Characterization of a small molecule inhibitor of the NLRP3 inflammasome and its potential use for acute lung injury

Abstract

Accumulating data support the key roles of the NLRP3 inflammasome, an essential component of the innate immune system, in human pathophysiology. As an emerging drug target and a potential biomarker for human diseases, small molecule inhibitors of the NLRP3 inflammasome have been actively pursued. Our recent studies identified a small molecule, MS-II-124, as a potent NLRP3 inhibitor and potential imaging probe. In this report, MS-II-124 was further characterized by an unbiased and comprehensive analysis through Eurofins BioMAP Diversity PLUS panel that contains 12 human primary cell-based systems. The analysis revealed promising activities of MS-II-124 on inflammation and immune functions, further supporting the roles of the NLRP3 inflammasome in these model systems. Further studies of MS-II-124 in mouse model of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and NLRP3 knockout mice demonstrated its target engagement, efficacy to suppress inflammatory cytokines and infiltration of immune cells in the lung tissues. In summary, the results support the therapeutic potential of MS-II-124 as a NLRP3 inhibitor and warrant future studies of this compound and its analogs to develop therapeutics for ALI/ARDS.

Graphical Abstract

1. Introduction

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is one of the cytosolic protein complexes known as inflammasomes that mediates the innate immune response in recognition of the pathogen-associated molecular patterns (PAMPs), and/or damage-associated molecular patterns (DAMPs).[1] This is achieved through regulating the maturation and release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 by the inflammasome and immediate downstream events.[2] Unlike other inflammasomes, the NLRP3 inflammasome can be activated by a plethora of stimuli, e.g., microbial products, endogenous molecules, and particulate matter.[3–7] Mechanistically, multiple molecular and cellular events have been linked to the activation of NLRP3, and this includes K⁺ efflux [8, 9], Ca²⁺ signaling [10–13], reactive oxygen species (ROS) [14], mitochondrial dysfunction [15, 16], and liposomal rupture [17]. Therefore, it not surprising that dysregulation of the NLRP3 inflammasome signaling pathway has been implicated in a variety of inflammatory diseases and neurological disorders.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a severe clinical disorder with high mortality. Unfortunately, effective treatments for ALI/ARDS are lacking due to the complex pathogenesis associated with this disease.[18] The outbreak of novel coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has evolved into a global pandemic and has been casting tremendous challenges and burdens on our societies.[19] Although the World Health Organization (WHO) has declared an end of COVID-19 as a public health emergency, due to the available vaccines and treatments, COVID-19 continues to impact our society nowadays. A subgroup of severely ill COVID-19 patients typically develop ALI/ARDS, and this is believed to be associated with the cytokine storm due to the dysregulated and exaggerated host immune responses triggered by the virus.[20] The NLRP3 inflammasome has been suggested to have critical roles in the development of ARDS/ALI in patients with COVID-19.[21, 22] Studies have also revealed the NLRP3 inflammasome to be an essential contributor to the development of ARDS/ALI.[23] A crosstalk between the production of extracellular histones and activation of NLRP3 inflammasome has also been suggested in ALI/ARDS.[18] In addition, mechanical ventilation to support patients with ALI/ARDSs causes ventilator-induced lung injury (VILI),[24] and the lung alveolar stretch can be sensed by NLRP3 inflammasome to induce lung inflammatory injury.[25] Studies from cellular and animal models established that viral proteins E, 3a, and ORF8b of SARS-CoV activate the NLRP3 inflammasome via different mechanisms.[26] Importantly, SARS-CoV and SARS-CoV-2 share an overall 82% sequence homology, and the three viral proteins E, 3a, and ORF8b that activate the NLRP3 inflammasome share amino acid identity of 95, 72, and 40%, respectively.[27, 28] This strongly supports the potential involvement of NLRP3 inflammasome in the development of ARDS/ALI in COVID-19 patients. Dysregulation of the NLRP3 inflammasome also correlates with lung infection caused by bacteria and viruses.[29] Furthermore, pharmacological suppression of NLRP3 inflammasome with small molecule inhibitors demonstrated protective effects in severe influenza A induced disease and COVID-19 in animal models.[30, 31]. Indeed, NLRP3 inhibitors are being tested in clinical trials for COVID-19 as well as other inflammatory disorders.[32]

