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. Author manuscript; available in PMC: 2019 Jun 15.
Published in final edited form as: J Immunol. 2018 May 4;200(12):4036–4043. doi: 10.4049/jimmunol.1700693

Small molecule mimetics of α-helical domain of IRAK2 attenuate the pro-inflammatory effects of IL-33 in asthma-like mouse models

Jinghong Li 1,3,§, Kunio Saruta 2, Justin P Dumouchel 1,3, Jenna M Magat 1,3, Joanna L Thomas 1, Dariush Ajami 2, Mitra Rebek 2, Julius Rebek Jr 2, Timothy D Bigby 1,3
PMCID: PMC5988972  NIHMSID: NIHMS959947  PMID: 29728508

Abstract

Interleukin-33 (IL-33) and its receptor ST2 play important roles in airway inflammation and contribute to asthma onset and exacerbation. IL-33/ST2 signaling pathway recruits adapter protein myeloid differentiation primary response 88 (MyD88) to transduce intracellular signaling. MyD88 forms a complex with interleukin receptor associated kinases (IRAKs), IRAK4 and IRAK2, called the Myddosome (MyD88-IRAK4-IRAK2). Myddosome subsequently activates downstream NF-kB, MAPKs p38 and JNK. We established an asthma-like mouse model by intratracheal administration of IL-33. The IL-33 model has very similar phenotype compared with the ovalbumin (OVA) induced mouse asthma model. The importance of MyD88 in the IL-33/ST2 signaling transduction was demonstrated by the MyD88 knock-out mice were protected from the IL-33 induced asthma. We synthesized small molecule mimetics of the α-helical domain of IRAK2 with drug-like characteristics based on the recent advances in the designing of α-helices compounds. The mimetics can competitively interfere the protein-protein interaction between IRAK2 and IRAK4, leading to disruption of Myddosome formation. A series of small molecules were screened using an NF-kB promoter assay in vitro. The lead compound, 7004, was further studied in the IL-33 induced and OVA induced asthma mouse models in vivo. Compound 7004 can inhibit the IL-33 induced NF-kB activity, disrupt Myddosome formation and attenuate the pro-inflammatory effects in asthma-like models. Our data indicate that the Myddosome may represent a novel intracellular therapeutic target for diseases in which IL-33/ST2 plays important roles, such as asthma and other inflammatory diseases.

Keywords: Interleukin-33, ST2, Myddosome, asthma, α-helical domain mimetics

Introduction

Interleukin-33 (IL-33) was identified as the functional ligand for the IL-1 receptor (IL-1R) family member ST2 (1). IL-33 is constitutively expressed in structural cells such as bronchial and alveolar epithelial cells, endothelium, bronchial smooth muscle cells, and fibroblast cells. IL-33 is also expressed in dendritic cells, macrophages, and mast cells (1). ST2 belongs to the toll-like receptor (TLR)/IL-1R (TIR) superfamily (2). The ST2 gene also encodes a soluble molecule by alternative splicing (3). Soluble ST2 (sST2) is not thought to induce signaling, and instead it acts as a decoy receptor for IL-33 (4). Polymorphisms of human IL-33 and ST2 genes are associated with asthma (5–7) and increased numbers of eosinophils (6).

The important roles for IL-33 and ST2 in asthma are further supported by recent studies that reveal IL-33 is a contributing factor to airway inflammation and asthma exacerbation (8, 9). IL-33 increases ST2 expression and IL-13 production in eosinophils (10). IL-33 dysregulates the regulatory T cells leading to impaired immunologic tolerance and promotes innate and adaptive type 2 immunity in the lung (11). In addition, IL-33 loss-of-function mutation reduces blood eosinophil counts and people with this mutation are protected from asthma (12). ST2 knock-out mice are partially protected from house dust mite induced asthma, especially in the peripheral lung (13). The serum concentrations of sST2 are increased during asthma acute exacerbation, suggesting that the host tries to attenuate IL-33 signaling (14). These findings suggest the IL-33/ST2 signaling pathway is involved in both the onset and the acute exacerbations of asthma (15). Therefore, blocking the IL-33/ST2 signaling pathway may have therapeutic potential for asthma (16, 17).

On a molecular level, IL-33/ST2 signaling pathway recruits adapter protein myeloid differentiation primary response 88 (MyD88) to transduce intracellular signaling. In animal allergic asthma models, IL-1R/MyD88 signaling is critical for the initiation of inflammatory response in the lung (18). MyD88 forms a complex with interleukin receptor associated kinases (IRAKs), IRAK4 and IRAK2, called the Myddosome (MyD88-IRAK4-IRAK2) (19). X-ray crystallography showed that the complete Myddosome structure is formed with 6 MyD88s, 4 IRAK4s, and 4 IRAK2s (19). After the formation of the Myddosome, phosphorylated IRAK-1 dissociates from MyD88 and then associates with TRAF-6, which in turn causes downstream activation of NF-kB, MAPKs p38 and JNK (20). The Myddosome structure has made it a possible target for blocking IL-33/ST2 signaling. In order for the Myddosome to signal, the MyD88-IRAK4 complex must bind IRAK2. MyD88-IRAK4 and IRAK4-IRAK2 protein-protein interactions have different binding properties, which allow for specific configurations of the Myddosome (21).

