KN3014, a piperidine-containing small compound, inhibits auto-secretion of IL-1β from PBMCs in a patient with Muckle–Wells syndrome

NLRP3, an intracellular pattern recognition receptor, recognizes numerous pathogens and/or its own damage-associated molecules, and forms complexes with the adaptor protein ASC. These complexes constitute the NLRP3 inflammasome, a platform for processing interleukin (IL)-1β and/or IL-18. Several NLRP3 mutations result in constitutive activation of the NLRP3 inflammasome, causing cryopyrin-associated periodic syndrome (CAPS). To the best of our knowledge, small compounds that specifically inhibit inflammasome activation through the pyrin domain (PYD) have not yet been developed. This study describes an attempt to develop small compounds targeting the NLRP3 inflammasome. A core chemical library of 9,600 chemicals was screened against reconstituted NLRP3 inflammasome in a cell-free system with an amplified luminescence proximity homogeneous assay and a cell-based assay by human peripheral blood mononuclear cells (PBMCs). Inflammasome activation was evaluated by ASC-speck formation in human PBMCs, accompanied by IL-1β secretion and processing, and by using IL-1β-based dual operating luciferase (IDOL) mice. The activity of these compounds was evaluated clinically using PBMCs from a patient with Muckle–Wells syndrome (MWS), a type of CAPS, with an R260W mutation in NLRP3. Screening identified KN3014, a piperidine-containing compound targeting the interaction between NLRP3 and ASC through the PYD. KN3014 reduced ASC-speck formation in human PBMCs, luminescence from IDOL mice, and auto-secretion of IL-1β by PBMCs from the patient with MWS. These findings suggest that KN3014 may be an attractive candidate for treatment of MWS, as well as other NLRP3 inflammasomopathies.


Results
Selection of candidate NLRP3 inflammasome inhibitors by screening with reconstituted NLRP3 inflammasome in a cell-free system. The amplified luminescence proximity homogeneous assay (ALPHA)-screen-based chemical library of 9,600 compounds was subjected to high-throughput screening using reconstituted NLRP3 inflammasome in a cell-free system 22,23 (Fig. 1a,b and Supplementary Figs. S1, S2, and S3). The sample layout is shown schematically (Supplementary Fig. S2). Screening did not involve use of a combination of constitutively active mutant C-terminal biotinylated full-length NLRP3 (NLRP3-FL-Btn) and FLAG-ASC-FL for the constructs, or a combination of wild-type NLRP3-FL-Btn and FLAG-ASC-FL with ligand, because a truncated form of ASC, containing a PYD but lacking a caspase recruitment domain (CARD), was able to interact with NLRP3 in the absence of NLRP3 ligand 22 . By contrast, a truncated form of ASC, containing a CARD but lacking a PYD, was unable to interact with NLRP3 22  In general, assays with a z-factor greater than 0.5 are considered accurate and suitable for high-throughput screening. The calculated z-factor was 0.87. Screening was performed at a final compound concentration of 1.0 µM. The inhibition rate (InH) (%) was calculated as 100 × {1 − (value of sample − value of negative control)/(value of positive control − value of negative control)}. The InH (%) of each compound is shown in a dot plot (Fig. 1c). Compounds with ± 15% InH were selected for further screening; these included two candidate inhibitors, KN3014 and KN8311, and three negative controls, KN1960, KN5330, and KN7644. The five selected compounds (KN3014, KN8311, KN1960, KN7644, and KN5330) are depicted by red, orange, green, blue, and dark blue dots, respectively (Fig. 1c).
Secondary screening of selected compounds by titration against reconstituted NLRP3 inflammasome in a cell-free system. The five selected compounds were subjected to secondary screening by quadruplicate screening against NLRP3 inflammasome ( Supplementary Fig. S3). The interaction between NLRP3-FL-Btn and FLAG-ASC-PYD was disrupted by both KN3014 and KN8311 in a dose-dependent manner. KN3014 and KN8311 had maximum InH (%) values of 56.42% and 39.24%, respectively, and IC50 values of 14.65 and 118.29 μM, respectively (Supplementary Table S1). By contrast, neither KN5330 nor KN7644 had an effect on the interaction between NLRP3-FL-Btn and FLAG-ASC-PYD. The maximum InH (%) of KN1960 was − 30.80%, promoting the interaction between NLRP3-FL-Btn and FLAG-ASC-PYD.