2. Results and discussion

2.1. Development and characterization of MS-II-124 as a potent and selective NLRP3 inflammasome inhibitor.

Glyburide, an FDA approved anti-diabetic medication, was identified to exhibit anti-inflammatory effects via the NLRP3 inflammasome. Our initial structure activity relationship (SAR) studies around the cyclohexylurea moiety of glyburide led to the discovery of sulfonamide compound, JC124, as a potent NLRP3 inflammasome inhibitor with an IC₅₀ of 3.25 μM [33]. Our recent structural optimization of JC124 via a scaffold hopping strategy identified MS-II-124 (Fig. 1) as a new lead NLRP3 inhibitor with significantly improved potency (IC₅₀ of 0.12 μM) [34]. Further characterization using human recombinant NLRP3 protein and a microscale thermophoresis (MST) assay demonstrated MS-II-124 as a direct NLRP3 binder with a K_d of 84 nM. Studies in wild type (WT) and nlrp3^−/− mice further confirmed MS-II-124 as a selective NLRP3 inhibitor in vivo [34]. Notably, positron emission tomography (PET) imaging studies in mice using a PET radiotracer of MS-II-24 demonstrated significant and rapid uptake in blood and lung, and this was followed by fast clearance [34]. The biological properties of MS-II-124 are summarized in Table 1. Herein, we further characterized MS-II-124 using an unbiased and comprehensive analysis by the Eurofins BioMAP Diversity PLUS panel that contains 12 human primary cell-based systems to investigate human pathophysiology of various organs and tissues. We also tested this compound in a mouse model of ALI to explore its potential as a treatment agent for ALI/ARDS.

Fig. 1.

Structural modification and discovery of MS-II-124.

Table 1.

Biological profile of MS-II-124 as a selective NLRP3 inhibitor

Biological and Physical profile of MS-II-124
NLRP3 inflammasome inh IC50, μM	0.12 ± 0.01
NLRC4 inflammasome inh IC50, μM	> 30
AIM2 inflammasome inh IC50, μM	> 30
TNF-α IC50, μM	> 30
IL-6 IC50, μM	> 30
Binding affinity, Kd (nM)	84 ± 14
In vivo selective target engagement	Selectively inhibit IL-1β
Brain penetration	~1.8% ID/cc
Organ with high biodistribution	Lung (22.6% ID/cc)

2.2. Systemic phenotypic profiling of MS-II-124 in human primary cell systems reveals its inflammation-related and immunomodulatory activities.

The BioMAP Diversity PLUS panel of 12 human primary cell-based systems are designed to model complex human tissue and disease biology of the vasculature, skin, lung and inflammatory tissues (Fig. S1).[35, 36] BioMAP profiling of MS-II-124 in the Diversity PLUS Panel used 148 biomarker readouts (7–17 per system) from the 12 primary human cell/co-culture systems to analyze the therapeutic and biological relevance of MS-II-124 and to validate its safety, specific drug effects and disease outcomes. MS-II-124 was tested at 110, 330, 1000 and 3000 nM concentrations. Notably, MS-II-124 is not cytotoxic at the concentrations tested in this study while it showed antiproliferative activities at 3 μM to human primary T cells and at 3 μM, 1 μM and 330 nM to human fibroblasts.

As shown in Fig. 2, changes in key biomarker activities upon MS-II-124 treatment were noted in the 4H system modeling a Th2 inflammatory environment, the LPS system recapitulating monocyte-driven Th1 inflammation, the BE3C system modeling Th1 related airway inflammation of the lung, the CASM3C system modeling the Th1 inflammatory state specific to arterial smooth muscle cells, the HDF3CGF system modeling wound healing, the MyoF system modeling myofibroblast-lung tissue remodeling, and the /Mphg system modeling chronic Th1 inflammation driven by macrophage activation. The majority of the observed activities by MS-II-124 treatment in this study are immune/inflammation related activities, which is expected given that MS-II-124 has demonstrated specific inhibition on the NLRP3 inflammasome, one essential component of the innate immune system that is involved in inflammatory responses.