It was reported that systemic and intra-nasal administration with IL-33 leads to eosinophilia, epithelial cell hypertrophy and increased mucus secretion in mice (22). IL-33 augments the production of Th2 cytokines IL-4, IL-5 and IL-13 (1, 23). In this study, we demonstrated that intratracheally administration of IL-33 for 4 days at a much lower dose (500ng) induces an asthma-like phenotype mouse model. The importance of MyD88 in the IL-33/ST2 signaling transduction was demonstrated by the MyD88 knock-out mice were protected from the IL-33 induced asthma. In order to disrupt the Myddosome formation, we synthesized a series of small molecule mimetics of the α-helical domain of IRAK2. The mimetics can competitively bind with IRAK4, therefore interfering the protein-protein interaction between IRAK2 and IRAK4. The design of the mimetics involves the scaffolds with mimicry side-chain residues on the α-helical domain. These residues play a key role in mediating IRAK4-IRAK2 protein-protein interactions and signaling transduction. The new generation of the scaffolds have more drug-like characteristics than the old generations (24). We first screened the effects of the small molecule mimetics on IL-33 induced NF-kB transcriptional activity in T cells. We further studied the lead compound 7004 in the IL-33 induced and OVA induced asthma-like mouse models. We found compound 7004 can attenuate the pro-inflammatory effects of IL-33 in vitro and in vivo.

Materials and Methods

Reagents, cell culture and reporter assays

EL4 murine thymoma cells were purchased from ATCC (Manassas, VA). Cells were grown in RPMI supplemented with 10% FBS. For promoter assay, EL4 cells were stably transfected with a NF-kB promoter-luciferase reporter, pGL4.32 (Promega, Sunnyvale, CA). The cells were exposed to varying concentrations of IL-33 (Cell Sciences, Canton, MA) to test its effect on NF-kB transcriptional activity. The cells were also preincubated with compound 7000 series for 30 minutes before IL-33 (10 nM) treatment of 4 hours. The effect of compound 7000 series on IL-33 induced NF-kB transcriptional activity was measured by the luciferase activity (Promega, Sunnyvale, CA).

Co-immunoprecipitation and Western blot analysis

For co-immunoprecipitation experiments, 100 μl of total cell lysate were incubated with 50 μl of anti-IRAK2 (abcam, Cambridge, MA) conjugated Dynabeads (Life Technologies, Grand Island, NY) for 1 hour at room temperature according to manufacturer’s manual. Proteins were separated in 10% SDS/PAGE and transferred to nitrocellulose membrane. The membrane was blocked for 1 hour in blocking buffer for fluorescent Western Blot (Rockland, Gilbertsville, PA). Proteins were detected by Western blot by using anti-IRAK4 antibody (abcam, Cambridge, MA). Fluorescent-labeled secondary antibody (Li-Cor Biosciences, Lincoln, NE) was used to detect the protein bands. An Odyssey imaging system imager was used to capture the images (Li-Cor Biosciences, Lincoln, NE).

Isolation of mouse primary spleen CD4+ cells

We isolated mouse primary spleen CD4+ cells using negative selection by magnetic-activated cell sorting system (Miltenyi Biotec Inc., Auburn, CA). The CD4+ T cells were grown in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum. CD4+ T cells were stimulated with PMA (100ng/ml) for 12 hours and then were preincubated for 30 minutes with vehicle or 7004. The cells were then treated with IL-33 (10 nM) for 4 hours. Cells culture supernatant was collected and IL-5, IL-13 were measured by ELISA.

IL-33 induced and OVA induced asthma-like phenotype in mouse models

All studies were performed according to NIH Guidelines for the Care and Use of Laboratory Animals, and approved by the Institutional Animal Care and Use Committee of the San Diego VA Healthcare System. Mouse strains studied included C57BL/6 from Jackson Laboratories (Bar Harbor, ME), MyD88 knockout from Dr. S. Akira (Research Institute for Microbial Disease, Osaka University, Japan). IL-33 Model: 6–8-week-old mice were intratracheally administrated with 500 ng of IL-33 per day for 4 consecutive days. OVA Model: 6–8-week-old mice were immunized and sensitized to OVA using a 21-day protocol. Mice were immunized by intraperitoneal injection of 50 μg OVA and 1 mg alum in PBS on day 0 and day 7. Followed by 4 intratracheal injections of 20 μg OVA in 50 μl of PBS on days 17, 18, 19 and 20. On day 5 for the IL-33 model or on day 21 for the OVA model, the mice were intubated with a 20 gauge IV catheter and placed on a computer-controlled small animal ventilator (Flexivent, SCRIEQ, Montreal, Canada) delivering 2% isoflurane continuously (25). Baseline airway resistance was measured. The animals were then challenged with an ultrasonic aerosol of 1.5, 3, 6, 12 and 24 mg/ml of methacholine. The peak airway resistance (Rrs) with each dose was obtained. Following the methacholine challenges, the animals were harvested. Bronchoalveolar lavage (BAL) fluid was obtained with 0.5 ml of PBS repeated for 3 times. The total cell counts and differentiation of the cells in BAL were studied. BAL was collected and IL-5, IL-13 levels were measured by ELISA.

Synthesis of small molecule mimetics of α-helical domain of IRAK2

In the MyD88-IRAK4-IRAK2 complex, W62, W63 and M66 in α-helix of IRAK2 were confirmed to interact with IRAK4 via X-ray crystallography study and furthermore, mutagenesis showed that two of these residues (W62 and M66) were critical for signal transduction (19). In order to mimic these three essential residues and inhibit signal transduction, we devised a pyrazinyl piperazine scaffold that resembles the IRAK2 interface in IRAK4-IRAK2 interactions. The scaffold allows for the presentation of side-chains in a similar orientation to the i, i+1, i+4 of the α-helix of IRAK2. A series of the small molecule mimetics of the α-helical domain of IRAK2 were created based on the pyrazinyl piperazine scaffold by adding a variety hydrophobic aromatic and alkyl groups (see results).

