β and IL-18 Output from Human Mononuclear Cells and Macrophages in Response to Lipopolysaccharide (LPS)/Adenosine Triphosphate (ATP) Activation

Human peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers were treated sequentially with LPS to stimulate transcription/translation of pro-IL-1β and ATP to promote the assembly of the NLRP3 inflammasome. In the context of this and subsequent cell-based assays, confirmation that IL-1β output is dependent on NLRP3 activation was established by subjecting the cells in question to a panel of control stimulations. Outcomes from these control panels are shown as inset plots adjacent to titration curves, and details of the control panels are provided in the methodology section of the Supporting Information document. PBMCs treated with LPS and ATP released IL-1β to the medium, whereas cultures treated with LPS or ATP individually did not (Figure 1B, inset). The output of IL-1β in response to LPS/ATP treatment was inhibited by the prototype NLRP3 inhibitor CP-456,773 but not by an inhibitor of p38 kinase (BIRB 796) when added to the cultures 60 min prior to ATP (Figure 1B, inset). The addition of NT-0249 (structure shown in Figure 1A) to the medium (containing 1% FBS) resulted in concentration-dependent inhibition of IL-1β output; the inhibitory concentration yielding 50% inhibition (IC₅₀) in this exemplar was 0.010 μM (Figure 1B). Table S1 provides a summary of IC₅₀ values observed on repeated testing in this context.

NT-0249 selectively inhibits cytokine output from human monocytes and macrophages. (A) The structure of NT-0249 as a sodium salt. (B) Human PBMCs were stimulated with LPS/ATP in a medium containing the indicated concentrations of NT-0249. The amount of IL-1β released to the medium is indicated as a function of NT-0249 concentration; each data point is the mean and SD of three replicate wells. Biological replicate testing with PBMCs isolated from different donors (n = 15) yielded mean and SEM IC₅₀ values of 0.012 and 0.00077 μM, respectively (Supporting Table 1). The inset shows IL-1β output from the assay control panel. (C) Human PBMCs were stimulated with LPS/ATP in a medium containing the indicated concentrations of NT-0249. The amount of IL-18 released to the medium is indicated as a function of NT-0249 concentration; each data point is the mean and SD of three replicate wells. Biological replicate testing with PBMCs from different donors (n = 4) yielded mean and SEM IC₅₀ values of 0.012 and 0.001 μM, respectively (Supporting Table 2). The inset shows IL-18 output from the assay control panel. (D) Heparin-stabilized blood was stimulated with LPS/ATP in the presence of the indicated concentrations of NT-0249. The amount of IL-1β released extracellularly is indicated as a function of NT-0249 concentration; each data point is the mean and SD of three replicate wells. Biological replicate testing with different blood isolates (n = 9) yielded mean and SEM IC₅₀ values for inhibition of IL-1β output of 1.0 and 0.11 μM, respectively (Supporting Table 3). The inset shows IL-1β output from the assay control panel. (E) Human Kupffer cells were treated sequentially with LPS and ATP in a medium containing the indicated concentrations of NT-0249. The amount of IL-1β released to the medium is indicated as a function of NT-0249 concentration; each data point is the mean and SD of three replicate wells. The inset shows IL-1β output from the assay control panel. (F) Human PBMCs were subjected to LPS stimulation in the presence of the indicated concentrations of NT-0249. Levels of IL-6 and TNFα recovered in the media are indicated as a function of NT-0249 concentration; each data point is the mean and SD of three replicate wells. The inset shows cytokine output from the assay control panel. (G) Human PBMCs were stimulated as indicated with LPS and/or ATP in a medium containing the indicated concentrations of NT-0249 or CP-456,773. Media fractions were probed for IL-1β by Western blot analysis. Migration of proteins of known molecular weight are indicated (kDa) on the left of the blot. The two left-hand lanes of the blot contained recombinant pro- and mature IL-1β, respectively, for reference. The adjacent plot shows the abundance of mature IL-1β as determined by densitometric analysis of the blot as a function of NT-0249 concentrations. IC₅₀ values noted in panels (B, C, D, E, G) were calculated using GraphPad Prism software.

IL-18 is also dependent on NLRP3 activation for post-translational processing. Like IL-1β, the release of IL-18 to the medium required stimulation with both LPS and ATP and was inhibited by CP-456,773 (Figure 1C, inset). The addition of NT-0249 to the medium led to a concentration-dependent inhibition of IL-18 output, yielding an IC₅₀ value of 0.012 μM in the example shown (Figure 1C). Table S2 provides a summary of IC₅₀ values observed against IL-18 output. In contrast to IL-1β and IL-18, tumor necrosis factor-α (TNFα) and IL-6 are released from LPS-stimulated cells via the traditional secretory pathway, which is not dependent on NLRP3 inflammasome function. PBMCs stimulated with LPS released both IL-6 and TNFα to the medium; their production was not affected by CP-456,773 but was inhibited by BIRB 796 (Figure 1F, inset). In this assay format, test agents are added prior to LPS, and BIRB 796 inhibition is expected as p38 kinase is involved in TLR4 signaling.³⁷ The addition of NT-0249 to LPS-treated PBMC cultures did not reduce IL-6 or TNFα output at concentrations ≤40 μM (Figure 1F).

The output of IL-1β from human blood is also achieved by sequential LPS/ATP activation (Figure 1D, inset). As with human PBMCs, stimulation with LPS or ATP individually leads to little IL-1β output. LPS/ATP-induced IL-1β output was inhibited by CP-456,773 but not by BIRB 796 (Figure 1D, inset). In the context of human blood, NT-0249 inhibited LPS/ATP-induced IL-1β output in a concentration-dependent manner, yielding complete inhibition at concentrations >7 μM; the IC₅₀ value in this example is 1.3 μM. Table S3 provides a summary of IC₅₀ values against IL-1β output from blood observed on repeated testing. The shift in the potency of NT-0249 observed in moving from a low to a high plasma protein environment is expected based on NT-0249 binding to plasma proteins, which reduces free drug concentrations (free fraction in human plasma measured as 1.9%).

To establish that NT-0249 inhibits the formation of mature IL-1β, human PBMCs were subjected to LPS/ATP activation in the presence and absence of NT-0249, after which media supernatants were subjected to Western blot analysis. LPS or ATP stimulation individually again were not sufficient to promote the release of IL-1β (Figure 1G; lanes 2 and 3). However, sequential LPS and ATP treatment led to the release of 17 kDa mature IL-1β; less of the 35 kDa pro-IL-1β species was detected extracellularly (Figure 1G; lane 4). The release of mature IL-1β was inhibited when 1 μM CP-456,773 was present in the culture medium (Figure 1G; lane 5). Likewise, NT-0249 blocked the production and release of mature 17 kDa IL-1β (Figure 1G; lanes 6–11); densitometric analysis of the blot yielded an estimated IC₅₀ value of 0.063 μM. Analysis of the same media supernatants for the presence of the 20 kDa subunit of active caspase-1 showed a similar dependence on LPS and ATP activation and sensitivity to NT-0249 inhibition (Figure S1).

Liver Kupffer cells represent the largest resident macrophage population in humans, and cytokine production by these cells is linked to numerous liver disorders.^38,39 Previous studies established that human Kupffer cells release IL-1β in response to NLRP3 activation.⁴⁰ Kupffer cells treated with LPS or ATP individually did not release IL-1β to the medium (Figure 1E, inset). However, when Kupffer cell cultures were sequentially treated with LPS followed by ATP, extracellular levels of IL-1β were enhanced. The prototype NLRP3 inhibitor CP-456,773 inhibited IL-1β output induced by LPS/ATP activation, while the p38 inhibitor BIRB 796 did not (Figure 1E, inset). Thus, the output of IL-1β from cultured Kupffer cells behaves as expected for an NLRP3-dependent process. Titration of NT-0249 into the medium reduced the output of IL-1β; an IC₅₀ value of 0.022 μM was determined, with NT-0249 concentrations ≥0.22 μM completely inhibiting cytokine release (Figure 1E).

NT-0249 Inhibits Inflammasome Assembly Dependent on NLRP3 but not NLR Family CARD Domain-Containing 4 (NLRC4)

In a nonactivated state, oligomers of NLRP3 examined by cryo-electron microscopy appear to exist in a double-ring cage-like structure with the N-terminal pyrin domain sequestered.⁴¹ Following activation, a conformational change in NLRP3 subunits is envisioned, exposing the pyrin domain and leading to recruitment of the adaptor PYD and CARD domain-containing protein (ASC) and, in turn, pro-caspase-1. Activation of NLRP3 inflammasome assembly in cells expressing fluorescently tagged ASC leads to aggregation of ASC monomers, which, when visualized by fluorescence microscopy, manifests as speck formation.⁴² Imaging THP-1 cells expressing green fluorescent protein (GFP)-tagged ASC thus affords an opportunity to assess the impact of test agents on NLRP3 inflammasome assembly. LPS-activated THP-1/ASC-GFP cells not exposed to an NLRP3 activator are largely free of specks. However, following the addition of nigericin to activate NLRP3 inflammasome assembly, many cells contain a single fluorescent speck readily detectable in proximity to an individual DAPI-staining nucleus (Figure 2A). Quantification indicated that 80% of the nigericin-treated cells possessed a fluorescent speck (Figure 2B). The addition of NT-0249 or CP-456,773 to the culture medium prior to nigericin treatment reduced the number of fluorescent specks (Figure 2A). Assessing impact over a range of NT-0249 concentrations yielded an IC₅₀ value of 0.028 μM (Figure 2B). In the same assay, CP-456,773 inhibited speck formation, yielding an IC₅₀ value of 0.068 μM (Figure 2B).

