AZD7762 | Mek signal

Repositioning of anti-cancer drug candidate, AZD7762, to an anti-allergic drug suppressing IgE-mediated mast cells and allergic responses via the inhibition of Lyn and Fyn
Young Hwan Park, Do Kyun Kim, Hyun Woo Kim, Hyuk Soon Kim, Dajeong Lee, Min Bum Lee, Keun Young Min, Jimo Koo, Su Jeong Kim, Changhee Kang, Young Mi Kim, Hyung Sik Kim, Wahn Soo Choi
PII: S0006-2952(18)30197-7
DOI: https://doi.org/10.1016/j.bcp.2018.05.012
Reference: BCP 13147

To appear in: Biochemical Pharmacology

Received Date: 23 March 2018
Accepted Date: 15 May 2018

Please cite this article as: Y.H. Park, D.K. Kim, H.W. Kim, H.S. Kim, D. Lee, M.B. Lee, K. Young Min, J. Koo,
S.J. Kim, C. Kang, Y. Mi Kim, H.S. Kim, W.S. Choi, Repositioning of anti-cancer drug candidate, AZD7762, to an anti-allergic drug suppressing IgE-mediated mast cells and allergic responses via the inhibition of Lyn and Fyn, Biochemical Pharmacology (2018), doi: https://doi.org/10.1016/j.bcp.2018.05.012

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Repositioning of anti-cancer drug candidate, AZD7762, to an anti-allergic drug suppressing IgE-mediated mast cells and allergic responses via the inhibition of Lyn and Fyn

Young Hwan Park a, Do Kyun Kim b, Hyun Woo Kim a, Hyuk Soon Kim a, Dajeong Lee

a, Min Bum Lee a, Keun Young Min a, Jimo Koo a, Su Jeong Kim a, Changhee Kang c,

Young Mi Kim d, Hyung Sik Kim e, Wahn Soo Choi a,*

a Department of Immunology, College of Medicine, Konkuk University, Chungju 27478,

Republic of Korea

b Mast Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA
c Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 05029, Republic of Korea.
d College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea

e Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of Korea

* Corresponding author at: School of Medicine, Konkuk University, Chungju 27478, Republic of Korea. E-mail address: [email protected] (W.S. Choi)

ABSTRACT

Mast cells are critical effector cells in IgE-mediated allergic responses. The aim of this study was to investigate the anti-allergic effects of 3-[(aminocarbonyl)amino]-5-(3- fluorophenyl)-N-(3S)-3-piperidinyl-2-thiophenecarboxamide (AZD7762) in vitro and in vivo. AZD7762 inhibited the antigen-stimulated degranulation from RBL-2H3 (IC50, ~
27.9 nM) and BMMCs (IC50, ~ 99.3 nM) in a dose-dependent manner. AZD7762 also inhibited the production of TNF-α and IL-4. As the mechanism of its action, AZD7762 inhibited the activation of Syk and its downstream signaling proteins, such as Linker of activated T cells (LAT), phospholipase (PL) Cγ1, Akt, and mitogen-activated protein (MAP) kinases (Erk1 / 2, p38, and JNK) in mast cells. In in vitro protein kinase assay, AZD7762 inhibited the activity of Lyn and Fyn kinases, which are important for the activation of Syk in mast cells. Furthermore, AZD7762 also suppressed the degranulation of LAD2 human mast cells (IC50, ~ 49.9 nM) and activation of Syk in a dose-dependent manner. As observed in experiments with mast cells in vitro, AZD7762 inhibited antigen-mediated passive cutaneous anaphylaxis in mice (ED50, ~ 35.8 mg/kg). Altogether, these results suggest that AZD7762 could be used as a new therapeutic agent for mast cell-mediated allergic diseases.

Keywords: AZD7762; Mast cells; Allergy; Anaphylaxis; Drug repositioning.