Fig. 2.

BioMAP profile of MS-II-124 in the Diversity PLUS Panel. The X-axis indicates the quantitative protein-based biomarker readouts measured in each system. The Y-axis represents a log-transformed ratio of the biomarker readouts for the drug-treated sample (n = 1) over vehicle controls (n ≥ 6). The grey region around the Y-axis represents the 95% significance envelope generated from historical vehicle controls. Biomarker activities are annotated when two or more consecutive concentrations change in the same direction relative to vehicle controls, are outside of the significance envelope, and have at least one concentration with an effect size > 20% (|log10 ratio| > 0.1). Biomarker key activities are described as modulated if these activities increase in some systems, but decrease in others. Cytotoxicity is indicated on the profile plot by a thin black arrow above the X-axis, and antiproliferative effects are indicated by a thick grey arrow.

Specifically, among the 14 annotated readouts upon MS-II-124 treatment (Table 2), three are immunomodulatory biomarkers and this includes reduced activity on sIL-10 (/Mphg system) and CD69 (LPS system) as well as increased activity on HLA-DR (CASM3C system). Notably, these three systems recapitulate Th1 inflammatory responses driven by immune cells. Changes were also annotated for five inflammation-related biomarkers, and this includes decreased activity of MCP-1 (4H system) and increased activity of ICAM-1/MIG (HDF3CGF system), and Esel/IL8 (/Mphg system). Treatment by MS-II-124 also led to tissue remodeling activities by increasing the activity of MMP1 (BE3C system) and EGFR (HDF3CGF system) and decreasing the activity of Col-I/Col-III (HDF3CGF system) and aSMA (MyoF system). Notably, the BEC3 and MyoF systems model lung-related functions.

Table 2.

Fourteen annotated readouts upon treatment with MS-II-124.

Biological and Disease Relevance Category	Decreased activity	Increased activity
Inflammation-related activities	MCP-1	ICAM-1, Esel, IL8, MIG
Immunomodulatory activities	slL-10, CD69	HLA-DR
Tissue remodeling activities	Col-I, Col-III, aSMA	MMP1, EGFR
Other activities		VEGFR2

2.3. Mechanistic and similarity analysis suggests a novel mechanism of action for MS-II-124

An unsupervised search for mathematically similar compound profiles from the BioMAP Reference Database of > 4,500 agents yielded no compound with a Pearson’s correlation coefficient score r > 0.7, which is the determined threshold by the Eurofin’s BioMAP Diversity PLUS panel. The similarity between agents is determined using a combinatorial approach that accounts for the characteristics of BioMAP profiles by filtering (Tanimoto metric) and ranking (BioMAP Z-Standard) the Pearson’s correlation coefficient between two profiles. Profiles are identified as having mechanistically relevant similarity if the Pearson’s correlation coefficient score r ≥ 0.7. Further Mechanism HeatMAP Analysis of the 148 biomarker readouts within the Diversity PLUS Panel upon MS-II-124 treatment in comparison to 19 consensus mechanism profiles also yielded no matches with mechanistic similarity (Fig. 3 and Table 3). Collectively, the results strongly suggest a novel and distinct mechanism of action for the observed activities by MS-II-124 in this study.

Fig. 3.

Mechanistic HeatMAP Analysis for MS-II-124. HeatMAP analysis of the 148 biomarker readouts (rows) within the Diversity PLUS Panel by MS-II-124 in comparison to 19 consensus mechanism class profiles (columns). Horizontal grey lines separate the 12 Diversity PLUS systems, while the vertical grey line separates MS-II-124 from the 19 consensus mechanism profiles. Biomarker activities outside of the significance envelope are red if protein levels are increased, blue if protein levels are decreased and white if levels are within the envelope or unchanged. Darker shades of color represent greater change in biomarker activity relative to vehicle control.

Table 3.

Top BioMAP Reference Database Matches for MS-II-124.