NT-0249 inhibits inflammasome assembly dependent on NLRP3. (A) THP-1 cells engineered to express ASC-GFP were stimulated with LPS only, LPS + nigericin, LPS + nigericin in the presence of 0.2 μM NT-0249, and LPS + nigericin in the presence of 0.25 μM CP-456,773. ASC-GFP speck formation was assessed by fluorescence image analysis. (B) Quantitation of speck formation: following sequential stimulation with LPS + nigericin, 80% of the THP-1/ASC-GFP cells were speck-positive. Horizontal bars indicate the mean from replicate treatment wells. Titration of NT-0249 or CP-456,773 into the culture medium inhibited speck formation in a concentration-dependent manner. Indicated IC₅₀ values were calculated using GraphPad Prism software. In 3 independent NT-0249 assessments, mean IC₅₀ and SEM values of 0.037 and 0.010 μM, respectively, were obtained.

The bacterial type III secretion system inner rod protein PrgJ is known to trigger NLRC4 inflammasome assembly in Salmonella-infected macrophages.⁴³ Intracellular delivery of PrgJ in the absence of bacterial infection can be achieved by treating cells simultaneously with Bacillus anthracis protective antigen (PA) and a fusion construct of PrgJ and the N-terminal domain of B. anthracis lethal factor (LFn).⁴⁴ Activation of human macrophage NLRC4 inflammasome following internalization of an LFn-PrgJ construct via PA has been demonstrated,⁴⁵ and IL-1β output induced by this system is unaffected by the prototype NLRP3 inhibitor CP-456,773.²³ LPS-activated human PBMCs treated with PA or LFn-PrgJ individually did not release IL-1β to the medium (Figure 3A). However, in combination, PA and LFn-PrgJ promoted IL-1β output, signifying that the two-component anthrax toxin delivery system was functioning as intended. Levels of IL-1β output achieved with PA and LFn-PrgJ were less than those achieved with nigericin activation of the NLRP3 inflammasome in the same LPS-activated PBMC preparation (Figure 3A). Importantly, the caspase inhibitor VX-765 blocked IL-1β output in response to PA and LFn-PrgJ treatment, in line with expectations that NLRC4 inflammasome assembly and subsequent caspase-1 activation promote IL-1β maturation and release (Figure 3A). When parallel LPS-activated PBMC cultures were treated with nigericin or PA/LFn-PrgJ in the absence or presence of CP-456,773, the test agent effectively inhibited nigericin-induced IL-1β output when present at 0.15 and 0.6 μM, but the same concentrations provided no significant inhibition of IL-1β release from PA/LFn-PrgJ-treated cultures (Figure 3B). Likewise, NT-0249 (tested at 0.1 or 0.4 μM) inhibited IL-1β output induced by LPS and nigericin but not by LPS and PA/LFn-PrJ (Figure 3C).

NT-0249 does not inhibit inflammasome assembly dependent on NLRC4. (A) The control panel conducted to ensure IL-1β output from LPS-activated human PBMCs was dependent on inflammasome activation; IL-1β output to the medium is indicated as a function of treatment with the indicated effector(s). (B) NLRP3-dependent IL-1β output from LPS + nigericin-treated PBMCs and NLRC4-dependent IL-1β output from LPS + PA + LFn-PrJ treated PBMCs is indicated as a function of treatment with the indicated concentrations of CP-456,773. (C) NLRP3-dependent IL-1β output from LPS + nigericin-treated PBMCs and NLRC4-dependent IL-1β output from LPS + PA + LFn-PrJ treated PBMCs as a function of treatment with the indicated concentrations of NT-0249. Levels of IL-1β in the media were measured by ELISA; the horizontal bar represents the mean of replicate treatment wells. In panels (B, C), indicated p-values for compound treatments versus no inhibitor controls were calculated in GraphPad Prism software using Welch’s ANOVA and Dunnett’s T3 test for multiple comparisons.

Crystal-Induced IL-1β Output is Sensitive to NT-0249 Inhibition

Endogenous DAMPs found in human disease that are reported to promote NLRP3 activation include monosodium urate (MSU), calcium pyrophosphate dihydrate (CPPD), and cholesterol crystals. To assess NT-0249's ability to suppress IL-1β output induced by crystal DAMPs, human PBMCs were activated with LPS and subsequently treated with preparations of individual crystals. For each crystal type, a set of controls was included to establish that IL-1β output is dependent on a 2-step activation protocol. Human PBMCs treated individually with LPS or cholesterol crystals released little IL-1β to the medium (Figure 4A, inset). However, when treated sequentially with LPS and cholesterol crystals, levels of extracellular IL-1β were enhanced. The addition of the phagocytosis inhibitor cytochalasin D reduced IL-1β output, consistent with the internalization of the cholesterol crystals being required to trigger NLRP3 activation. Titration of NT-0249 into medium prior to cholesterol crystal addition led to a concentration-dependent reduction in IL-1β output (Figure 4A), resulting in an exemplar IC₅₀ value of 0.038 μM. Similar studies were conducted in which LPS-activated PBMCs were treated with MSU or CPPD crystals; NT-0249 inhibited IL-1β output from LPS-treated human PBMCs induced by MSU or CPPD (Figure S2).

Inhibition of crystal-induced IL-1β output was also studied in the context of human blood. Stimulation of heparin-stabilized blood with LPS or MSU crystals individually resulted in little IL-1β output, but in combination, they stimulated much greater levels of cytokine output (Figure 4B, inset). Titration of NT-0249 into the blood matrix prior to MSU crystal addition reduced the level of IL-1β output in a concentration-dependent manner, resulting in an exemplar IC₅₀ value of 0.70 μM (Figure 4B). In parallel studies, NT-0249 inhibited IL-1β output from LPS-activated human blood treated with cholesterol or CPPD crystals (Figure S2).

NT-0249 Demonstrates NLRP3 Target Engagement in a Cell-Based Competitive Binding Assay

Cell-based assays employing bioluminescence resonance energy transfer (BRET) technologies have been employed to demonstrate CP-456,773 binding to NLRP3.^46,47 In one iteration, HEK293 cells expressing NLRP3 tagged with nanoluciferase are exposed to a fluorescent tracer ligand based on CP-456,773. Titration of the fluorescent tracer leads to a concentration-dependent enhancement of a BRET signal. This assay format provides a facile means to assess whether other potential NLRP3 inhibitors bind to NLRP3 and disrupt the binding of the fluorescent tracer.⁴⁷ NT-0249 was profiled in this target engagement assay alongside CP-456,773. IC₅₀ values estimated for the inhibition of fluorescent tracer binding corresponded to 0.011 and 0.013 μM, respectively, for NT-0249 and CP-456,773 (Figure S3). The IC₅₀ value observed for CP-456,773 is in line with what has been reported previously in the target engagement assay.⁴⁷

NT-0249 Inhibits IL-1β Output from Wild-Type and D305N Mouse Blood

Heparinized blood isolated and pooled from wild-type (WT) mice was subjected to LPS/ATP activation. As with human blood, no significant IL-1β output was observed from WT mouse blood treated with LPS or ATP individually (Figure 5A, inset). However, sequential treatment with LPS and ATP promoted the release of IL-1β in a process inhibited by CP-456,773 but not BIRB 796. Titration of NT-0249 into the blood matrix resulted in concentration-dependent inhibition of LPS/ATP-induced IL-1β output (Figure 5A). An IC₅₀ value of 0.24 μM was determined, with NT-0249 concentrations ≥3 μM providing complete inhibition (Figure 5A).

IL-1β output from D305N mouse blood was also examined for sensitivity to NT-0249 inhibition. In contrast to WT mouse blood, however, LPS alone is sufficient to promote IL-1β release from blood isolated from mice carrying the D305N GoF NLRP3 mutation; this behavior is comparable to that seen with human blood isolated from CAPS patients.⁴⁸ Titration of NT-0249 into the D305N blood matrix prior to LPS addition resulted in concentration-dependent inhibition of IL-1β output (Figure 5B). The observed IC₅₀ value of 0.25 μM is comparable to that seen in WT mouse blood, suggesting that the D305N mutation in NLRP3 does not alter the sensitivity of monocytes to NT-0249 inhibition. Titration of CP-456,773 into the D305N blood assay also inhibited LPS-induced IL-1β output, yielding an IC₅₀ value of 0.87 μM (Figure 5B).

NT-0249 Inhibits IL-1β Output following LPS/ATP Challenge In Vivo

Before assessing in vivo pharmacology, the pharmacokinetic profile of NT-0249 in WT mice was established (Table S4). NT-0249 has good oral bioavailability (41%) and a plasma half-life of 0.74 h. An acute peritonitis model was employed to allow a correlation of plasma drug levels with NT-0249 pharmacological impact.²³ In this model, WT mice are dosed orally with NT-0249, followed by sequential intraperitoneal injections of LPS (1 h-post NT-0249 dosing) and ATP (3 h-post NT-0249 dosing). Thirty minutes post-ATP injection, the mice are euthanized, and the levels of cell-free IL-1β recovered in peritoneal cavity lavage fluids are assessed by ELISA. Peritoneal lavage fluid from mice treated with vehicle-only contained a mean value of 800 pg/mL of IL-1β (Figure 6A). Mice treated with increasing doses of NT-0249 ranging from 0.1 to 10 mg/kg yielded progressively less IL-1β. At 1, 3, and 10 mg/kg, levels of IL-1β were reduced significantly relative to the control value, with the 10 mg/kg dose reducing cytokine levels to near baseline (Figure 6A). In contrast, levels of IL-6 recovered in the peritoneal lavage fluids were unaffected by NT-0249 treatment (Figure 6B). The lack of an effect on IL-6 in the context of an acute challenge model is expected and is consistent with the specificity seen in vitro. Dosing with CP-456,773 (10 mg/kg) also reduced IL-1β output while sparing IL-6 (Figure 6A and B). Plasma levels of NT-0249 recovered from blood samples collected at termination indicated a dose-proportional increase (Figure 6C). Plotting levels of IL-1β in relationship to concentrations of plasma NT-0249 (Figure 6D) or dose (Figure 6E) yields half-maximal effective (EC₅₀) and median effective dose (ED₅₀) values of 77 ng/mL (0.17 μM) and 2.2 mg/kg, respectively.