⦁ Introduction

Mast cells are located in the connective tissues of skin, gastrointestinal tract, and the mucus layer of the airway tract; and are involved in the induction of innate immunity [1]. In allergic diseases, mast cells act as effector cells that secrete a variety of allergy- inducing substances [1, 2]. In allergic patients, an exposure to certain antigen induces various Th2 immune responses including the production of Th2 cytokines and immunoglobulin (Ig)E. IgE binds to FcεRI, an IgE-high affinity receptor expressed on the surface of mast cells. When antigen is bound to the IgE/FcεRI complex on mast cells, mast cells are activated and secrete many allergic mediators including histamine and cytokines, resulting in diseases such as allergic rhinitis, pruritus, allergic diarrhea, and anaphylaxis [3, 4]. Activated mast cells secrete biogenic amines, proteases, and lipid-derived mediators that are present in intracellular granules within minutes. Over time, mast cells produce and secrete pro-inflammatory cytokines and other chemokines. The secreted allergic mediators induce allergic inflammation by recruiting immune cells to target tissue [3, 5].
The activation mechanism of mast cells begins with the attachment of antigen to IgE bound to extracellular α chain of FcεRI, the high affinity IgE receptor. An initial signaling event following aggregation of FcεRIs is mediated by Lyn and other Src family kinases including Fyn. Lyn, which is present in the membrane of mast cells, is activated by antigen and phosphorylates the immunoreceptor tyrosine-based motifs (ITAMs) of β and γ subunits of FcεRI. Syk, located in the cytosol, is fully activated by recruiting to phosphorylated ITAMs of FcεRI. Activated Syk phosphorylates LAT and

PLCγ1. Subsequently, the concentration of Ca2+ is then increased to induce degranulation of mast cells. In addition, the activation of three typical mitogen-activated protein (MAP) kinases such as JNK, Erk1/2, and p38 MAP kinases causes synthesis and secretion of pro-inflammatory cytokines [6, 7]. The pivotal signaling pathway is known as the Lyn/Syk/LAT pathway and there is also a Fyn/Gab2/ PI3K pathway activated by antigen stimulation. The signaling pathway is also important for the optimal activation of mast cells as a complementary pathway [8, 9].
Currently, the pharmaceutical industry, along with research to discover new compounds, is actively studying drug repositioning of substances that fail to enter clinical trials due to its efficacy or drugs already on the market [10, 11]. AZD7762 is a urea-based check point kinase inhibitor developed by AstraZeneca (Waltham, MA, USA) and is currently being clinically tested for use as an anti-cancer drug. Interestingly to us, it was also reported that AZD7762 inhibited Src-family kinases including Lyn and Fyn kinases, which are critical for the activation of mast cells, in an in vitro enzyme assay [12]. However, there is currently no report on whether AZD7762 inhibits mast cells and allergic responses by antigen stimulation. In this study, we investigated whether AZD7762 inhibited the activation of mast cells and IgE-mediated allergic response in mice. Now, we demonstrate for the first time that AZD7762 effectively suppresses the degranulation of mast cells and allergic response in vivo via the inhibition of Lyn and Fyn in mast cells.

⦁ Materials and methods

⦁ Antibodies and Reagent

AZD7762 (3-[(Aminocarbonyl)amino]-5-(3-fluorophenyl)-N-(3S)-3-piperidinyl-2-thio phene carboxamide) was purchased from Selleckem (catalog no. s1532; Houston, TX, USA). Monoclonal dinitrophenol (DNP)-specific IgE (catalog no. D8406), streptavidin (catalog no. S0677), DNP-human serum albumin (HSA, catalog no. A6661), Evans blue (catalog no. E2129), and toluidine blue O (catalog no. 198161) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against phosphorylated forms of ZAP-70Tyr319/SykTyr352 (catalog no. 2701), SykTyr525/526 (catalog no. 2711), LATTyr191 (catalog no. 3584), PLCγTyr783 (catalog no. 2821), Aktser473 (catalog no. 9271), Erk1/2Thr202/Tyr204 (catalog no. 9106), p38Thr180/Tyr182 (catalog no. 9211), and SAPK/JNKThr183/Tyr185 (catalog no. 9251) were purchased from cell signaling Technology, Inc. (Danvers, MA, USA). Antibodies against Lyn (catalog no. sc-7274), Fyn (catalog no. sc-434), and Syk (catalog no. sc-51703) were from Santa Cruz Biotechnology (Dallas, Texas, USA). Human IgE (catalog no. 401152) and antibodies against LAT (catalog no. 06-807) and Actin (catalog no. MAB1501) were obtained from EMD Millipore (Billerica, MA, USA). The media used for cell culture were obtained from GIBCO/Life Technology, Inc. (Rockville, MD, USA).