In vivo osmotic minipump delivery of compound 7004

To reduce the toxicity due to vehicle, different solvents were tested to dissolve 7004. A solution of 50% DMSO, 30% Polyethylene glycol, 15% ethanol, and 5% PBS was found to be the least toxic vehicle without reducing the solubility of compound 7004. The Alzet model 2001 osmotic minipump (Alzet Corporation, Palo Alto, CA) has a reservoir of 200 μl capacity with a mean pumping rate of 1.0 μl/h for 1 week duration. The minipump device was implanted subcutaneously in mice on the back. Treatment with compound 7004 started 1 day before the first dose of IL-33 or on day 16 of the OVA protocol (the day before starting intratracheal challenges). The minipump delivered 1 μl/h of 7004 at 20 mM (9.5μg/h) or vehicle. This dose is equivalent to 0.8 μmol/kg/h by infusion therapy. In all cases, vehicle solution was used as control. Compound 7004 or vehicle was continuously delivered subcutaneously until harvest.

Statistical analysis

ANOVA was performed using GraphPad Prism software (La Jolla, CA). Statistical analyses of data were performed using paired and unpaired Student’s t-tests or using one-way ANOVA. Statistical significance is defined by p<0.05.

Results

IL-33 induces mouse asthma-like phenotype and MyD88 is necessary to mediate IL-33 signaling

The comparison of the IL-33 induced and OVA induced mouse asthma models is in Figure 1A. It takes 21 days to establish the OVA model. It takes 4 days to establish the IL-33 model. IL-33 induced models have airway hyperresponsiveness (AHR) very similar compared to OVA models (Figure 1B). We found that IL-33 induced models had high cell counts in their BAL, indicating a large inflammatory cell infiltrate with eosinophilia infiltration (Figure 1C). Next, we looked for Th2 cytokine expression in the BAL. Increases in the Th2 cytokines, IL-5 and IL-13, were more robust in IL-33 induced models compared with OVA models (Figure 1D). This is likely mediated by the type 2 innate lymphoid cells (ILC2). ILC2 cells are known to produce high levels of IL-5 and IL-13 in response to IL-33 treatment (26). MyD88 knock-out mice given IL-33 did not develop an asthma-like phenotype. They had no significant differences in airway resistance (Figure 1E), total cell counts, and cell differentials compared to the saline controls (Figure 1F).

Figure. 1. IL-33 induces mouse asthma-like phenotype and MyD88 is necessary to mediate IL-33 signaling.

Figure. 1

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A. Comparison of IL-33 and ovalbumin (OVA) induced asthma-like phenotype models. OVA Model: Mice are immunized on day 0 and day 7, then undergo intratracheal challenge with OVA on day 17, 18, 19, and 20. The mice are studied on day 21. IL-33 Model: Mice undergo intratracheal challenge with IL-33 on day 0, 1, 2, and 3. The mice are studied on day 4.

B. IL-33 induced airway hyperresponsiveness (AHR) is similar to OVA induced AHR. The peak airway resistance (Rrs) with series of methacholine was obtained. * P < 0.01 compared with Saline control (n=8).

C. The bronchoalveolar lavage (BAL) fluids total cell counts and differentials of macrophages, neutrophils, lymphocytes, and eosinophils were obtained. IL-33 increases the inflammatory cell infiltrate in the lung, specifically causes eosinophilia, comparable to OVA induced model. * P < 0.01 compared with Saline control (n=8).

D. IL-33 induces a more robust Th2 cytokine response in BAL. IL-5 and IL-13 are both elevated. * P < 0.01 compared with Saline control (n=8).

E. Wildtype C57BL/6 or MyD88 knock-out mice were intratracheally administrated with IL-33. In the wildtype mice, IL-33 group has increased airway resistance compared with Saline group (n=8). * P < 0.01 compared with Saline control. In the MyD88 knock-out mice, no significant differences of airway resistance between IL-33 and Saline groups (n=8).

F. The BAL total cell counts and differentials of macrophages, neutrophils, lymphocytes, and eosinophils were obtained. In the wildtype mice, IL-33 increased total cell counts and eosinophils compared with Saline group (n=8). * P < 0.01 compared with Saline control. In the MyD88 knock-out mice, no significant differences of cell infiltration or eosinophilia between IL-33 and Saline groups (n=8).

Design and synthesis of small molecule mimetics of α-helical domain of IRAK2

For IL-33/ST2 signaling, MyD88 first binds to IRAK4 via V43, A44, E52, Y58, I61 and R62 residues to form a binary complex. The interacting amino acids of IRAK4 are R12, V16, R20, E69, T76 and N78 (19). The MyD88-IRAK4 complex recruits IRAK2 to form a ternary Myddosome complex (19). Structure-based mutagenesis indicates that E25, Q50, F51, R54, and A94 residues in IRAK4, and Y6, W62, M66, R67 in IRAK2 are critical residues for IRAK4-IRAK2 engagement. The protein-protein interaction at the IRAK4-IRAK2 interface involves α-helices. Three amino acids in the α-helical domain of IRAK2, including W62, W63 and M66, are essential for the IRAK2-IRAK4 protein-protein interaction (Figure 2A). Furthermore, mutagenesis showed that two of these residues (W62 and M66) were critical for signal transduction (Figure 2A). A pyrazinyl piperazine scaffold that resembles the IRAK2 interface involved in the IRAK2-IRAK4 interaction was synthesized (Figure 2B). A series of the small molecule mimetics of the α-helical domain of IRAK2 were created based on the pyrazinyl piperazine scaffold by adding side-chains in order to improve solubility, absorption, distribution, metabolism, and excretion (ADME) profile. (Figure 2C).