MS-II-124	Database Match	BioMAP Z-Standard	Pearson’s Score	# of Common Readouts	Mechanism Class
3 μM	Conivaptan Hydrochloride, 10 μM	7.735	0.567	148	Vasopressin Inhibitor
	Canagliflozin, 17 μM	7.608	0.559	148	SGLT2 Inhibitor
	Butenafine HCI, 10 μM	7.378	0.546	148	Antifungal Agent
1 μM	Rimonabant, 3.3 μM	8.152	0.591	147	CB1 Inverse Agonist
	CP 55,940, 1.1 μM	8.139	0.589	148	Cannabinoid Receptor Agonist
	Amiodarone, 3 μM	8.125	0.588	148	Anti-arrhythmic Agent
330 nM	Oleoylethanolamide, 3.3 μM	4.864	0.383	148	PPARα Agonist
	Dibucaine HCI, 1.1 μM	4.678	0.377	142	Anesthetic Agent
	Benztropine Mesylate, 1.1 μM	4.639	0.367	148	Muscarinic Acetylcholine Receptor Inhibitor
110 nM	Nialamine, 3.3 μM	4.506	0.358	148	Monoamine Oxidase Inhibitor
	MRT67307, 80 nM	4.442	0.353	148	IL-10 Receptor Agonist
	FumaricAcid, 200 μM	4.418	0.351	148	Dicarboxylic Acid

2.4. MS-II-124 attenuates pulmonary inflammation and reduce immune cell infiltration in a mouse ALI/ARDS model

Given the observed inflammation-related and immunomodulatory effects of MS-II-124 from the studies of BioMAP Diversity PLUS Panel of human primary cell systems, especially its lung-related activities, its accumulation in lung tissues,[34] and the pathophysiological roles of the NLRP3 inflammasome in ALI/ARDS, we next tested its protective activity in a mouse model of ALI/ARDS induced by intranasal (i.n.) instillation of LPS. The short-term LPS challenge of mice can stimulate mixed inflammatory response in the airway and lung, including lung epithelial and endothelial barriers disruption, inflammatory cells infiltration and proinflammatory cytokines release. These phenotypes are clinically relevant for both COVID-19 associated and classical ALI/ARDS.[37–39] When compared to the basal levels of the cytokines (ranging from 2.79–6.60 pg/mL) in the bronchoalveolar lavage fluids (BALF), LPS challenge significantly increased the release of IL-1β, TNF-α and IL-6 (Fig. 4). Comparison of LPS-treated NLRP3^−/− mice and their wild type (WT) counterparts showed that deletion of NLRP3 led to a significantly reduced production of the proinflammatory cytokine IL-1β (Fig. 4A). While a reduction of TNF-α production was also observed in LPS-exposed NLRP3^−/− mice compared to their WT counterparts (Fig. 4B), no statistical significance was achieved between these two groups. Notably, both cytokines have been shown to be elevated in patients infected with COVID-19.[40] Preventive treatment of WT C57BL/6 mice with MS-II-124 at 10 mg/kg by intraperitoneal injection (i.p.) significantly suppressed LPS-induced production of IL-1β in the BALF of lung, comparable to the effects of NLRP3 deletion. MS-II-124 treatment did not show significant effect on the level of TNF-α in the BALF of lung in the WT mice. When the level of IL-6 is compared, MS-II-124 treatment significantly suppressed its production in the BALF of lung in WT mice while NLRP3 deletion did not show significant suppression (Fig. 4C). The discrepancy observed between MS-II-124 treatment in WT mice and NLRP3 deletion may be due to the fact that MS-II-124 treatment represents a short-term and acute treatment compared to the genetic depletion of NLRP3 gene during their development stage, reflecting a long-term inhibition. More importantly, no significant difference was observed for the level of IL-1β, TNF-α and IL-6 in the BALF of lung from NLRP3^−/− mice between MS-II-124-treated and vehicle-treated groups. Collectively, these results strongly support target engagement and selectivity of the compound in vivo.

Fig. 4. MS-II-124 attenuates pulmonary inflammation in ALI/ARDS mice.