⦁ Animal

The mice (5 to 6 week old BALB/c male mice) were obtained from Orient Bio, Inc. (Gyeonggi-do, Korea). The mice were used to prepare bone marrow-derived mast cells

(BMMC) and to perform passive cutaneous anaphylaxis (PCA) experiments. The animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) at Konkuk University.
⦁ Culture of rat basophilic leukemia (RBL)-2H3 cells, mouse bone marrow-derived mast cells (BMMCs), and LAD2 human mast cells
RBL-2H3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and were cultured in complete MEM medium that contained 4 mM L- glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 15% fetal bovine serum (FBS) in a humidified incubator at 37 °C, 5% CO2. The experiments with RBL-2H3 cells were performed in the passage of 5–20. For BMMCs, bone marrow cells were isolated from femurs of mouse and then cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium that contains 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES, 10% FBS, and 10 ng/ml interleukin (IL)-3. After 4 to 6 weeks of culture, the purity of mast cells reached 95% or more. The experiments with BMMCs were carried out in the passage of 4–8. LAD2 cells were maintained in StemPro-34 SFM Complete Medium supplemented with 100 ng/ml human recombinant stem cell factor, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. LAD2 cells were fed by hemi-depletion of media once a week and used in the passage of 5–20.

⦁ Measurement of β-hexosaminidase in mast cells

This experiment was performed as described previously [13]. Briefly, cells were sensitized with 50 ng/ml DNP-specific IgE in media overnight. The cells were transferred into Siraganian buffer (25 mM PIPES, pH 7.2, 119 mM NaCl, 5 mM KCl,
0.4 mM MgCl2, 1 mM CaCl2, 5.6 mM glucose, and 0.1% fatty acid-free fraction V BSA) for RBL-2H3s or Tyrode buffer (20 mM HEPES, pH 7.4, 135 mM NaCl, 5 mM KCl,
1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA) for BMMCs. The cells were incubated with or without AZD7762 for 30 min, the cells were subsequently stimulated with 50 ng/ml of DNP-HSA for 15 min. LAD2 cells were sensitized with 100 ng/ml biotinylated human IgE overnight. LAD2 cells were transferred into HEPES buffer (10 mM HEPES, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na2HPO4, 5.6 mM glucose, 1.8 mM CaCl2, 1.3 mM MgSO4, and 0.04% BSA). LAD2 cells were incubated with or without AZD7762 for 30 min, and then the cells were stimulated with 100 ng/ml streptavidin for 30 min. The degree of degranulation of mast cells was expressed as % of β-hexosaminidase secreted out of total β-hexosaminidase.

⦁ Measurement of cell viability

RBL-2H3 cells (2 × 104 cells/well) and BMMCs (5.0 x 104 cells/well) were cultured in a 96-well plate for 4 h and subsequently treated with AZD7762 for 4 h. Cell viability was measured with a Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol.

⦁ Measurement of secreted TNF-α and IL-4 by ELISA

IgE-sensitized RBL-2H3 cells (1.0 × 106 cells/well) were stimulated with 50 ng/ml DNP-HSA for 3 h, with or without AZD7762. The amount of tumor necrosis factor (TNF)-α and IL-4 in cultured media were measured using BDTM ELISA (BD Biosciences, San Jose, Calif, USA) according to the manufacturer’s protocol.

⦁ Western blot analysis

This experiment was performed as described previously [13]. Briefly, cells were sensitized with 50 ng/ml DNP-IgE (for RBL-2H3 cells and BMMCs) or 100 ng/ml human IgE (for LAD2 cells) overnight and washed twice with phosphate-buffered saline (PBS) and changed with fresh media. After incubating with or without AZD7762 at 37 °C for 30 min, the cells were subsequently stimulated with 50 ng/ml DNP-HSA (for RBL-2H3 cells and BMMCs) or 100 ng/ml SA (for LAD2 cells) for 10 min. The reaction was then stopped by ice. Cells were lysed using RIPA buffer (Thermo Fisher scientific, Waltham, MA, USA) in 1 mM phenylmethylsulfonyl fluoride, 2.5 mM p- nitrophenyl phosphate, 0.7 µg/ml pepstatin, and protease-inhibitor cocktail. Cell lysates were centrifuged at 15,000 ×g for 5 min. The supernatants were mixed with a NuPAGETM LDS sample buffer (4×) (Thermo Fisher scientific) and denatured by heating for 5 min at 100 °C. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. After incubating with a primary antibody in tris-buffered saline with 0.1% tween 20 buffer that contained 5% BSA or skim milk. Immuno-reactive proteins were detected by a use of horse-radish peroxidase-coupled secondary antibodies. The blots

were detected using chemiluminescence reagents (Thermo Fisher scientific) according to the manufacturer’s protocol.