Figure. 2. Design and synthesis of small molecule mimetics of α-helical domain of IRAK2.

Figure. 2

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A. X-ray structure of MyD88-IRAK4-IRAK2 complex (PDB: 3MOP) indicated that W62, W63 and M66 in α-helix of IRAK2 (shown in magenta) interacted with IRAK4.

B. Pyrazinyl piperazine as a novel α-helical mimetic scaffold to allow for the presentation of side-chains in a similar orientation to the i, i+1, i+4 on α-helix.

C. α-helical domain of IRAK2 mimetics (7000 series) were synthesized based on diversity of hydrophobicity and improvement of solubility and absorption, distribution, metabolism, and excretion (ADME) profile.

Compound 7004 and 7009 inhibit IL-33 induced NF-kB transcriptional activity in a dose-dependent manner by disrupting Myddosome formation

In order to examine if IL-33 can induce NF-kB transcriptional activity in vitro, we analyzed a dose-dependent curve of IL-33 in an NF-kB promoter reporter assay. EL4 T cells stably transfected with an NF-kB promoter luciferase reporter, pGL4.32, were used for the in vitro studies. As shown in Figure 3A, IL-33 induced NF-kB transcriptional activity in a dose-dependent manner. We then studied our small molecule mimetics of α-helical domain of IRAK2 for their effects on IL-33 induced NF-κB transcriptional activity. The cells were preincubated with each small molecule compound for 30 minutes before IL-33 treatment (Figure 3B). Dose response curves of the most effective compounds, 7004 and 7009, were generated and IC50s were calculated. Lipophilic compounds 7004 and 7009, inhibited IL-33 induced NF-kB reporter activity with IC50s of 9.7μM and 22 μM, respectively (Figure 3C). Water-soluble compounds 7002, 7005, and 7011 had modest inhibitory activities and higher IC50s (data not shown).

Figure. 3. Compound 7004 and 7009 inhibit IL-33 induced NF-kB transcriptional activity in a dose-dependent manner by disrupting Myddosome formation.

Figure. 3

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A. IL-33 induced NF-kB transcriptional activity in dose-dependent manner. EL4 cells stably transfected with a NF-kB promoter-luciferase reporter, pGL4.32 were exposed to IL-33 (0, 10 nM, 100 nM) for 4 hours. Cells were lysed and NF-kB transcriptional activity was measured by the luciferase activity (n=6). * P < 0.01 compared with control. ** P < 0.01 compared with lower concentrations.

B. Screening of the small molecule mimetics of α-helical domain of IRAK2 (7000 series). All compounds were screened at 100 μM concentrations. EL4 cells were preincubated for 30 minutes with individual compound, and then were exposed to IL-33 (10 nM) for 4 hours. Cells were lysed and NF-kB transcriptional activity was measured by the luciferase activity (n=6). * P < 0.01 compared with IL-33 or IL-33+DMSO controls.

C. Dose dependent curves and IC50 of compounds 7004 and 7009. EL4 cells were preincubated for 30 minutes with the varying concentrations of 7004 and 7009 (0–100 μM), and then were exposed to IL-33 (10 nM) for 4 hours. The IC50 values for compounds 7004 and 7009 were 9.7 and 22.1 μM, respectively (n=6).

D. Compound 7004 disrupted the Myddosome formation. The EL4 cells were preincubated for 30 minutes with vehicle or 7004 and then were treated with IL-33 for 1 minute, 5 minutes, and 30 minutes. IL-33 exposure of EL4 T cells induced a significant increase in IRAK2-IRAK4 association at 5-minute and 30-minute (lane 4, 6). Preincubation of the cells with compound 7004 decreased the IRAK2-IRAK4 association (lane 5, 7) (n=6). * P < 0.05 compared with vehicle control.

E. Isolated mouse primary spleen CD4+ cells were preincubated for 30 minutes with vehicle or compound 7004. The cells were then treated with IL-33 (10 nM) for 4 hours. Cells culture supernatant was collected and IL-5, IL-13 were measured by ELISA. Compound 7004 partially decreases the IL-5, IL-13 levels in culture supernatant (n=6). *P < 0.01 compared with control without IL-33. ** P < 0.05 compared with DMSO and compoind 7004 groups.

To analyze the specificity of compound 7004 in disrupting Myddosome formation, we performed co-immunoprecipitation by using antibodies specific for IRAK2 and IRAK4. Treatment of IL-33 results in a significant increase in IRAK2-IRAK4 association in EL4 T cells, demonstrated by co-immunoprecipitation studies. The IL-33 induced increase of IRAK2-IRAK4 association can be seen 5 minutes after receiving IL-33, and the effect remained after 30 minutes (Figure 3D, lane 4, 6). Preincubation of the cells with compound 7004 for 30 minutes decreases the IRAK2-IRAK4 association (Figure 3D, lane 5, 7). We further isolated mouse primary spleen CD4+ cells. The cells were preincubated for 30 minutes with compound 7004 before treatment with IL-33. In the cell culture supernatants, preincubation with compound 7004 decreases the IL-5, IL-13 production (Figure 3E).

Compound 7004 attenuates the pro-inflammatory effects in vivo in IL-33 induced and OVA induced asthma-like mouse models

Next, we analyzed the effects of compound 7004 in asthma-like mouse models. Compound 7004 was continuously delivered by osmotic minipump subcutaneously. We found that compound 7004 partially blocked the IL-33 induced and OVA induced AHR (Figure 4A). Additionally, inflammatory cell infiltration, including eosiniophils, were reduced in BAL (Figure 4B). Compound 7004 also partially decreased IL-5 and IL-13 in BAL (Figure 4C).