NT-0249 selectively inhibits cytokine output in an acute LPS/ATP peritonitis model. Cohorts of WT mice (all male, n = 8 per cohort) were dosed orally with NT-0249 (0.1, 0.3, 1, 3, and 10 mg/kg), CP-456,773 (10 mg/kg), or vehicle, followed by sequential IP injections of LPS and ATP. (A) Levels of IL-1β recovered in peritoneal lavage fluids as a function of the dose of NT-0249, CP-456773, or vehicle. Values for individual mice assigned to each study cohort are indicated. Statistical significance (relative to vehicle-treated cohort) was determined in GraphPad Prism using ordinary one-way ANOVA with Dunnett’s Multiple Comparisons test and is indicated. In this analysis, one outlier value was removed from the vehicle-control cohort based on both ROUT and Grubb outlier analyses. (B) Levels of IL-6 recovered in peritoneal lavage fluids as a function of the dose of NT-0249, CP-456,773, or vehicle. Values for individual mice assigned to each study cohort are indicated. NS = nonsignificant relative to vehicle cohort. (C) Plasma levels of NT-0249 detected at study termination by LC-MS/MS analysis. (D) A plot correlating levels of IL-1β recovered in peritoneal lavage fluids with plasma levels of NT-0249 for each mouse in the study. The indicated EC₅₀ value was calculated in GraphPad Prism, setting the maximum level to 800 (mean of LPS/ATP cohort) and the lower level to 0 pg/mL IL-1β. (E) A plot correlating levels of IL-1β recovered in peritoneal lavage fluids with the dose of NT-0249 administered. The indicated ED₅₀ value was calculated in GraphPad Prism, setting the upper level to 800 and the lower level to 0 pg/mL IL-1β.

A similar acute challenge model was conducted in the context of the lung. In this case, WT mice were dosed orally with NT-0249, followed by sequential instillation of LPS and ATP. Two hours after ATP instillation, the animals were euthanized and IL-1β levels in lung lavage fluids were assessed by ELISA. Mice treated with LPS alone yielded low levels of IL-1β in lung lavage fluids, but the combination of LPS plus ATP resulted in high levels of IL-1β output from vehicle-treated mice (Figure 7A). Treatment with doses of NT-0249 ranging from 6 to 30 mg/kg resulted in significant reductions in IL-1β output with near-complete inhibition achieved at 30 mg/kg (Figure 7A). Plasma levels of NT-0249 recovered from terminal blood samples again indicated a dose-proportional increase (Figure 7B). Plotting levels of IL-1β in relationship to dose of NT-0249 (Figure 7C) or plasma concentrations of NT-0249 (Figure 7D) yields ED₅₀ and EC₅₀ values of 6.7 mg/kg and 140 ng/mL (0.30 μM), respectively.

Dose-dependent inhibition of IL-1β output in the context of an acute LPS/ATP lung challenge model. Cohorts of WT male mice were dosed with NT-0249 (1.2, 6, 10, and 30 mg/kg) or vehicle, followed by sequential instillation of LPS and ATP to the lung. This study was performed on two separate days. On the first day, cohorts corresponded to LPS/ATP + vehicle (n = 5) and LPS/ATP dosed with 10 mg/kg NT-0249 (n = 6). Based on outcomes from this round, a follow-up round was conducted a week later with cohorts consisting of LPS only (n = 2), LPS/ATP + vehicle (n = 5), LPS/ATP dosed with 1.2 mg/kg NT-0249 (n = 6), LPS/ATP dosed with 6 mg/kg NT-0249 (n = 6), and LPS/ATP dosed with 30 mg/kg NT-0249 (n = 6). As a result of running a vehicle-control cohort in both rounds, the n value for this cohort is larger than the other cohorts in the combined data set. (A) Levels of IL-1β recovered in lung lavage fluids as a function of the dose of NT-0249 (or vehicle). Values for individual mice assigned to each study cohort are indicated. P-values, where indicated (relative to vehicle-treated cohort), were calculated in GraphPad Prism using ordinary one-way ANOVA with Dunnett’s Multiple Comparisons test and are indicated. (B) Plasma levels of NT-0249 were detected by LC-MS/MS analysis at study termination as a function of the dose administered. (C) A plot correlating levels of IL-1β recovered in lung lavage fluids with the dose of NT-0249 administered. The indicated ED₅₀ value was calculated in GraphPad Prism, setting the upper level to 4200 (mean of LPS/ATP cohort) and the lower level to 0 pg/mL IL-1β. (D) A plot correlating levels of IL-1β recovered in lung lavage fluids with plasma levels of NT-0249 for each mouse in the study. The indicated EC₅₀ value was calculated in GraphPad Prism, setting the upper level to 4200 and the lower level to 0 pg/mL IL-1β.

Therapeutic Impact in the Context of the D305N Syntenic Mouse Model of Inflammation

The generation of mice (NLRP3) in which the mouse Nlrp3 locus was removed and replaced with the corresponding human DNA, including the entire human NLRP3 gene, has been described.³⁵ When the human DNA is engineered to carry a D305N GoF mutation, the mice develop inflammation and arthropathy as they age.³⁶ While appearing normal at birth, D305N mice begin to display disease manifestations shortly after weaning, evidenced by visual changes in the autopods. These changes progressively worsen throughout the mouse’s lifespan, accompanied by a rise in levels of inflammatory mediators measurable in tissue extracts.

To explore whether NT-0249 provides a therapeutic benefit in the context of an ongoing inflammatory disease model, D305N mice were treated with NT-0249 after the onset of disease manifestations. Cohorts of age-matched D305N female mice (mean age of 20.5 weeks), randomized by litter age and starting weight, were fed control chow or the same chow formulated with NT-0249 for 8 days after which they were euthanized; harvested tissues and blood were then assessed for the presence of a panel of inflammatory markers. NT-0249-containing chow was formulated at two different levels (733 or 73 mg of NT-0249/kg of chow) to approximate doses of 100 (high) and 10 (low) mg/kg, respectively. Cohorts of mice (NLRP3) expressing the common NLRP3 allele were included in the study; these mice, like WT mice, do not develop an inflammatory phenotype. A comparison of the age and weight of the various cohorts is shown in Figure S4.

As previously reported, D305N mice develop enlarged spleens relative to NLRP3 mice (Figure 8A). After 8 days of NDT-0249 treatment, significant reductions in spleen weight were observed in both the low and high NT-0249 dose cohorts relative to D305N mice on the control chow (Figure 8A). NT-0249 treatment did not affect the spleen weights of NLRP3 animals. D305N mice develop arthritic tails, and tissue analysis indicates that this change is accompanied by neutrophil infiltration, which can be gauged by measuring the activity of the neutrophil-specific marker myeloperoxidase (MPO) in tissue homogenates. Relative to tail homogenates generated from NLRP3 mice, D305N tail homogenates possessed much higher levels of MPO activity (Figure 8B). Following 8 days of treatment with NT-0249, levels of tail homogenate MPO activity were significantly reduced in both the low and high-dose cohorts (Figure 8B). Within the plasma compartment, levels of IL-1ra were elevated in D305N relative to NLRP3 mice and reduced by NT-0249 treatment, though the reductions did not achieve statistical significance (Figure 8C).

NT-0249 therapeutic impact in the context of the D305N syntenic mouse model of inflammation. Female NLRP3 or D305N mice were allowed ad libitum access to control or NT-0249-formulated chow for 8 days. Following study completion, a panel of inflammatory outcome measures was assessed. Values for individual mice assigned to each study cohort are indicated in the plots as a function of treatment with control chow or chow formulated to deliver NT-0249 at doses of 10 and 100 mg/kg; horizontal bars mark the mean. P-values, where indicated, were calculated in GraphPad Prism using ordinary one-way ANOVA and Tukey’s multiple comparisons test. (A) Spleen weights. (B) Levels of MPO, as determined by enzyme activity recovered in tail homogenates (normalized to total protein). (C) Plasma IL-1ra levels as determined by ELISA. (D) Levels of IL-1β, as determined by ELISA recovered in autopod homogenates (normalized to total protein). Levels of IL-1β from NLRP3 mice were below the limit of detection and are plotted as 0. (E) Levels of IL-6, as determined by ELISA recovered in autopod homogenates (normalized to total protein). Levels of IL-6 from NLRP3 mice were below the limit of detection and are plotted as 0. (F) Levels of MPO, as determined by enzyme activity recovered in autopod homogenates (normalized to total protein).