Wild type C57BL/6 (n=6) or NLRP3^−/− (n=4) mice were treated with MS-II-124 (10 mg/kg) by i.p. 1 h before i.n. instillation of LPS (40 μg in 40 μL PBS). BALF was collected 18 h after LPS exposure. Levels of cytokines in the BALF were measured by ELISA. Data as mean ± SD. Statistics by student t-test. **p < 0.01; *p < 0.05; ns, not significant.

Treatment of WT mice with a single dose of MS-II-124 (10 mg/kg, i.p.) significantly reduced the pulmonary inflammation (Fig. 5), evidenced by the decrease of total immune cells (Fig. 5A), neutrophils (Fig. 5B), and γδT cells (Fig. 5C) in the BALF of lung. Similarly, no statistical difference was observed between vehicle- and MS-II-124-treated NLRP3^−/− mice, suggesting that MS-II-124 selectively engaged the NLRP3 inflammasome in vivo and can potentially reduce lung injury under inflammatory conditions.

Fig. 5. MS-II-124 reduces immune cell infiltration into the airway in ALI/ARDS mice.

Wild type C57BL/6 (n=6) or NLRP3^−/− (n=4) mice were treated with MS-II-124 (10 mg/kg) by i.p. 1 h before i.n. instillation of LPS (40 μg in 40 μL PBS). BALF was collected 18 h after LPS exposure and the number of total cells as well as indicated immune cells in BALF were measured by FACS. Data as mean ± SD. Statistics by student t-test. **p < 0.01; *p < 0.05; ns, not significant.

We also examined the histology of the lung tissues by the hematoxylin and Eosin (H&E) staining. The infiltration of immune cells into the alveolar was evident in the lung tissues of LPS-exposed WT mice, whereas this was greatly reduced in NLRP3^−/− mice (Fig. 6A). Consistent with the results from the BALF analysis, treatment of WT mice with MS-II-124 profoundly suppressed infiltration of immune cells (Fig. 6A), while no difference was observed between vehicle-treated and MS-II-124-treated NLRP3^−/− mice. Thickening or congestion of alveolar walls was also seen in the pulmonary sections of WT mice following LPS exposure, but not in NLRP3^−/− mice or MS-II-124 treated mice (Fig. 6A). We also noticed that the body weight of animals started to drop 24 hours after dosing LPS and recovered on day 5. MS-II-124 treatment did not affect the body weight of mice when compared to vehicle-treated control group. Additionally, there were no significant differences among these groups (WT vs NLRP3^−/−) (Fig. 6B). Taken together, the results in this study not only confirm the role of the NLRP3 inflammasome in the development of ALI/ARDS, but also support the protective activity of MS-II-124.

Fig. 6. MS-II-124 reduces pulmonary inflammation in ALI/ARDS mice.

WT or NLRP3^−/− mice (n=4) were given MS-II-124 (10 mg/kg, i.p.) one hour before i.n. instillation of LPS (40 μg in 40 μL PBS). (A) Lungs were fixed in 10% formalin and subjected to H&E staining. The blue arrows depict the infiltrating neutrophils. The black arrows indicate the alveolar wall (Bar = 20 μm). (B). Body weight was monitored and measured for 5 days.

3. Conclusion

As the first line defender and one of the critical components of the innate immune system, the NLRP3 inflammasome plays important roles in regulating the maturation and release of proinflammatory cytokines IL-1β and IL-18. Recent advances in understanding the molecular basis of its activation have provided evidence to shed light on its functions in the inflammatory responses. Notably, emerging studies have demonstrated the pathophysiological roles of this inflammasome in multiple human diseases including ALI/ARDS and COVID-19. The active pursuit of small molecule inhibitors of the NLRP3 inflammasome by both Pharma and academic laboratories also attests that the NLRP3 inflammasome serves as a viable drug target to develop potential therapeutics. In this study, one of our recently developed NLRP3 inhibitors, MS-II-124, was further characterized in the BioMAP Diversity PLUS panel from Eurofins and in a mouse model of ALI/ARDS.