Figure. 4. Compound 7004 attenuates the pro-inflammatory effects in vivo in IL-33 induced and OVA induced asthma-like mouse models.

Figure. 4

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A. In the IL-33+Vehicle group, the mice have increased airway resistance compared with Saline+Vehicle group (n=6). In the IL-33+7004 group, the mice have mildly increased airway resistance compared with Saline+vehicle group at low doses of methacholine, but no significant differences at high doses of methacholine (12, 24 mg/ml). At high doses of methacholine, IL-33+7004 group has significantly lower airway resistance compared with IL-33+Vehicle group (n=6). * P < 0.05 between IL-33+Vehicle and IL-33+7004 groups. In the OVA+Vehicle group, the mice have increased airway resistance compared with Saline+Vehicle group (n=6). The OVA+7004 group has significantly lower airway resistance compared with OVA+Vehicle group (n=6). ** P < 0.05 between OVA+Vehicle and OVA+7004 groups.

B. The BAL total cell counts and differentials of macrophages, neutrophils, lymphocytes, and eosinophils were obtained. IL-33+Vehicle group has increased total cell counts and eosinophils compared with Saline+Vehicle group (n=6). IL-33+7004 group has increased total cell counts and eosinophils compared with Saline+Vehicle group, but the numbers are significantly lower than the IL-33+Vehicle group (n=6). * P < 0.05 between IL-33+Vehicle and IL-33+7004 groups. OVA+Vehicle group has increased total cell counts and eosinophils compared with Saline+Vehicle group (n=6). OVA+7004 group has increased total cell counts and eosinophils compared with Saline+Vehicle group, but the numbers are significantly lower than the OVA+Vehicle group (n=6). ** P < 0.05 between OVA+Vehicle and OVA+7004 groups.

C. Compound 7004 partially decreased IL-5 and IL-13 levels in BAL. * P < 0.05 between IL-33+Vehicle and IL-33+7004 groups.

Discussion

In the United States, the burden of asthma continues to grow with an increase in the prevalence from 7.3% in 2001 to 8.4% in 2010 (27). During the last decade, anti-IgE antibody has been the biologic treatment for severe asthmatic patients with elevated IgE levels (28). Anti-IL-5 antibody and anti-IL-5 receptor α antibody became available on the market recently. The anti-IL-5 biologics treatment is effective in highly selected patient populations with specific phenotypes of severe asthma with persistent, glucocorticosteroid-resistant eosinophilia (28). Antibodies against IL-13, IL-4, and IL-17 are still under evaluation (28). There are varieties of inflammatory cells and cytokines that contribute to the onset and exacerbation of asthma. Therefore, the therapies targeting single cytokines are only effective in selected patient populations but not general asthmatic patients. Recent understanding of the innate immune response in the pathogenesis of asthma, such as IL-33, IL-25 and thymic stromal lymphopoietin (TSLP) pathways, provide potential for therapeutic targets other than Th2 cytokines.

Interrupting the IL33/ST2 signaling pathway has been previously demonstrated to be effective in mouse asthma-like models (29). Overexpression of sST2, which functions as a decoy receptor for IL-33, in an OVA induced mouse asthma model results in decreased eosinophils, IL-4 and IL-5 in the BAL (30). Intraperitoneal injection of an ST2 antibody on day 25, 27, and 29 of an OVA induced asthma model can decrease AHR (31). Treatment with anti-IL-33 antibody before the sensitization and challenge of OVA can prevent the development of asthma in a mouse model (32). So far, no attempt has been made to evaluate Myddosome as a potential therapeutic target for asthma. We anticipate that disrupting the Myddosome complex formation to block the IL-33/ST2 signaling pathway may provide more effective treatment options compared with the antibodies against IL-33 or ST2. In addition, since many TLR and IL-1R signaling use MyD88 as the adapter protein, disrupting the Myddosome complex formation is expected to have widespread anti-inflammatory properties that include other signaling pathways utilizing IRAK4 and IRAK2.

In general, antibodies interfere with protein-protein interactions in the extracellular region, but they cannot reach the intracellular region due to their poor cell membrane permeability. Small molecular compounds, on the other hand, can penetrate cell membranes and can function intracellularly where most of the signaling transduction occurs. Our previous work showed that a synthetic small molecule mimetic of the BB-loop of the TIR domain of MyD88 could disrupt MyD88 and IL-1R association, and reduce fever and inflammation caused by IL-1 in mice (33). Synthetic small molecules that can inhibit protein-protein interaction have proven effective in several disease models (34, 35).

In this study, we focused on disrupting the Myddosome formation by interfering the IRAK2-IRAK4 protein-protein interaction. The roles of IRAKs have been implicated in certain disease models such as human immunodeficiencies and inflammatory diseases (36, 37). IRAK4 was studied as the therapeutic target in LPS induced shock, autoimmune diseases and malignancy (38–41). There are studies targeting the Myddosome formation in order to decrease inflammation in LPS induced mouse septic shock model (42). A small molecule analogue of brazilin (IinQ) was identified as a potent inhibitor of IRAK1-dependent NF-κB activation by disrupting the MyD88-IRAK1-TRAF6 complex formation (42).

We found that small molecule mimetics of the α-helical domain of IRAK2 were capable of inhibiting the IL-33 induced NF-κB activation in vitro, and attenuating the pro-inflammatory responses in asthma-like mouse models in vivo. The small molecule compound 7004 specifically reduces the IRAK2-IRAK4 protein-protein interaction. The therapeutic effects of compound 7004 are observed not only in the IL-33 induced model, but also in the OVA induced model. We have examined the IL-33 and ST2 expression levels in the OVA induced model and found they were both elevated (data not shown). This explains the effectiveness of compound 7004 in OVA induced model and shed light on the interaction between innate and adaptive immunities in asthma. Our data indicate that the Myddosome may represent a novel intracellular therapeutic target for diseases in which IL-33/ST2 plays important roles, such as asthma and other inflammatory diseases. Efforts are underway to refine the small molecule compounds in order to increase the potency and drug-like characteristics.