Homogenates prepared from D305N mouse autopods collected at the end of the study possessed higher levels of inflammatory cytokines IL-1β and IL-6 and elevated MPO activity relative to levels seen in autopods recovered from NLRP3 mice (Figure 8D–F). Following 8 days of treatment with NT-0249, levels of these three analytes were reduced relative to levels seen in vehicle-control-treated D305N mice. While greater statistical significance relative to vehicle-control-treated mice was observed for each of the 3 autopod analytes at the 100 mg/kg NT-0249 dose, only the MPO outcome measure achieved a statistically significant difference between the 100 and 10 mg/kg dose cohorts themselves (Figure 8F). Mean NT-0249 plasma levels derived from blood harvested at termination were 5.5, 3.5, 0.29, and 0.26 μM for the 100 mg/kg D305N (n = 12), 100 mg/kg NLRP3 (n = 6), 10 mg/kg D305N (n = 12), and 10 mg/kg NLRP3 (n = 6) cohorts, respectively.

To provide evidence of target-proximal impact, autopod tissue homogenates were assessed for the presence of pro- and mature IL-1β by Western blotting. Anticipating the detection of mature IL-1β would be difficult due to its low abundance, prompted an enrichment strategy to be employed. A soluble type I IL-1 receptor-Fc construct (IL-R-Fc) was added to the tissue homogenates to bind mature IL-1β, after which receptor–ligand complexes were recovered. Evidence that this receptor capture approach provides a valid assessment of the levels of mature IL-1β within the tissue homogenates is presented in Figure S6. As shown in Figure 9A, when 50 μg of total autopod homogenates were analyzed by direct Western blotting, a prominent band was detected migrating at 35 kDa in homogenates recovered from D305N mice but not NLRP3 mice; mobility of this species is consistent with that of pro-IL-1β. No polypeptide species migrating as mature IL-1β (17 kDa) was detected in any of the autopod homogenates as assessed by the direct Western blot. However, a blot of IL-1R-Fc isolates derived from 2 mg of input autopod homogenate demonstrated the presence of mature IL-1β in D305N but not NLRP3 homogenates (Figure 9B). Importantly, NT-0249 treatment reduced levels of mature IL-1β recovered in the IL-1R-Fc capture isolates, confirming that the test agent inhibits NLRP3 function in the context of the disease model. Levels of mature IL-1β were reduced at both the 10 and 100 mg/kg doses of NT-0249 relative to D305N mice on control chow, with greater impact at the 100 mg/kg dose of NT-0249. Therapeutic treatment with NT-0249 also reduced levels of pro-IL-1β in the treated mice (Figure 9A).

Western blot detection of IL-1β in autopod homogenates. Two autopods from each of the following cohorts were analyzed: NLRP3 mice on control chow, D305N mice on control chow, D305N mice on 10 mg/kg dose NT-0249-formulated chow, and D305N mice on 100 mg/kg dose NT-0249-formulated chow. (A) Western blot corresponding to direct application of 50 μg of autopod homogenate; the arrow on the right points to the band attributed to pro-IL-1β. (B) Western blot corresponding to IL-1R-Fc pull-down isolates derived from 2 mg of the indicated autopod homogenate; the arrow on the right points to the migration position of mature IL-1β. Each gel contained a lane loaded with 10 pg of mature recombinant mouse IL-1β for reference. Migration of proteins of known molecular weight are indicated (kDa) on the left of the blots.

A separate eight-day therapeutic study was conducted in which D305N mice were allowed access to NT-0249 formulated chow (73 mg NT-0249/kg chow) on days 1, 3, 5, and 7 and control chow on alternate days over the course of the study. When the same panel of biochemical analytes was assessed at the study end, no significant changes were observed between D305N mice administered control chow or alternate day NT-0249-formulated chow (Figure S5). Terminal plasma samples (collected following a day off formulated chow) were analyzed for NT-0249; levels were below the limit of quantification (0.5 nM) in 8/13 animals in the D305N cohort, with a mean of 12.4 nM being seen in the other 5 animals. In the NLRP3 cohort, levels of NT-0249 were below the limit of quantification in 3/5 animals, with an average value of 1.3 nM in the other two.

Discussion

In vitro and in vivo profiling of NT-0249 establish a mechanism of action based on selective inhibition of NLRP3 activation. Like the prototype NLRP3 inhibitor CP-456,773, NT-0249 contains a sulfonylurea core, which is ionized at neutral pH to yield an anionic species. This same core is found in glyburide, the lead structure employed in the discovery of CP-456,773.⁴⁹ The pK_a of NT-0249 is 5.67, in line with a value of 5.3 for glyburide; although acidic, their weak acid nature may facilitate diffusion across phospholipid bilayers.^50,51 Glyburide and other sulfonylureas have been used safely in the clinic for the treatment of type II diabetes for decades.^52,53 Their use in this indication is attributed to high affinity binding to a sulfonylurea receptor (SUR) found in pancreatic β-cells.⁵⁴ In contrast, prior studies demonstrated that CP-456,773’s inhibition of NLRP3 activation is not dependent on SURs.^23,55 Rather, evidence that CP-456,773 and similar structures bind directly to NLRP3 to prevent a conformational change required for its activation implicates NLRP3 itself as the target responsible for inhibition of IL-1β post-translational processing.²⁶⁻²⁸ In terms of cellular pharmacology, NT-0249 and CP-456,773 demonstrate similar functionality. The ability of NT-0249 to inhibit the binding of a CP-456,773 surrogate fluorescent tracer to the NACHT domain of NLRP3 in a cell-based target engagement assay implicates NLRP3 as the molecular target of NT-0249.

Pharmacological profiling of NT-0249 in vitro was accomplished using a panel of cell-based assays designed to assess both on-target and off-target activities. IL-1β output from human PBMCs stimulated with LPS and ATP provided a proximal downstream assessment of NLRP3 activation. In the presence of a low-serum environment, NT-0249 potently inhibited IL-1β output, yielding a mean IC₅₀ value of 0.012 μM. Within the context of the same human PBMC assay, NT-0249 inhibited IL-18 output with a mean IC₅₀ value of 0.012 μM. The comparable potency achieved against IL-1β and IL-18 output is totally consistent with NT-0249 disrupting NLRP3 activation, which, in turn, disables downstream cytokine post-translational processing. The output of IL-1β from human Kupffer cells was also dependent on LPS/ATP activation, and NT-0249 potently inhibited Kupffer cell IL-1β production (IC₅₀ = 0.022 μM). Thus, in a low-serum environment, NT-0249 inhibited NLRP3-dependent cytokine output equally well, whether from monocytes or macrophages. In the presence of a high protein (blood) environment, NT-0249 inhibited LPS/ATP-induced IL-1β output, yielding an IC₅₀ value of 1.0 μM. The shift in potency in moving from a low to a high protein environment is attributed to NT-0249 binding to plasma proteins, which results in reduced levels of free drug available for target impact. Human PBMCs also provided a system to assess NT-0249 selectivity with respect to cytokine output. Following LPS-only stimulation, PBMCs released both IL-6 and TNFα to the medium via a process effectively inhibited by a p38α kinase inhibitor. However, concentrations of NT-0249 up to 40 μM did not inhibit the output of either IL-6 or TNFα, consistent with their production being independent of NLRP3.

Selectivity was also assessed with respect to the inhibition of inflammasome platforms. LPS-stimulated human THP-1 cells engineered to express GFP-tagged ASC assemble fluorescent specks when treated with nigericin, signifying the assembly of NLRP3 inflammasome complexes. NT-0249 inhibited speck formation in a concentration-dependent manner. Likewise, sequential LPS/nigericin treatment promoted IL-1β output from human PBMCs, which was inhibited by NT-0249. In contrast, IL-1β release from LPS-activated human PBMCs in response to NLRC4 inflammasome activation with PA and LFn-PrgJ was not inhibited by NT-0249. Thus, NT-0249 acts as a selective inflammasome inhibitor.

In vivo, a variety of disease-specific DAMPs are postulated to function as activators of NLRP3. For example, crystals composed of cholesterol, MSU, and CPPD are found in atherosclerosis, gout, and pseudogout patients, respectively, and are reported to promote NLRP3 activation. The application of each of these individual crystal types to LPS-activated human PBMCs promoted IL-1β output. The release of IL-1β was inhibited by cytochalasin D, in line with phagocytosis of the crystals being required for NLRP3 activation.⁵⁶ NT-0249 also effectively inhibited IL-1β output induced by each of the crystal types. Likewise, the individual crystals served as effective triggers to promote IL-1β output from LPS-activated human blood; as in the case of LPS/ATP-induced IL-1β output, the potency of NT-0249 as an inhibitor of crystal-induced cytokine release was attenuated moving from a low to high plasma protein environment. The ability of NT-0249 to inhibit IL-1β output in response to diverse stimuli, including crystals, nigericin, and ATP, signifies that its mechanism of action is downstream of the triggering insult.

Prior to conducting in vivo mouse studies, NT-0249 was assessed as an inhibitor of IL-1β output from mouse blood. IL-1β output from WT mouse blood was dependent on sequential LPS and ATP treatment, as seen in human blood. NT-0249 inhibited LPS/ATP-induced IL-1β release from WT mouse blood, yielding an IC₅₀ value of 0.22 μM. The release of IL-1β from D305N mouse blood, on the other hand, only required stimulation with LPS. Lack of a trigger 2 requirement (e.g., ATP) is attributed to the D305N mutation, causing NLRP3 to assume a constitutively activated state such that LPS alone is sufficient to promote cytokine output.⁵⁷ Importantly, NT-0249 inhibited LPS-induced IL-1β output from D305N mouse blood with an IC₅₀ value (0.25 μM) comparable to that seen in WT mouse blood. Thus, the D305N GoF mutation does not alter sensitivity to NT-0249.