Cell-based screening of the five compounds with peripheral blood mononuclear cells. The ability of these five compounds to affect cytokine secretion by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) was assessed by cell-based screening with each compound tested in triplicate wells. Briefly, 1 × 10 5 human PBMCs were stimulated with 0.1 ng/mL LPS in the presence of 5.0 or 50 μM of one of the selected compounds or DMSO for 8 h, and the concentrations of IL-1β (Fig. 2a) and TNF-α (Fig. 2b) in the culture supernatants were measured in an enzyme-linked immunosorbent assay (ELISA). KN3014 was the only compound that markedly inhibited IL-1β secretion from PBMCs but had no effect on TNF-α secretion without serious cytotoxicity (Fig. 2c). KN8311 slightly reduced IL-1β secretion but had no effect on TNF-α secretion without serious cytotoxicity.  (Fig. 2e). KN5330 was identified as 3-(4-bromo-2-chlorophenylsulfonamido)-3-p-tolylpropanoic acid, with a molecular formula of C 16 H 15 BrClNO 4 S and a molecular weight of 433 daltons (Fig. 2f). KN1960 was identified as methyl 2-((3-bromo-4-methylbenzoyl)amino)benzoate, with a molecular formula of C 16 H 14 BrNO 3 and a molecular weight of 348 daltons (Fig. 2g). KN7644 was identified as 3-(5-(cyclohexylamino)-1,3,4-thiadiazol-2-yl)-6-methoxy-2H-chromen-2-one, with a molecular formula of C 18 H 19 N 3 O 3 S and a molecular weight of 357 daltons (Fig. 2h). KN3014 reduced ASC-speck formation accompanied by IL-1β processing and secretion without affecting LPS-induced pro-IL-1β production. We tested whether KN3014 or KN8311 could inhibit inflammasome activation in LPS-stimulated human PBMCs. First, we evaluated whether KN3014 or KN8311 could inhibit IL-1β processing and secretion, finding that the induction of pro-IL-1β in PBMCs treated with 0.1 ng/mL LPS was unchanged by KN3014, KN8311, or MCC950 (Fig. 3a, upper panel), whereas processing of IL-1β was inhibited by KN3014 in a dose-dependent manner (Fig. 3a, middle panel), without affecting protein expression (Fig. 3a, lower panel). The concentrations of IL-1β in the supernatants of PBMCs cultured with 0.1 ng/mL LPS were dose-dependently reduced by 5.0 and 50 μM KN3014, but not by KN8311. In comparison, 5.0 and 50 μM MCC950 completely inhibited the secretion of IL-1β into culture supernatants (Fig. 3b). www.nature.com/scientificreports/ Because ASC-speck formation is regarded as a simple upstream readout of inflammasome activation 24 , we assessed the effects of KN3014, KN8311, and MCC950 on ASC-speck formation. Consistent with their effects on IL-1β processing, KN3014 and MCC950 dose-dependently reduced ASC-speck formation by 0.1 ng/mL LPStreated PBMCs, whereas KN8311 had no effect (Fig. 3c,d). None of these compounds had an effect on NF-κB p65 nuclear translocation, as observed by immunofluorescence microscopy (Fig. 3e,f).

KN3014 reduced inflammasome activation in IL-1β-based dual operating luciferase mice.
We further tested whether KN3014 and KN8311 could inhibit inflammasome activation in a mouse model, using IL-1β-based dual operating luciferase (IDOL) mice to image IL-1β-related inflammation. Mouse splenocytes were incubated with 1.0 μg/mL LPS, in the presence of 1.0 or 10 μM KN3014 or KN8311. Both concentrations of KN3014 dose-dependently reduced the fluorescence signal from splenocytes, as did 10 μM MCC950, whereas KN8311 had no effect (Fig. 4a). Intraperitoneal injection of 0.1 μg/g body weight of LPS and 1.0 μmol/g body weight KN3014 reduced system fluorescence signal compared with intraperitoneal injection of LPS and vehicle (DMSO) (Fig. 4b,c).