Acknowledgments

This work was supported by: Department of Veterans Affairs Merit Review Grant (TDB). NIH T32-HL098062 (JL, JPD)

References

  • 1.Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, Zurawski G, Moshrefi M, Qin J, Li X, Gorman DM, Bazan JF, Kastelein RA. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity. 2005;23:479–490. doi: 10.1016/j.immuni.2005.09.015. [DOI] [PubMed] [Google Scholar]
  • 2.Tominaga S. A putative protein of a growth specific cDNA from BALB/c-3T3 cells is highly similar to the extracellular portion of mouse interleukin 1 receptor. FEBS Lett. 1989;258:301–304. doi: 10.1016/0014-5793(89)81679-5. [DOI] [PubMed] [Google Scholar]
  • 3.Tominaga S, Kuroiwa K, Tago K, Iwahana H, Yanagisawa K, Komatsu N. Presence and expression of a novel variant form of ST2 gene product in human leukemic cell line UT-7/GM. Biochem Biophys Res Commun. 1999;264:14–18. doi: 10.1006/bbrc.1999.1469. [DOI] [PubMed] [Google Scholar]
  • 4.Hayakawa H, Hayakawa M, Kume A, Tominaga S. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation. J Biol Chem. 2007;282:26369–26380. doi: 10.1074/jbc.M704916200. [DOI] [PubMed] [Google Scholar]
  • 5.Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, von Mutius E, Farrall M, Lathrop M, Cookson WO. A large-scale, consortium-based genomewide association study of asthma. N Engl J Med. 2010;363:1211–1221. doi: 10.1056/NEJMoa0906312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Gudbjartsson DF, Bjornsdottir US, Halapi E, Helgadottir A, Sulem P, Jonsdottir GM, Thorleifsson G, Helgadottir H, Steinthorsdottir V, Stefansson H, Williams C, Hui J, Beilby J, Warrington NM, James A, Palmer LJ, Koppelman GH, Heinzmann A, Krueger M, Boezen HM, Wheatley A, Altmuller J, Shin HD, Uh ST, Cheong HS, Jonsdottir B, Gislason D, Park CS, Rasmussen LM, Porsbjerg C, Hansen JW, Backer V, Werge T, Janson C, Jonsson UB, Ng MC, Chan J, So WY, Ma R, Shah SH, Granger CB, Quyyumi AA, Levey AI, Vaccarino V, Reilly MP, Rader DJ, Williams MJ, van Rij AM, Jones GT, Trabetti E, Malerba G, Pignatti PF, Boner A, Pescollderungg L, Girelli D, Olivieri O, Martinelli N, Ludviksson BR, Ludviksdottir D, Eyjolfsson GI, Arnar D, Thorgeirsson G, Deichmann K, Thompson PJ, Wjst M, Hall IP, Postma DS, Gislason T, Gulcher J, Kong A, Jonsdottir I, Thorsteinsdottir U, Stefansson K. Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet. 2009;41:342–347. doi: 10.1038/ng.323. [DOI] [PubMed] [Google Scholar]
  • 7.Torgerson DG, Ampleford EJ, Chiu GY, Gauderman WJ, Gignoux CR, Graves PE, Himes BE, Levin AM, Mathias RA, Hancock DB, Baurley JW, Eng C, Stern DA, Celedon JC, Rafaels N, Capurso D, Conti DV, Roth LA, Soto-Quiros M, Togias A, Li X, Myers RA, Romieu I, Van Den Berg DJ, Hu D, Hansel NN, Hernandez RD, Israel E, Salam MT, Galanter J, Avila PC, Avila L, Rodriquez-Santana JR, Chapela R, Rodriguez-Cintron W, Diette GB, Adkinson NF, Abel RA, Ross KD, Shi M, Faruque MU, Dunston GM, Watson HR, Mantese VJ, Ezurum SC, Liang L, Ruczinski I, Ford JG, Huntsman S, Chung KF, Vora H, Calhoun WJ, Castro M, Sienra-Monge JJ, del Rio-Navarro B, Deichmann KA, Heinzmann A, Wenzel SE, Busse WW, Gern JE, Lemanske RF, Jr, Beaty TH, Bleecker ER, Raby BA, Meyers DA, London SJ, Gilliland FD, Burchard EG, Martinez FD, Weiss ST, Williams LK, Barnes KC, Ober C, Nicolae DL. Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations. Nat Genet. 2011;43:887–892. doi: 10.1038/ng.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kurowska-Stolarska M, Stolarski B, Kewin P, Murphy G, Corrigan CJ, Ying S, Pitman N, Mirchandani A, Rana B, van Rooijen N, Shepherd M, McSharry C, McInnes IB, Xu D, Liew FY. IL-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation. J Immunol. 2009;183:6469–6477. doi: 10.4049/jimmunol.0901575. [DOI] [PubMed] [Google Scholar]
  • 9.Prefontaine D, Lajoie-Kadoch S, Foley S, Audusseau S, Olivenstein R, Halayko AJ, Lemiere C, Martin JG, Hamid Q. Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells. J Immunol. 2009;183:5094–5103. doi: 10.4049/jimmunol.0802387. [DOI] [PubMed] [Google Scholar]
  • 10.Stolarski B, Kurowska-Stolarska M, Kewin P, Xu D, Liew FY. IL-33 exacerbates eosinophil-mediated airway inflammation. J Immunol. 2010;185:3472–3480. doi: 10.4049/jimmunol.1000730. [DOI] [PubMed] [Google Scholar]