Two acute in vivo challenge models in WT mice were employed to develop PK/PD relationships. In the first, sequential LPS and ATP administration to the peritoneal cavity of WT mice promoted NLRP3 activation and release of IL-1β. Prior dosing with NT-0249 led to a dose-dependent inhibition of IL-1β output. In contrast, NT-0249 did not affect the production of IL-6 in this acute challenge model, confirming the selectivity seen in vitro. An EC₅₀ value of 0.17 μM was calculated based on the peritonitis model, which is comparable to the IC₅₀ value of 0.22 μM observed in the ex vivo mouse blood LPS/ATP induction assay. This comparability suggests that target coverage in the peritonitis model correlates well with the target impact achieved in the blood assay. In a second model, sequential LPS and ATP instillation to the lungs of WT mice was used to assess NT-0249 impact within an isolated tissue compartment. LPS/ATP instillation led to enhanced levels of IL-1β being recovered in lung lavage fluid, which were reduced by prior oral dosing with NT-0249. In this model, an EC₅₀ value of 0.30 μM was achieved. The slightly higher EC₅₀ value observed in the context of a lung challenge model may reflect a lower distribution of NT-0249 to the lung than to the peritoneal cavity.

To demonstrate NT-0249’s therapeutic impact in a disease model, we employed a mouse model of CAPS. While the range of potential clinical indications that can be envisioned for an NLRP3 inhibitor is wide and not restricted to CAPS, including both peripheral and central indications, mice engineered to possess human D305N NLRP3 in place of endogenous Nlrp3 afford an exceptional model for establishing proof of concept. First, the model is driven by a GoF mutation of human NLRP3 identified in clinical studies. D305N mice naturally develop inflammatory disease outcomes as they age, mimicking those seen in CAPS patients; no exogenous stimulant is required to elicit disease. Second, because D305N mice are born in a normal state and only develop the disease over time, the model allows for a true therapeutic study to be conducted. Animals are allowed to age to the point where disease manifestations are sufficient to distinguish them from animals carrying the common NLRP3 allele and then subjected to therapeutic intervention; this type of model is expected to translate better to the clinic than prevention-type models where test agents are coadministered along with the insult required to promote disease manifestations. Lastly, given inhibition of NLRP3 activation is the pharmacodynamic target of NT-0249, the D305N model provides a target-centric approach for assessing PK/PD relationships.

D305N mice treated with NT-0249 for only 8 days displayed reduced levels of multiple inflammatory biomarkers. Relative to NLRP3 mice, spleens in D305N mice are enlarged and NT-0249 treatment reduced spleen weight. Likewise, D305N mice possessed elevated plasma levels of the acute phase reactant IL-1ra⁵⁸ and tail homogenate MPO activity relative to NLRP3 mice. Eight days of NT-0249 treatment reduced plasma levels of IL-1ra, in line with IL-1β’s known role in promoting the acute phase response.⁵⁹ Similarly, the observed reduction in tail homogenate MPO activity is consistent with IL-1β’s known role in promoting neutrophil trafficking.⁶⁰ Homogenates prepared from autopods of D305N mice contained elevated levels of IL-1β, IL-6, and MPO activity relative to those from NLRP3 mice, signifying an ongoing inflammatory response. Eight days of NT-0249 treatment reduced levels of each of these analytes. The reduction in MPO activity again is indicative of a reduced neutrophil presence. While NT-0249 does not directly inhibit IL-6 generation, the reduction in IL-6 levels observed in tissue homogenates is consistent with IL-1β signaling leading to IL-6 production.⁶¹ Indeed, human CAPS patients possess elevated plasma IL-6 levels, which are rapidly reduced by treatment with an anti-IL-1β antibody.⁶²

Importantly, NT-0249 treatment reduced levels of mature IL-1β recovered from the autopod homogenates, providing direct evidence of NLRP3 function disruption within the context of the model. When analyzed by Western blot, D305N autopod homogenates contained a prominent polypeptide corresponding to pro-IL-1β, which was not present in NLRP3 tissues. Mature IL-1β, on the other hand, was not detected on a direct Western blot but was readily detected following the use of an IL-1R-Fc pull-down strategy to enrich for the active cytokine species and input of a larger amount of total homogenate protein. NT-0249 treatment dose-dependently reduced levels of mature IL-1β recovered in D305N autopod homogenates. Interestingly, NT-0249 treatment also reduced the levels of pro-IL-1β. This correlation is consistent with mature IL-1β driving further expression of pro-IL-1β and aligns with what has been observed in the clinic where canakinumab treatment of CAPS patients reduced IL-1β expression.⁶² It is also notable that the steady-state levels of pro-IL-1β in D305N autopod homogenates exceed those of mature IL-1β, perhaps suggesting the presence of cells that express pro-IL-1β but not D305N NLRP3 or that IL-1β-producing cells that express GoF D305N NLRP3 do not all spontaneously activate.

In general, both low (10 mg/kg) and high (100 mg/kg) dose NT-0249 chow formulations resulted in significant reductions in the panel of inflammatory markers. Although the magnitude of changes trended greater at the higher dose, generally, there were no statistically significant differences achieved between the low- and high-dose cohorts. Average levels of NT-0249 in plasma collected at termination from D305N-treated mice were 5.5 μM and 0.29 μM, respectively, for the high- and low-dose cohorts. Thus, the therapeutic benefit observed in the D305N model with the low NT-0249 dosing regimen was achieved when plasma NT-0249 levels were in the range of the IC₅₀ value observed in the ex vivo mouse blood assay (0.22 μM) and EC₅₀ values observed in acute LPS/ATP challenge models (0.17–0.30 μM). This correspondence indicates that sustained complete target coverage is not required to provide biological benefit in the context of a relevant disease model. However, the same low-dose NT-0249 chow formulation did not reduce the inflammatory biomarkers when administered every other day for 8 days, suggesting that intermittent target coverage is less beneficial.

In summary, the cellular pharmacology presented in this study indicates that NT-0249 is a potent and selective inhibitor of both human and mouse NLRP3 activation. Following oral administration to WT mice, NT-0249 dose-dependently inhibited IL-1β output in response to acute LPS/ATP challenge models conducted locally within the lung and peritoneal cavity. Plasma drug levels yielding efficacy in these acute models aligned well with the potency of NT-0249 measured in an ex vivo mouse blood assay. Most importantly, when tested in the context of a CAPS autoinflammatory disease model, NT-0249 reduced the formation of mature IL-1β and a panel of biochemical outcome measures linked to IL-1β signaling. The degree of pharmacological impact achieved in the D305N model was affected by both dose and dosing frequency, providing a translational framework to aid the selection of NT-0249 dosing strategies to be employed in future NT-0249 clinical studies.

Methods

Materials

NT-0249 and CP-456,773 were synthesized by NodThera; the synthesis of NT-0249 has been described previously.⁶³ BIRB 796 was obtained from Tocris. Freshly drawn human heparin-stabilized blood was sourced from Bloodworks NW in Seattle, WA. Primary human Kupffer cells were obtained from BioIVT and human THP-1-ASC-GFP cells from Invitrogen. Antibodies employed included: rabbit polyclonal anti-human IL-1β (Abcam; AB2105), donkey anti-rabbit IgG-DyLight 800 conjugate (Rockland; 611–1458–002), donkey anti-goat (H&L) Alexa Fluor Plus 800 highly cross-adsorbed antibody (ThermoFisher; A32930), and goat anti-mouse IL-1β (R&D Systems; AF-401-NA). Recombinant proteins employed included: human pro-IL-1β (NovoPro, 501519), human IL-1β (Novus; NBC1–18478), mouse IL-1β (R&D Systems; 401-ML), and murine IL-1R-Fc (R&D Systems; 771-MR-100). ELISA kits employed to measure cytokines within in vivo derived samples were obtained from ThermoFisher to measure human IL-1β (88–7261–88), human TNFα (88–7346–88), and human IL-6 (88–7066–88). Kits employed to measure cytokines derived from in vivo mouse studies were obtained from R&D Systems to measure murine IL-1β (SMLB00C), murine IL-6 (MB000B-1), and murine IL-1ra (MRA00). A murine IL-1β ELISA kit from ThermoFisher (88–7013–88) was also employed for assessing cytokine output from murine blood. DNA encoding B. anthracis lethal factor N-terminal domain fused to Salmonella typhimurium Type III secretion system inner rod protein PrgJ was custom synthesized at GeneWiz. For expression of the fusion construct, a pET-28a+ vector and Escherichia coli BL21(D3)pLysS cell system was employed from Sigma-Aldrich (70777). Mouse lines employed (all bred at UNC) included 129S6/SvEv Tac (Taconic), 129-NLRP3 D305N (hD305N; B.H. Koller, UNC), and 129-NLRP3 (B.H. Koller, UNC). Other reagents included Ficoll-Paque Plus (Cytivia; 17144003), RPMI GlutaMax (61870–036), heat-inactivated FBS (10082–147), penicillin/streptomycin (15140–122), HEPES (15630–080), protease inhibitor tablets (A32955), dithiothreitol (B0009), nickel-NTA-beads (88221), BCA protein assay (23225), bolt 4–12% minigels (NW041125), and Protein A- Plus Agarose (22812) from ThermoFisher; LPS (L4391), ATP (A6419), nigericin (481990), Z-VAD-FMK (627610), VX-765 (5313720001), cholesterol (C8667), 1-propanol (402893), cytochalasin D (C8273), human serum albumin (A8763), O-dianisidine hydrochloride (D9143–5G), hexadecyltrimethylammonium (H6269), and iodoacetamide (I1149) from Sigma-Aldrich; MSU crystals (tlrl-msu-25) and CPPD crystals (tlrl-cppd) from InvivoGen; heparin (25021–400–10) from Sargent Pharmaceuticals; and myeloperoxidase standard (475911) from Calbiochem.