KN3014 reduced spontaneous IL-1β secretion from PBMCs of a patient with Muckle-Wells syndrome, a cryopyrin-associated periodic fever syndrome. To investigate the possible clinical activity of KN3014, we assessed whether this compound could ameliorate symptoms in a patient with CAPS. This patient was a 21-year-old Japanese woman diagnosed with MWS at age 7 years, with an arginine-to-tryptophan www.nature.com/scientificreports/ mutation at position 260 (R260W) of NLRP3 25 . She was being treated with injections of 150 mg canakinumab every 2 months. A blood sample was collected immediately before canakinumab administration. PBMCs isolated from this patient and from a healthy volunteer were incubated with or without 0.1 ng/mL LPS. The PBMCs from the MWS patient spontaneously secreted IL-1β, with secretion enhanced by incubation with 0.1 ng/mL LPS, whereas the PBMCs from the healthy volunteer required LPS treatment for IL-1β secretion (Fig. 5a,b). Incubation of the patient's PBMCs with either KN3014 or MCC950 at concentrations of 0.1 μg/mL and 10 μg/ mL almost completely inhibited the spontaneous secretion of IL-1β secretion (Fig. 5a) and completely inhibited IL-1β secretion by PBMCs from the healthy volunteer (Fig. 5b). The PBMCs from the patient with MWS also spontaneously secreted TNF-α in the absence of LPS, whereas the PBMCs from the healthy volunteer did not (Fig. 5c). LPS stimulated TNF-α secretion from PBMCs of the patient with MWS and the healthy volunteer, with the amount of TNF-α secreted being lower in culture supernatants of PBMCs from the patient with MWS than the healthy volunteer (Fig. 5d). KN3014 dose-dependently reduced TNF-α secretions from PBMCs of the patient with MWS and the healthy volunteer, whereas MCC950 dose-dependently enhanced TNF-α secretion from PBMCs of the patient with MWS (Fig. 5d).   Fig. S4a-d), suggesting that KN3014 may interfere with ASC function through PYD ( Supplementary Fig. S5a-b).

Discussion
Activation of the NLRP3 inflammasome has been associated with the pathology of various diseases and with physiological aging 18 . Although innate defenses against pathogens based on the NLRP3 inflammasome-IL-1β axis are thought to be important, excess activation can lead to cell and tissue damage 21 . Therapeutic agents targeting IL-1β, such as anakinra, rilonacept, and canakinumab, have been used to treat patients with autoinflammatory diseases, such as CAPS, which involve gain of function mutations of NLRP3; however, IL-1β-targeting biologic agents affect various types of signals from inflammasomes and/or non-specific proteases that result in the accumulation of IL-1β 21 . Patients with CAPS and/or inflammasomopathies require treatment with small compounds that target the inflammasome. The present study describes a piperidine-containing small compound that targets the interaction between NLRP3 and ASC through PYD. This compound also inhibited the constitutive autosecretion of IL-1β by PBMCs from a patient with MWS. A total of 9,600 compounds were screened to identify those that could reduce the interaction between NLRP3 and the PYD of ASC, called the NLRP3 inflammasome, in a cell-free system with ALPHA 22 (Fig. 1a-c and Supplementary Figs. S1, S2, and S3). Because this reconstituted NLRP3 inflammasome is highly customized for drug discovery based on protein-protein interactions between the PYDs of NLRP3 and ASC, the identified candidates may be highly specific inhibitors targeting the NLRP3 inflammasome itself (Fig. 1a,b). Previous drug screening using ALPHA in the Proteo-Science Center of Ehime University identified small compounds with high InH (> 50%) that could disrupt a broad range of interactions non-specifically. Two compounds, KN3014 and www.nature.com/scientificreports/ KN8311, targeting the NLRP3 inflammasome were selected. As controls for secondary screening, three additional compounds, KN1960, KN5330, and KN7644, with InH values (< 0%) were selected (Fig. 1c). Titration experiments on secondary screening revealed that both KN3014 and KN8311 inhibited cell-free reconstituted NLRP3 inflammasome in a dose-dependent manner. Because KN3014 has an IC50 of approximately 14.65 μM, it may have clinical potential. By contrast, the IC50 of KN8311 was approximately 118.29 μM even in the cell-free model. Because KN5330 and KN7644 did not show functional curves, and KN1960 was likely to act as a ligand of the NLRP3 inflammasome, these compounds were not further analyzed ( Supplementary  Fig. S3 and Supplementary Table S1).
Cell-based screening using normal human PBMCs found that 50 μM KN3014 strongly inhibited IL-1β secretion, consistent with the results of primary and secondary screening with the NLRP3 inflammasome in the cell-free system. By contrast, 50 μM KN3014 did not inhibit TNF-α secretion by LPS-stimulated human PBMCs without serious cytotoxicity (Fig. 2a-c).
KN3014 and KN8311 each contain an internal methyl phenyl amine, which may be essential for targeting the NLRP3 inflammasome, with KN3014 containing a piperidine structure (Fig. 2d-h). Similar to the NLRP3 inflammasome inhibitor MCC950, neither KN3014 nor KN8311 affected Nod2-and RIPK2-dependent or intrinsic NF-κB activation, suggesting that neither KN3014 nor KN8311 affected the secretion of other cytokines induced by NF-κB, such as TNF-α (Fig. 2i).