  • 11.Chen CC, Kobayashi T, Iijima K, Hsu FC, Kita H. IL-33 dysregulates regulatory T cells and impairs established immunologic tolerance in the lungs. J Allergy Clin Immunol. 2017 doi: 10.1016/j.jaci.2017.01.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Smith D, Helgason H, Sulem P, Bjornsdottir US, Lim AC, Sveinbjornsson G, Hasegawa H, Brown M, Ketchem RR, Gavala M, Garrett L, Jonasdottir A, Sigurdsson A, Magnusson OT, Eyjolfsson GI, Olafsson I, Onundarson PT, Sigurdardottir O, Gislason D, Gislason T, Ludviksson BR, Ludviksdottir D, Boezen HM, Heinzmann A, Krueger M, Porsbjerg C, Ahluwalia TS, Waage J, Backer V, Deichmann KA, Koppelman GH, Bonnelykke K, Bisgaard H, Masson G, Thorsteinsdottir U, Gudbjartsson DF, Johnston JA, Jonsdottir I, Stefansson K. A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma. PLoS Genet. 2017;13:e1006659. doi: 10.1371/journal.pgen.1006659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zoltowska AM, Lei Y, Fuchs B, Rask C, Adner M, Nilsson GP. The interleukin-33 receptor ST2 is important for the development of peripheral airway hyperresponsiveness and inflammation in a house dust mite mouse model of asthma. Clin Exp Allergy. 2016;46:479–490. doi: 10.1111/cea.12683. [DOI] [PubMed] [Google Scholar]
  • 14.Oshikawa K, Kuroiwa K, Tago K, Iwahana H, Yanagisawa K, Ohno S, Tominaga SI, Sugiyama Y. Elevated soluble ST2 protein levels in sera of patients with asthma with an acute exacerbation. Am J Respir Crit Care Med. 2001;164:277–281. doi: 10.1164/ajrccm.164.2.2008120. [DOI] [PubMed] [Google Scholar]
  • 15.Makrinioti H, Toussaint M, Jackson DJ, Walton RP, Johnston SL. Role of interleukin 33 in respiratory allergy and asthma. Lancet Respir Med. 2014;2:226–237. doi: 10.1016/S2213-2600(13)70261-3. [DOI] [PubMed] [Google Scholar]
  • 16.Lee HY, Rhee CK, Kang JY, Byun JH, Choi JY, Kim SJ, Kim YK, Kwon SS, Lee SY. Blockade of IL-33/ST2 ameliorates airway inflammation in a murine model of allergic asthma. Exp Lung Res. 2014;40:66–76. doi: 10.3109/01902148.2013.870261. [DOI] [PubMed] [Google Scholar]
  • 17.Mitchell PD, O’Byrne PM. Biologics and the lung: TSLP and other epithelial cell-derived cytokines in asthma. Pharmacol Ther. 2017;169:104–112. doi: 10.1016/j.pharmthera.2016.06.009. [DOI] [PubMed] [Google Scholar]
  • 18.Agoro R, Piotet-Morin J, Palomo J, Michaudel C, Vigne S, Maillet I, Chenuet P, Guillou N, Le Berichel J, Kisielow M, Flodby P, Borok Z, Crandall ED, Le Bert M, Quesniaux V, Muller M, Di Padova F, Ryffel B, Gabay C, Couturier-Maillard A. IL-1R1-MyD88 axis elicits papain-induced lung inflammation. Eur J Immunol. 2016;46:2531–2541. doi: 10.1002/eji.201646366. [DOI] [PubMed] [Google Scholar]
  • 19.Lin SC, Lo YC, Wu H. Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature. 2010;465:885–890. doi: 10.1038/nature09121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.McGettrick AF, O’Neill LA. The expanding family of MyD88-like adaptors in Toll-like receptor signal transduction. Mol Immunol. 2004;41:577–582. doi: 10.1016/j.molimm.2004.04.006. [DOI] [PubMed] [Google Scholar]
  • 21.Gay NJ, Gangloff M, O’Neill LA. What the Myddosome structure tells us about the initiation of innate immunity. Trends Immunol. 2011;32:104–109. doi: 10.1016/j.it.2010.12.005. [DOI] [PubMed] [Google Scholar]
  • 22.Kondo Y, Yoshimoto T, Yasuda K, Futatsugi-Yumikura S, Morimoto M, Hayashi N, Hoshino T, Fujimoto J, Nakanishi K. Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system. Int Immunol. 2008;20:791–800. doi: 10.1093/intimm/dxn037. [DOI] [PubMed] [Google Scholar]
  • 23.Kurowska-Stolarska M, Kewin P, Murphy G, Russo RC, Stolarski B, Garcia CC, Komai-Koma M, Pitman N, Li Y, Niedbala W, McKenzie AN, Teixeira MM, Liew FY, Xu D. IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4. J Immunol. 2008;181:4780–4790. doi: 10.4049/jimmunol.181.7.4780. [DOI] [PubMed] [Google Scholar]
  • 24.Saraogi I, Hamilton AD. alpha-Helix mimetics as inhibitors of protein-protein interactions. Biochem Soc Trans. 2008;36:1414–1417. doi: 10.1042/BST0361414. [DOI] [PubMed] [Google Scholar]
  • 25.Thomas JL, Dumouchel J, Li J, Magat J, Balitzer D, Bigby TD. Endotracheal intubation in mice via direct laryngoscopy using an otoscope. J Vis Exp. 2014 doi: 10.3791/50269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Bartemes KR, Iijima K, Kobayashi T, Kephart GM, McKenzie AN, Kita H. IL-33-responsive lineage- CD25+ CD44(hi) lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs. J Immunol. 2012;188:1503–1513. doi: 10.4049/jimmunol.1102832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Akinbami LJ, Moorman JE, Bailey C, Zahran HS, King M, Johnson CA, Liu X. Trends in asthma prevalence, health care use, and mortality in the United States, 2001-2010. NCHS Data Brief. 2012:1–8. [PubMed] [Google Scholar]