IL-1β Output from Human PBMCs and Kupffer Cells

Heparin-stabilized blood from healthy volunteers was obtained from Bloodworks NW (Seattle); blood from both male and female donors was employed with no noticeable differences in assay performance. PBMCs were prepared using Ficoll-Paque Plus centrifugation. Total cell numbers were determined using a hemacytometer and adjusted to 2.6 × 10⁶ cells/mL in RPMI GlutaMAX medium containing 1% FBS, 1% penicillin/streptomycin, and 20 mM HEPES, pH 7.3. The LPS/ATP challenge assay was conducted as previously detailed.³⁰ Briefly, 0.1 mL (2.6 × 10⁵ cells) of the PBMC suspension was added to each well of 96-well plates. After a 2 h incubation at 37 °C in a 5% CO₂ incubator to allow adherence of monocytes, media and nonadherent cells were aspirated. Fresh RPMI GlutaMAX medium containing 5% FBS, 1% penicillin/streptomycin, and 20 mM HEPES (pH 7.3, Base Medium) was added and the plates were incubated overnight at 37 °C in a 5% CO₂ incubator. The following day, LPS was added to designated wells (final concentration of 100 ng/mL) and the plates were incubated at 37 °C for 2 h to allow transcription/translation of pro-IL-1β. At this point, media were removed by aspiration and 0.143 mL of fresh RPMI GlutaMAX medium containing 1% FBS and 1% penicillin/streptomycin were added; the presence of LPS was maintained in wells previously exposed to this stimulus. The test compound at various concentrations (or 0.2% DMSO) was added to designated wells, and plates were placed at 37 °C in a 5% CO₂ incubator for 60 min. To designated wells, 0.0075 mL of a 100 mM ATP solution (final concentration = 5 mM) was then introduced to promote NLRP3 activation, and the plates returned to a 37 °C/5% CO₂ incubator. After a 60 min stimulation, plates were subjected to centrifugation, after which media supernatants were harvested for the assessment of IL-1β levels by ELISA.

Human Kupffer cells were thawed and plated into wells of 96-well collagen-coated plates following the supplier’s instructions. Individual wells were seeded with 1 × 10⁵ cells suspended in RPMI GlutaMAX medium containing 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 20 mM HEPES, pH 7.3. The plates were incubated overnight at 37 °C in a 5% CO₂ incubator. LPS was introduced to achieve a final concentration of 100 ng/mL, and the IL-1β output assay was conducted as indicated above for PBMCs.

IL-1β output from PBMCs was also assessed by Western blotting. Human PBMCs were seeded at a density of 5 × 10⁶ per ml in RPMI medium containing 0.1% FBS in 6 well plates (3 mL/well). Cells were primed with LPS (100 ng/mL) for 2.5 h at 37 °C. NT-0249 and CP-456,773 were prepared in DMSO and added to designated wells to achieve the desired final concentrations and 0.2% DMSO. The cultures were incubated for an additional 30 min at 37 °C, after which activation of NLRP3 was induced by the addition of 5 mM ATP. The cultures were incubated at 37 °C for 1 h, after which media were collected and clarified by centrifugation. Media proteins were precipitated using methanol/chloroform extraction. Precipitated proteins were resuspended in SDS buffer (containing 50 mM DTT) and heated at 90 °C for 5 min. The disaggregated samples were subjected to SDS gel electrophoresis in MES buffer. Following electrophoresis, proteins were transferred from the gels to nitrocellulose membranes and subjected to Western blot analysis. The nitrocellulose membranes were blocked with Licor Intercept block for 2 h and then incubated with a rabbit antibody against IL-1β overnight at 4 °C. Membranes were washed and incubated with a donkey anti-rabbit secondary antibody DyLight 800 conjugate for 1 h at room temperature. Resulting membrane blots were imaged using the Licor Odyssey System at 800 nm. For quantitation, blot images were uploaded to Licor Image Studio Lite software (version 5.2) as Tiff files. Bands corresponding to mature IL-1β were manually boxed using a drawing tool in each lane of interest; the total areas of each boxed region were equivalent. A total of 800 nm relative fluorescence units (RFU) corresponding to each boxed region were captured and normalized to a total protein fluorescent stain RFU associated with the entire lane. The latter, which is attributed to the presence of serum in the medium, was obtained by staining the nitrocellulose blot with Licor Revert 700 Total Stain. The total protein staining area of each lane was manually boxed using the imaging software, and a total 700 nm RFU signal was captured; the sizes of the boxed areas were constant across all lanes. IL-1β RFU intensities were normalized to total stain RFUs and plotted in GraphPad Prism as a function of NT-0249 concentration using a four-parameter logistic, variable slope mode to estimate an IC₅₀ value.

IL-6 and TNFα Output from Human PBMCs

0.14 mL of a suspension of freshly isolated PBMCs (2.8 × 10⁵ cells) were added to each well of 96-well plates. To wells designated to receive the test compound, 0.05 mL of Base Medium containing 4× the desired final test agent concentration was added; control wells received 0.05 mL of Base Medium without the compound. Plates were placed at 37 °C in a 5% CO₂ incubator for 30 min. To wells designated to receive LPS, 0.01 mL of Base Medium containing 2 μg/mL LPS was added to achieve a final LPS concentration of 100 ng/mL, and the plates were returned to a 37 °C/5% CO₂ incubator. After 4 h of LPS stimulation, plates were subjected to centrifugation, and media supernatants were recovered and assessed for cytokine content using TNFα- and IL-6-specific ELISA kits.

IL-1β Output from Blood

Heparin-stabilized blood from healthy volunteers was subjected to a 2-step activation protocol as previously described.^23,30 Blood samples were diluted with RPMI GlutaMAX medium containing 20 mM HEPES, pH 7.3 (2 parts blood to 1 part medium), after which 0.14 mL of the diluted samples were placed in individual wells of a 96-well plate designated not to receive LPS. To the remaining diluted blood sample, an appropriate volume of 1 μg/mL LPS was added to achieve a final concentration of 100 ng/mL. 0.14 mL of the LPS-containing diluted blood sample was then placed into wells of the 96-well plate designated to receive LPS. The plates were incubated for 3 h at 37 °C in a 5% CO₂ incubator to allow activation of pro-IL-1β transcription/translation, after which 0.05 mL of RPMI GlutaMAX media containing 1% FBS, 1% penicillin/streptomycin, 20 mM HEPES (pH 7.3), and test agent (at a 4-fold higher concentration than final) were added to designated wells. Wells designated not to receive the test agent received 0.05 mL of RPMI GlutaMAX media containing 1% FBS, 1% penicillin/streptomycin, 20 mM HEPES (pH 7.3), and 1.6% DMSO. The plates were returned to the 37 °C/5% CO₂ incubator for an additional 30 min. At this point, 0.01 mL of 100 mM ATP and 20 mM HEPES (pH 7.3) were added to designated wells to promote NLRP3 activation. The plates were incubated for an additional 60 min at 37 °C/5% CO₂, after which they were subjected to centrifugation and plasma supernatants were harvested and assessed for IL-1β content by ELISA. A similar protocol was employed to assess IL-1β output from heparin-stabilized mouse blood with the following changes: blood samples from multiple mice were pooled, the LPS concentration was increased to 1 μg/mL, and cytokine levels in plasma supernatants were assessed using an ELISA specific for murine IL-1β.

Crystal-Induced IL-1β Output

Cholesterol crystals were prepared as previously detailed.⁶⁴ Briefly, ultrapure cholesterol (100 mg) was dissolved in 1-propanol (50 mL). The solution was mixed with distilled water (1:1.5) and rested for at least 10 min for monohydrate crystals to stabilize. 1-Propanol was removed by evaporation, and the crystals were resuspended in PBS/0.05% human serum albumin. All steps were performed at room temperature.

MSU and CPPD crystal-induced IL-1β output from human PBMCs was assessed using cells aged overnight in culture as described above in the ATP-induced assay format. On the day of assay, LPS was added to designated wells at a final concentration of 100 ng/mL. Plates were incubated at 37 °C for 2.5 h to allow transcription/translation of pro-IL-1β. Cytochalasin D was added to designated control wells at the time of LPS addition (final concentration of 2 μM). After incubation, the medium was removed by aspiration and replaced by fresh RPMI GlutaMAX medium containing 1% FBS with LPS and cytochalasin being maintained in the designated wells. The test agent or 0.2% DMSO was added to designated wells, and plates were incubated at 37 °C for 0.5 h. MSU (final concentration 200 μg/mL) or CPPD crystals (final concentration 100 μg/mL) were added, and the plates were returned to a 37 °C incubator for 4 h. Supernatants were harvested by centrifugation, and levels of IL-1β were assessed by ELISA.

Cholesterol crystal-induced IL-1β output was assessed using PBMCs isolated from heparin-stabilized human whole blood on the day of their isolation. Total cell numbers were determined using a hemacytometer and adjusted to 2.6 × 10⁶ cells/mL. A total of 2.6 × 10⁵ cells (0.1 mL) was added to each well of 96-well plates. LPS was added to designated wells at a final concentration of 100 ng/mL. Cytochalasin D (final concentration 2 μM) was added to designated control wells at the time of LPS addition. Plates were incubated at 37 °C for 2.5 h, after which the test agent or 0.2% DMSO was added to designated wells, and plates were incubated at 37 °C for 0.5 h. Cholesterol crystals (final concentration 500 μg/mL) were added to designated wells, and the plates were returned to the 37 °C incubator for 4 h. Supernatants were harvested by centrifugation, and levels of IL-1β were assessed by ELISA.