Next, we tested whether KN3014 and KN8311 could inhibit inflammasome activation. Because ASC-speck formation with IL-1β processing is an indicator of inflammasome activation 24 , both immunoblotting and immunofluorescence microscopy were utilized to evaluate ASC-speck formation. KN3014 reduced ASC-speck formation, accompanied by IL-1β processing and secretion, without affecting LPS-induced pro-IL-1β production or activation of NF-κB ( Fig. 3a-f). This finding confirmed that KN3014, as well as MCC950, could inhibit inflammasome activation.
We also tested whether KN3014 or KN8311 could ameliorate IL-1β-dependent systemic inflammation in an animal model. Consistent with the above results, both KN3014 and MCC950 dose-dependently reduced the luminescence of splenocytes obtained from IDOL mice incubated with 1 μg/mL LPS; however, KN8311 had no effect. KN3014 also reduced systemic luminescence of splenocytes from IDOL mice treated with LPS ( Fig. 4a-c). The results suggest that KN3014 actually inhibits the NLRP3 inflammasome, ameliorating IL-1β-dependent systemic inflammation in this animal model.
Residual signals were observed in IDOL mice treated with maximum concentrations of KN3014 and MCC950. LPS activation of the mouse IL-1β promoter fused to the luciferase-IDOL-construct in IDOL mice can result in the detection of signal, even when the luciferase-IDOL construct is spliced to the inflammasome. However, the luminescence signal of the unspliced form may persist. The difference between human and mouse sequences of the NLRP3 inflammasome may result in a residual signal, even after treatment with KN3014 or MCC950 ( Supplementary Fig. S1).
Finally, we incubated KN3014 with PBMCs obtained from a patient with MWS. Because this patient had last been treated with canakinumab 2 months earlier, the serum concentration of canakinumab had decreased to the extent that IL-1β secretion from her PBMCs had increased. In the absence of stimulation, PBMCs from this patient secreted IL-1β and TNF-α. Both KN3014 and MCC950 dose-dependently reduced the spontaneous auto-secretion of both IL-1β and TNF-α, suggesting that KN3014 may be a candidate therapeutic agent for the treatment of patients with CAPS ( Fig. 5a-d).
We previously reported that PBMCs from this patient with MWS spontaneously secreted IL-1β, whereas TNF-α secretion was inhibited even when these PBMCs were stimulated with LPS 25 . Because this patient was receiving the anti-IL-1β agent with canakinumab, the secretion of TNF-α by her PBMCs may indicate a dysfunction in a negative feedback loop 25 .
PBMCs from this patient showed a greater response to LPS stimulation than PBMCs from a normal volunteer. Secretion of IL-1β and TNF-α was not completely inhibited by 10 μg/mL KM3014 or MCC950, although both appeared effective in this MWS patient with an R260W NLRP3 mutation. These findings were consistent with results showing that MCC950 was effective in a mouse model of CAPS 26 .
Interestingly, MCC950 dose-dependently altered TNF-α secretion from LPS-stimulated PBMCs from the patient with MWS but not from the normal volunteer (Fig. 5d), suggesting that TNF-α secretion may be a tradeoff between IL-1β secretion and TNF-α secretion in response to an NLRP3 inflammasome-dependent mechanism 25 . KN3014 did not markedly affect TNF-α secretion, suggesting that the mechanisms of inhibition by KN3014 and MCC950 differ.
Indeed, KN3014 targets the NLRP3 inflammasome by inhibiting the interaction between the PYD domains of NLRP3 and ASC, whereas MCC950 targets the Walker B ATP-hydrolysis motif located in the nucleotide-binding oligomerization domain 27,28 . This difference in target site may explain the difference in physiological responses to KN3014 and MCC950, with KN3014 interfering with ASC function (Supplementary Figs. S4 and S5). The results provide new insight into mechanisms regulating the NLRP3 inflammasome.
In summary, this study identified KN3014 as a new small compound targeting the interaction between NLRP3-PYD and ASC-PYD of the NLRP3 inflammasome. Identification of this compound could lead to potential new therapeutic agents for patients with CAPS, as well as offer new insights into the mechanisms regulating the NLRP3 inflammasome and its inflammasomopathies.