The results from the BioMAP profiling revealed the compound’s anti-inflammatory and immunomodulatory activities in several human primary cell systems that model monocyte and macrophage driven Th1 inflammation pathways, consistent with the fact that MS-II-124 is a selective and potent NLRP3 inhibitor. Notably, no cytotoxicity was observed for this compound in all of the systems at tested concentrations, while antiproliferative activities were observed for human fibroblasts and primary T cells. Further mechanistic analysis against the BioMAP Reference Database of > 4,500 compounds yielded no matches with mechanistic similarity, reflected by the Pearson’s correlation coefficient score r < 0.7, the threshold defined by the BioMAP Diversity PLUS panel to be significant. The results strongly indicate that the observed activities of MS-II-124 on inflammation and immune modulation may be via a novel and distinct mechanism of action, not captured by the reference compounds in the BioMAP Reference Database. Studies of MS-II-124 in a mouse ALI/ARDS model induced by LPS instillation clearly demonstrated its anti-inflammatory activities, evidenced by its significant suppression of IL-1β and IL-6 in the BALF of lungs and infiltration of immune cells into the lungs in WT mice. No significant difference was observed between the vehicle- and MS-II-124-treated groups in NLRP3^−/− mice, further supporting the in vivo target engagement and selectivity of this compound. Histology analysis of the lung tissues also demonstrated the inhibitory activity of MS-II-124 on the infiltration of immune cells into the alveolar, consistent with results from BALF analysis. Collectively, the results from both in vitro studies in human primary cell systems and in vivo studies in a mouse ALI/ARDS model strong support the anti-inflammatory and immunomodulatory activities of MS-II-124 as a NLRP3 inhibitor, and encourage further development of this compound in preclinical and clinical studies as a potential therapeutic agent for ALI/ARDS.

4. Experimental section

4.1. General notes

Mice. C57BL/6 mice were purchased from Envigo (Somerset, NJ). B6.129S6-Nlrp3tm1Bhk/J (NLRP3^−/−) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All experiments and procedures involving mice were approved by the Institutional Animal Care and Use Committees of Virginia Commonwealth University and Hunter Holmes McGuire VA Medical Center.

Reagents and antibodies. ELISA kits for mouse TNF-α and IL-6 were purchased from BioLegend (San Diego, CA). ELISA kits for mouse IL-1β were purchased from R&D Systems (Minneapolis, MN). Fluorochrome-conjugated mouse monoclonal Abs against CD11b (M1/70), Ly6G (1A8), Ly6C (HK1.4), CD3 (17A2) and γδTCR (UC7–13D5) for flow cytometry analysis were purchased from BioLegend.

4.2. Eurofins BioMAP Diversity PLUS panel analysis

Detailed protocols for the BioMAP Diversity PLUS panel of 12 human primary cell/co-culture systems and related analysis, including benchmark, similarity and mechanism HeatMAP analysis, have previously been published.[35, 36, 41–47]

4.3. ALI/ARDS model

Age-matched (6–8 weeks) WT or NLRP3^−/− mice were treated with one dose of MS-II-124 (10 mg/kg, i.p.) for one hour followed by i.n. administration of 40 μg LPS (diluted in 40 μL PBS). Bronchoalveolar lavage fluids (BALF) was collected 18 h after LPS exposure. The airway of the lung was rinsed with 1 mL PBS each time, repeated 3 times. Levels of IL-1β, TNF-α and IL-6 in the first 1 mL BALF were determined by ELISA. Total cell number or numbers of immune cell subsets in the total BALF were recorded by Flow Cytometry. Lungs were fixed in 10% formalin and subjected to H&E staining.

Highlights.

• MS-II-124 was identified as a potent, selective NLRP3 inhibitor in vitro and in vivo.

• Positron emission tomography (PET) imaging studies of MS-II-124 demonstrated significant and rapid uptake in blood and lung.

• BioMAP profiling revealed the MS-II-124 exhibited anti-inflammatory and immunomodulatory activities and may be via a novel and distinct mechanism of action.

• MS-II-124 showed potential as a treatment agent for ALI/ARDS.