Crystal-induced IL-1β output was also assessed in the context of human blood. Heparin-stabilized blood was diluted with RPMI GlutaMAX containing 20 mM HEPES, pH 7.3 (2 parts blood to 1 part RPMI). 0.14 mL of the diluted blood mixture was placed into wells of 96-well plates not designated to receive LPS. To the remainder of the diluted blood, LPS was added to achieve a final concentration of 100 ng/mL, and 0.14 mL of the resulting blood mix was added to designated wells. Plates were incubated for 3 h at 37 °C after which 0.05 mL of RPMI GlutaMAX media containing 1% FBS, 20 mM HEPES (pH 7.3), 1% penicillin–streptomycin, and test agent (at 4× final concentration) was added to appropriate wells. Wells not designated to receive the test agent instead received 0.05 mL RPMI GlutaMAX containing 1.6% DMSO. Plates were returned to the incubator for 0.5 h. MSU crystals (200 μg/mL), CPPD crystals (100 μg/mL), or cholesterol crystals (500 μg/mL) subsequently were added to designated wells. Plates containing MSU or CPPD crystals were incubated for 2 h at 37 °C, whereas plates containing cholesterol crystals were incubated for 4 h at 37 °C. Supernatants were harvested by centrifugation, and levels of IL-1β were assessed by ELISA.

ASC-GFP Speck Assay

A suspension of THP-1-ASC-GFP cells was incubated in a culture medium containing 10% FBS with or without 1 μg/mL LPS for 2 h at 37 °C. Cells were collected by centrifugation, resuspended in an assay medium containing 1% FBS, and dispensed into a 96-well plate (0.1 mL containing 2 × 10⁵ cells/well). Diluted compounds or equivalent dilutions of vehicle (DMSO) were then added to the cells, and the cells were incubated for 30 min at 37 °C; the final DMSO concentration in all wells was 0.4%. At this point, the caspase inhibitor z-VAD-FMK was added to all wells to yield a final concentration of 20 μM, and the cells were incubated for 5 min at 37 °C; z-VAD-FMK was included to prevent pyroptosis and preserve speck detection. Next, 1 μL of a 500 mM nigericin solution (in 10% EtOH) was added to yield a final concentration of 5 μM. Control wells not treated with nigericin received an equivalent dilution of ethanol. After a 1 h incubation at 37 °C, cells were fixed with a final concentration of 1% paraformaldehyde and then incubated with Hoechst 33342 trihydrochloride to fluorescently label DNA. Images of the cells were acquired using an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices) and analyzed using CellProfiler software.

NLRC4 Inflammasome Assay

Peripheral blood mononuclear cells (PBMCs) isolated from human whole blood were resuspended in a culture medium containing 1% FBS and dispensed into 96-well plates. After a 2 h incubation at 37 °C, media and unattached cells were removed by aspiration, and a culture medium containing 10% FBS was added to each well. The cells were incubated at 37 °C overnight.

The following morning, the media were replaced with an assay medium containing 1% FBS and 100 ng/mL LPS, and the cells were incubated for 2.5 h at 37 °C. Next, a control or test article was added to the cells. Concentrations of CP-456,773 tested in the NLRC4 assay represent 5- and 20-fold multiples above its IC₅₀ value observed in the PBMC LPS/ATP-induced IL-1β output assay, while those for NT-0249 represent 10- and 40-fold multiples. Negative-control wells received an equivalent dilution of DMSO, and the CASP1/4 inhibitor VX-765 was used as a positive-control compound predicted to inhibit NLRC4-mediated IL-1β release. The cells were then incubated for 30 min at 37 °C, after which appropriate secondary stimuli were added to designated wells. For activation of NLRP3, nigericin was introduced to achieve a final concentration of 5 μM. For activation of NLRC4, B. anthracis Protective Antigen (PA) and a fusion construct composed of the N-terminal domain of B. anthracis lethal factor and the bacterial type III secretion protein PrgJ (LFn-PrgJ; expressed and isolated at NodThera) were added to yield final concentrations of 1 and 4 μg/mL, respectively.⁴⁵ To confirm both proteins were required for NLRC4 inflammasome activation, control wells containing each individual protein were also prepared. After 4 h of incubation at 37 °C, culture supernatants were harvested and stored at 4 °C. IL-1β levels in the culture supernatants were determined by ELISA following the manufacturer’s instructions.

Isolation of an LFn-PrgJ fusion protein and its use to activate NLRC4 inflammasomes when delivered into the cytoplasm of mammalian cells have been described previously.^45,65 To implement this approach in our laboratory, a DNA sequence encoding the N-terminal domain of B. anthracis lethal factor fused to the S. typhimurium Type III Secretion System inner rod protein PrgJ was synthesized and cloned into the pET-28a+ plasmid for expression in E. coli BL21(DE3)pLysS. The pET-28a+ vector introduced an N-terminal poly-His purification tag onto the fusion protein. The expressed fusion protein was isolated by chromatography on nickel-NTA beads, dialyzed extensively into PBS, and stored in single-use aliquots at −80 °C.

IL-1β Western Blotting and IL-1R-Fc Capture from Mouse Autopod Homogenates

Harvested mouse autopods collected at the end of the 8-day therapeutic study were stored at −80 °C. A subset of individual front autopods were pulverized in liquid nitrogen and then homogenized in PBS containing 1% TX-100 supplemented with a protease inhibitor tablet per the manufacturer’s instructions and 1 mM iodoacetamide (to inhibit caspase activity). Iodoacetamide was prepared fresh from the solid immediately before use. The homogenates were stored at −80 °C.

Prior to Western analysis, the homogenates were thawed and subjected to a 30 min centrifugation at 4 °C in a microcentrifuge (16,000g). The resulting supernatants were collected and stored on ice. The total protein concentration of each supernatant was determined via the BCA assay. For a direct Western blot, 50 μg of total homogenate protein was disaggregated in SDS sample buffer, 100 mM DTT, heated to 95 °C for 5 min, centrifuged briefly, and loaded onto a single lane of a Bolt 4–12% minigel. For IL-1R-Fc capture of mature IL-1β, 2 mg 2 mg of each homogenate was transferred to a new microcentrifuge tube, and the total volume was adjusted to 0.5 mL with homogenization buffer. Two μg of murine IL-1R1-Fc (20 μL of a 100 μg/mL stock prepared in sterile PBS) were added to each tube, after which the resulting solutions were mixed well and incubated on ice for 2 h. To capture receptor–ligand complexes, 50 μL of a 20% suspension of Protein A Plus Agarose was added to the mixtures and incubated for an additional 1 h at 4 °C with continuous mixing. The agarose beads were then recovered by centrifugation and washed 3 times with 0.5 mL of PBS, 1% TX-100 by repeated centrifugation. The washed beads were suspended in 40 μL of SDS sample buffer (containing 100 mM DTT) and heated to 95 °C for 5 min. After removal of the beads by centrifugation, the entire supernatant fractions were loaded directly onto lanes of Bolt 4–12% minigels. The gels were subjected to electrophoresis in MES running buffer, after which proteins were transferred to nitrocellulose using a Power Blotter device. The nitrocellulose blots were blocked in Licor Intercept buffer for 1 h at RT and then exposed to goat anti-mouse IL-1β at 0.1 μg/mL in Intercept buffer containing 0.1% Tween-20 overnight at 4 °C with continuous shaking. Blots were washed with PBS and 0.1% Tween-20 and then incubated with donkey anti-goat (H + L) Alexa Fluor Plus 800 highly cross-adsorbed antibody at 0.1 μg/mL in Intercept buffer containing 0.1% Tween-20 and 2% donkey serum for 1 h in the dark at RT (with mixing). After washing, images were acquired using a LiCor Odyssey instrument.

In Vivo LPS/ATP Peritonitis Model

These studies were performed under contract with HD Biosciences in Shanghai, P.R. China, conducted at an AAALAC-accredited facility and approved by the Institutional Animal Care and Use Committee (IACUC) of HDB. Cohorts of WT C57BL/6 male mice (8 weeks of age) were dosed by oral gavage with NT-0249 (dissolved in 0.2% Tween 80/0.5% methylcellulose) or vehicle alone. One hour postdosing, the mice received an intraperitoneal injection of LPS (1 μg/mouse) followed 2 h later by ip injection of 0.5 mL of 30 mM ATP (in PBS adjusted to pH 7). Thirty minutes later, the mice were euthanized (CO₂ inhalation) and their peritoneal cavities were lavaged with 3 mL of ice-cold PBS containing 25 U/mL heparin and 10% heat-inactivated FBS. A protease inhibitor tablet (for every 50 mL of solution) was added to the lavage buffer just prior to use. Terminal EDTA-stabilized blood samples were also collected, from which plasma fractions were isolated and analyzed for NT-0249 by LC-MS/MS analysis. Harvested lavage fluids were centrifuged to remove cells and cell debris, and the clarified samples were stored at −80 °C for cytokine measurements.