Chemical library screening. Details of the screening of the ALPHA screen-based chemical library are schematically described and depicted in Fig. 1. In brief, 9,600 synthesized chemicals established by the Drug Discovery Initiative (University of Tokyo, Tokyo, Japan) were dissolved in DMSO, and 250 nL of each, at a concentration of 120 μM, was added to each well of an Optiplate-384. To each well was added 9.8 µL of the NLRP3-FL mixture containing 1 µL biotinylated NLRP3-FL using a FlexDrop dispenser (PerkinElmer). In addition, 10 µL of an ASC-PYD mixture containing 1 µL FLAG-ASC-PYD was added to sample wells and positive control wells using a FlexDrop dispenser, and 10 µL of an ASC-CARD mixture containing 1 µL FLAG-ASC-CARD was added to negative control wells using a Picus NxT dispenser (Sartorius, Goettingen, Germany). Subsequently, 10 µL of a mixture of donor beads and acceptor beads was added to each well using a Cytotoxicity assay. Compound cytotoxicity was assessed using CytoTox96 non-radioactive cytotoxicity assays (Promega), which measure the release of lactate dehydrogenase (LDH). LDH concentration was measured based on the absorbance of the solution at 490 nm.
Criteria supporting inflammasome activation. Because ASC-speck formation is regarded as a simple upstream readout for inflammasome activation, the ratio of ASC-speck formation was evaluated as a criterion supporting inflammasome activation 24  IL-1β-based dual operating luciferase mice. Inflammasome activation in vivo was evaluated using IDOL mice (TansGenic Inc., Kobe, Japan) 32 . The IDOL gene contains a mouse IL-1β promoter, luciferase, mouse IL-1β partial cDNA, and the CL1-PEST degradation signal in frame 32  Isolation of splenocytes from IDOL mice. Spleens were harvested from IDOL mice, placed in 10 mL RPMI 1640 medium in a 100 mm plastic culture dish, and ground up using the rough side of an autoclaved slide glass. Whole cells were collected and passed through a 40 μm cell strainer (Greiner Bio-One GmbH, Kremsmünster, Austria) and collected in a 50 mL round tube. Cells were spun down, and the supernatant was removed. The cell pellet was resuspended (final concentration, 1 × 10 6 cells/mL) for 4 h in RPMI 1640 medium containing 1.0 μg/mL LPS, and 1.0 or 10 μM KN3014 or KN8311.
Cytokines secreted by cultured human peripheral mononuclear cells. Cytokine secretion assays were performed as described 25 . Briefly, PBMCs were cultured at a density of 1 × 10 5 /mL or 1 × 10 6 /mL in 1 mL RPMI1640 containing 10% heat-inactivated FBS per well of a 24-well flat-bottom plate (BD Biosciences, San Jose, CA, USA) in the presence or absence of lipopolysaccharides (LPS) from Escherichia coli O55:B5, cellculture tested and purified by phenol extraction (Sigma-Aldrich, St. Louis, MO, USA) for 8 h at 37 °C in a humidified atmosphere with 5% CO 2 . Concentrations of IL-1β and TNF-α in the supernatants were measured by ELISA with specific antibodies (BD Biosciences) 25 .
Evaluation of ASC-speck formation in peripheral blood mononuclear cells. ASC-speck formation was evaluated by immunofluorescence microscopy using anti-human ASC mAb. Briefly, separated human mononuclear cells were incubated with 0.1 ng/mL LPS plus KN3014, KN8311, MCC950, or vehicle (DMSO) for 8 h, and fixed with 70% ethanol at − 20 °C for 30 min. The fixed cells were incubated with anti-ASC mAb followed by goat-anti-mouse Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch, 115-546-146).

Statistical analysis.
All results are presented as the mean ± standard deviation (SD) of data from three independent experiments and compared by Mann-Whitney U-tests. A p-value < 0.05 was considered statistically significant.
Ethics approval and guidelines for human experiments. Analysis of samples from the healthy volunteer and the patient were approved by the Human Research Ethical Committees of Ehime University (No. 1712006) and Shinshu University with the patient-supplying written informed consent (No. 476). All experiments were performed in accordance with the relevant guidelines and regulations for human samples. All normal human PBMCs used in the study were separated from a healthy male volunteer. Human PBMCs from the healthy control and the patient with MWS were separated by Ficoll-gradient centrifugation, according to the manufacturer's instructions (GE Healthcare Biosciences AB, Piscataway, NJ, USA). The patient with MWS is a 21-year-old Japanese woman diagnosed with a CAPS/MWS at the age of 7 years; she harbors an arginine-to-tryptophan mutation at position 260 (R260W) of NLRP3 25 . She was being treated with injections of canakinumab (150 mg every 2 months); therefore, her PBMCs were separated just before injection to avoid the effects of medication.

Ethics approval and guidelines for animal experiments.
Mouse experiments were approved by the animal ethics committee of Ehime University and were performed in accordance with the relevant guidelines for care and use of animals.