  • 28.Mitchell PD, El-Gammal AI, O’Byrne PM. Emerging monoclonal antibodies as targeted innovative therapeutic approaches to asthma. Clin Pharmacol Ther. 2016;99:38–48. doi: 10.1002/cpt.284. [DOI] [PubMed] [Google Scholar]
  • 29.Theoharides TC, Petra AI, Taracanova A, Panagiotidou S, Conti P. Targeting IL-33 in autoimmunity and inflammation. J Pharmacol Exp Ther. 2015;354:24–31. doi: 10.1124/jpet.114.222505. [DOI] [PubMed] [Google Scholar]
  • 30.Oshikawa K, Yanagisawa K, Tominaga S, Sugiyama Y. Expression and function of the ST2 gene in a murine model of allergic airway inflammation. Clin Exp Allergy. 2002;32:1520–1526. doi: 10.1046/j.1365-2745.2002.01494.x. [DOI] [PubMed] [Google Scholar]
  • 31.Kearley J, Buckland KF, Mathie SA, Lloyd CM. Resolution of allergic inflammation and airway hyperreactivity is dependent upon disruption of the T1/ST2-IL-33 pathway. Am J Respir Crit Care Med. 2009;179:772–781. doi: 10.1164/rccm.200805-666OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Liu X, Li M, Wu Y, Zhou Y, Zeng L, Huang T. Anti-IL-33 antibody treatment inhibits airway inflammation in a murine model of allergic asthma. Biochem Biophys Res Commun. 2009;386:181–185. doi: 10.1016/j.bbrc.2009.06.008. [DOI] [PubMed] [Google Scholar]
  • 33.Bartfai T, Behrens MM, Gaidarova S, Pemberton J, Shivanyuk A, Rebek J., Jr A low molecular weight mimic of the Toll/IL-1 receptor/resistance domain inhibits IL-1 receptor-mediated responses. Proc Natl Acad Sci U S A. 2003;100:7971–7976. doi: 10.1073/pnas.0932746100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Cochran AG. Protein-protein interfaces: mimics and inhibitors. Curr Opin Chem Biol. 2001;5:654–659. doi: 10.1016/s1367-5931(01)00262-9. [DOI] [PubMed] [Google Scholar]
  • 35.Berg T, Cohen SB, Desharnais J, Sonderegger C, Maslyar DJ, Goldberg J, Boger DL, Vogt PK. Small-molecule antagonists of Myc/Max dimerization inhibit Myc-induced transformation of chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 2002;99:3830–3835. doi: 10.1073/pnas.062036999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Flannery S, Bowie AG. The interleukin-1 receptor-associated kinases: critical regulators of innate immune signalling. Biochem Pharmacol. 2010;80:1981–1991. doi: 10.1016/j.bcp.2010.06.020. [DOI] [PubMed] [Google Scholar]
  • 37.Bahia MS, Kaur M, Silakari P, Silakari O. Interleukin-1 receptor associated kinase inhibitors: potential therapeutic agents for inflammatory- and immune-related disorders. Cell Signal. 2015;27:1039–1055. doi: 10.1016/j.cellsig.2015.02.025. [DOI] [PubMed] [Google Scholar]
  • 38.Park SH, Baek SI, Yun J, Lee S, Yoon DY, Jung JK, Jung SH, Hwang BY, Hong JT, Han SB, Kim Y. IRAK4 as a molecular target in the amelioration of innate immunity-related endotoxic shock and acute liver injury by chlorogenic acid. J Immunol. 2015;194:1122–1130. doi: 10.4049/jimmunol.1402101. [DOI] [PubMed] [Google Scholar]
  • 39.Kelly PN, Romero DL, Yang Y, Shaffer AL, 3rd, Chaudhary D, Robinson S, Miao W, Rui L, Westlin WF, Kapeller R, Staudt LM. Selective interleukin-1 receptor-associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy. J Exp Med. 2015;212:2189–2201. doi: 10.1084/jem.20151074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Dou H, Song Y, Liu X, Yang L, Jiang N, Chen D, Li E, Tan R, Hou Y. A novel benzenediamine derivate rescued mice from experimental sepsis by attenuating proinflammatory mediators via IRAK4. Am J Respir Cell Mol Biol. 2014;51:191–200. doi: 10.1165/rcmb.2013-0411OC. [DOI] [PubMed] [Google Scholar]
  • 41.Chaudhary D, Robinson S, Romero DL. Recent advances in the discovery of small molecule inhibitors of interleukin-1 receptor-associated kinase 4 (IRAK4) as a therapeutic target for inflammation and oncology disorders. J Med Chem. 2015;58:96–110. doi: 10.1021/jm5016044. [DOI] [PubMed] [Google Scholar]
  • 42.Kang K, Won M, Yuk JM, Park CY, Byun HS, Park KA, Lee SR, Kang YG, Shen HM, Lee IY, Hur GM. IinQ attenuates systemic inflammatory responses via selectively impairing the Myddosome complex formation upon TLR4 ligation. Biochem Pharmacol. 2016;121:52–66. doi: 10.1016/j.bcp.2016.09.017. [DOI] [PubMed] [Google Scholar]

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