In Vivo LPS/ATP Lung Challenge Model

Mice (male, 129S6, 8.6–13.3 weeks of age) were dosed with vehicle or NT-0249 as a suspension in 0.5% methylcellulose at 1.2, 6, 10, and 30 mg/kg by oral gavage. After 60 min, the mice were administered 1 μg of LPS in PBS by oropharyngeal deposition (anesthesia drop method). Two hours later, the mice received 0.5 mL of 30 mM ATP (pH 7.2) in PBS by oropharyngeal deposition (anesthesia drop method). Two hours post-ATP administration, the mice were euthanized and blood was collected via cardiac puncture; harvested blood was stabilized with heparin and used to prepare plasma. The pulmonary circulation was perfused with saline (in order to avoid contamination of blood leukocytes), after which lungs were lavaged with 3 mL of ice-cold PBS containing 25 U/mL heparin and 10% heat-inactivated FBS. A protease inhibitor mixture was added to the lavage buffer just prior to use at one tablet per 50 mL of buffer. From the cell-free recovered bronchoalveolar lavage fluids (BALFs), levels of IL-1β were assessed by ELISA. Plasma samples were sent to Cyprotex Discovery Limited (Abingdon, U.K.) for LC-MS/MS determination of NT-0249 levels. The samples were subjected to organic solvent (3 part to 1 part plasma) protein precipitation and clarified supernatants were diluted with HPLC grade water (2 parts water/1 part supernatant) prior to LC-MS/MS analysis. Levels of NT-0249 were calculated based on comparison to a standard curve generated with authentic NT-0249.

Therapeutic Evaluation of NT-0249 in a D305N Mouse Model of CAPS

The acute LPS/ATP lung challenge model described above and the D305N mouse model studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee guidelines for the University of North Carolina at Chapel Hill. The generation and characterization of the D305N model were detailed previously.³⁴ After randomization based on litter of origin, weight, and age, female mice expressing either human NLRP3 or the D305N functional variant were placed on Open Standard Diet (Research Diets, New Brunswick, NJ) or Open Standard Diet formulated with NT-0249 at 73 mg/kg and 733 mg/kg. Different dyes were blended into the formulated chows to allow visual distinction. Mice on study were allowed ad libitum access to the designated chow for eight consecutive days, after which they were euthanized, and blood and tissues were harvested. Blood samples were stabilized with heparin and used to prepare plasma. Autopods and a segment of the tails were homogenized in 0.5% hexadecyltrimethylammonium bromide, and MPO activity was measured using O-dianisidine hydrochloride as a substrate.⁶⁶ IL-1β and IL-6 levels in tissue homogenates were assessed using ELISA kits specific for the murine cytokines.⁶⁷ IL-1ra levels in plasma were assessed by ELISA. In these studies, only female mice were employed as this allowed the cohousing of mice from different litters (litters from NLRP3 and D305N breeding cages). Males from different litters, and thus different genotypes, are challenging to cohouse due to the development of hierarchies and displays of aggressive behavior, which is not observed with female mice. The use of female mice permitted cohousing of NLRP3 and D305N mice in the same cage and limited the differential response attributed to the “cage effect.″ This effect is especially significant when mice are housed on ventilated racks as employed here. Cohousing ensures the microbiome is shared among all mice, regardless of their genotype.

Statistical analysis

Data presented were graphed and analyzed using GraphPad Prism software (version 10 sourced from Graphpad Software, Boston, MA). Individual points corresponding to compound titration curves represent the mean and standard deviation of triplicate determinations. Concentration titration curves were fit and IC₅₀ values were determined using a four-parameter logistic, variable slope model. Statistical analyses were conducted using ordinary one-way ANOVA with Dunnett’s multiple comparison test or ordinary one-way ANOVA and Tukey’s multiple comparison test.

Acknowledgments

The authors would like to thank Ed Miao and Alan Stubbs for their guidance in the generation and use of the LFn-PrgJ fusion protein employed in the NLRC4 inflammasome activation assay. Microscopy and image analysis was supported by the Cellular Imaging Shared Resource RRID:SCR_022609 of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium (P30 CA015704), and the authors particularly thank Julio Vazquez, Lena Schroeder, and Jin Meng of the Cellular Imaging Shared Resource for their assistance. Special thanks to John N. Snouwaert at UNC for his assistance in assembling of the graphical abstract (Figure Made in BioRender.com). Dr. Beverly H. Koller received grant support from NodThera.

Glossary

Abbreviations

ATP: adenosine triphosphate
ASC: PYD and CARD domain-containing protein
CAPS: cryopyrin-associated periodic syndrome
CC: cholesterol crystals
CPPD: calcium pyrophosphate dihydrate
DAMPs: danger-associated molecular patterns
EC₅₀: half-maximal effective concentration
ED₅₀: median effective dose
GFP: green fluorescent protein
GoF: gain-of-function
IL: interleukin
IL-1R-Fc: type I interleukin-1 receptor-Fc construct
IC₅₀: concentration causing 50% inhibition
LFn: lethal factor
LPS: lipopolysaccharide
MPO: myeloperoxidase
MSU: monosodium urate
NLRP3: NOD-like receptor pyrin domain-containing protein 3
NLRC4: NLR family CARD domain-containing 4
NT-0249: [(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide
PA: protective antigen
PrgJ: type III secretion inner rod protein
PAMPs: pathogen-associated molecular patterns
PBMCs: peripheral blood mononuclear cells
SUR: sulfonylurea receptor
TNFα: tumor necrosis factor α

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00061.

Methodology relating to assay control panels; compilation of NT-0249 IC₅₀ values vs IL-1β output observed in the LPS/ATP PBMC assay (Table S1); compilation of NT-0249 IC₅₀ values vs IL-18 output observed in the LPS/ATP PBMC assay (Table S2); compilation of NT-0249 IC₅₀ values vs IL-1β output observed in the LPS/ATP human blood assay (Table S3); NT-0249 PK/disposition parameters in wild-type mice (Table S4); NT-0249 impact on the release of mature caspase-1 induced by LPS/ATP (Figure S1); NT-0249 impact on IL-1β output induced by crystals (Figure S2); NT-0249 impact in an NLRP3 target engagement assay (Figure S3); baseline characteristics of mice in the NT-0249 therapeutic study (Figure S4); outcome measures from alternate day dosing study (Figure S5); validation of IL-1R-Fc pull-down approach (Figure S6) (PDF)

Author Contributions

Conceptualization: B.H.K., A.P.W., and C.A.G. Methodology: B.H.K. and J.R.D. Formal analysis: J.R.D., P.S., H.W., A.B., and M.N. Investigation: M.N., J.R.D., P.S., H.W., C.D., and K.S. Resources: D.H. and B.H.K. Writing—Original draft: C.A.G. Writing—Review and editing: J.R.D., P.S., H.W., C.D., D.H., A.P.W., B.H.K., and C.A.G. Visualization: J.R.D., P.S., H.W., and A.B. Supervision: C.A.G., B.H.K., and A.P.W.

Nodthera funded all studies.

The authors declare no competing financial interest.

Notes

J.R.D., P.S., H.W., C.D., K.S., A.B., D.H., A.P.W., and C.A.G. are current or former members of NodThera. NodThera holds a US patent relating to NT-0249, with applications pending in other jurisdictions. A.B. is currently employed by Harness Therapeutics (Babraham Cambridge, CB22 3AT, U.K.). B.H.K. and M.N. declare that they have no financial interest in NodThera.

Supplementary Material

pt4c00061_si_001.pdf^{(477KB, pdf)}

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PERMALINK

Pharmacological Analysis of NLRP3 Inflammasome Inhibitor Sodium [(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl][(1-methyl-1H-pyrazol-4-yl)({[(2S)-oxolan-2-yl]methyl})sulfamoyl]azanide in Cellular and Mouse Models of Inflammation Provides a Translational Framework

John R Doedens

Pamela Smolak

MyTrang Nguyen

Heather Wescott

Christine Diamond

Ken Schooley

Andy Billinton

David Harrison

Beverly H Koller

Alan P Watt

Christopher A Gabel

Abstract

Results

NT-0249 Selectively Inhibits IL-1β and IL-18 Output from Human Mononuclear Cells and Macrophages in Response to Lipopolysaccharide (LPS)/Adenosine Triphosphate (ATP) Activation

Figure 1.

NT-0249 Inhibits Inflammasome Assembly Dependent on NLRP3 but not NLR Family CARD Domain-Containing 4 (NLRC4)

Figure 2.

Figure 3.

Crystal-Induced IL-1β Output is Sensitive to NT-0249 Inhibition

Figure 4.

NT-0249 Demonstrates NLRP3 Target Engagement in a Cell-Based Competitive Binding Assay

NT-0249 Inhibits IL-1β Output from Wild-Type and D305N Mouse Blood

Figure 5.

NT-0249 Inhibits IL-1β Output following LPS/ATP Challenge In Vivo

Figure 6.

Figure 7.

Therapeutic Impact in the Context of the D305N Syntenic Mouse Model of Inflammation

Figure 8.

Figure 9.

Discussion

Methods

Materials

IL-1β Output from Human PBMCs and Kupffer Cells

IL-6 and TNFα Output from Human PBMCs

IL-1β Output from Blood

Crystal-Induced IL-1β Output

ASC-GFP Speck Assay

NLRC4 Inflammasome Assay

IL-1β Western Blotting and IL-1R-Fc Capture from Mouse Autopod Homogenates

In Vivo LPS/ATP Peritonitis Model

In Vivo LPS/ATP Lung Challenge Model

Therapeutic Evaluation of NT-0249 in a D305N Mouse Model of CAPS

Statistical analysis

Acknowledgments

Glossary

Abbreviations

Supporting Information Available

Author Contributions

Notes

Supplementary Material

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

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