⦁ Immunoprecipitation and in vitro tyrosine kinase assay

The cells were lysed in a NP-40 based lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet p-40, 10% glycerol, 60 mM octyl-β-glucoside, 10 mM NaF, 1 mM Na3VO4, 1mMphenylmethylsulfonylfluoride, 2.5mMnitrophenylphosphate, 0.7 µg/ml pepstatin, and a protease inhibitor cocktail tablet). Lysates were centrifuged at 15,000 ×g at 4°C for 5 min. The supernatant was precleared by the addition of 50 μl protein G-agarose. One milligram of protein was incubated overnight with 5 μg specific antibody at 4°C and followed by an addition of protein G agarose for 1 hour. Then the agarose was washed 5 times with a washing buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Nonidet p-40, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF,
2.5 mM nitrophenylphosphate, 0.7 µg/ml pepstatin, and a protease inhibitor cocktail tablet). The activity of tyrosine kinase was analyzed using a Universal Tyrosine Kinase Assay kit (GenWay Biotech Inc., San Diego, CA, USA) according to the manufacturer’s recommendation.

⦁ Passive cutaneous anaphylaxis (PCA)

PCA was induced in mice as demonstrated previously [13]. Briefly, mice (n = 5) were injected intradermally in the ear with 50 ng of DNP-specific IgE, After 24 h,

AZD7762 was orally administered. After 1h, 100 μg of DNP-HSA in PBS containing 5 mg/ml Evans blue was injected into the tail vein. The mice were euthanized 1 h after the DNP-HSA injection, followed by the removal of the ear. The dye was extracted overnight from ear tissues in 1 ml of formamide at 63°C. The absorbance was measured at 620 nm.

⦁ Histological analysis

The mice ears were cut and fixed in 4% paraformaldehyde. The ear tissue was dehydrated with ethanol and subsequently embedded in paraffin. The paraffinized tissue was cut into 4 μm section and stained with toluidine blue. To determine the percentage of degranulated mast cells in ear tissues, the number of degranulated mast cells in three sections per ear tissue were measured (n = 5 mice, 3 sections per ear).

⦁ Statistical analysis

All values were presented as the mean ± s.e.m. from three independent experiments, at least in triplicate, for in vitro cell experiments and from five mice for PCA experiments. Kruskal-Wallis test by ranks (nonparametric one-way analysis of variance), followed by Dunn’s post hoc tests was carried out if more than one group was being compared. Due to the nonnormal distribution of the data, nonparametric Mann-Whitney U test when two groups were being compared. All statistical calculations (*p < 0.05 and

**p < 0.01) were done with the SigmaStat software version 4.0 (Systat Software Inc., San Jose, CA, USA).

⦁ Results

⦁ Effect of AZD7762 on degranulation from mast cells by antigen

We assessed the activity of β-hexosaminidase, a granule marker, to determine whether AZD7762 (Fig. 1) inhibits degranulation in mast cells. AZD7762 inhibited the degranulation of RBL-2H3 (IC50, ~ 27.9 ± 3.3 nM) and BMMC (IC50, ~ 99.3 ± 8.9 nM) by antigen stimulation (Fig 2A). When RBL-2H3 and BMMC were cultured in 10 µM AZD7762 for 4 h, any cytotoxicity of AZD7762 was not observed (Fig. 2B). To evaluate the reversibility of the AZD7762 effect on mast cell degranulation, 1 µM of AZD7762 was pretreated with RBL-2H3 cells for 60 min and washed out 5 times. Then, the degranulation of mast cells by antigen stimulation was measured. As a result, 76.9 ± 3.4% of mast cell degranulation was recovered by washing (Fig. 2C), indicating that AZD7762 reversibly inhibits mast cell degranulation.

⦁ Effect of AZD7762 on expression and secretion of cytokines from mast cells

Pro-inflammatory cytokines are important for inducing various symptoms in allergic patients [14]. Mast cells secrete cytokines such as TNF-α and IL-4 by antigen stimulation in allergic conditions [15, 16]. In this study, secretion of TNF-α and IL-4

from mast cells was inhibited by AZD7762 in a dose-dependent manner (Fig. 3). These results suggest that AZD7762 could suppresses allergic symptoms caused by cytokines as well as acute symptoms by degranulation in mast cells in allergic diseases.

⦁ Mechanism of action of AZD7762 in IgE-mediated mast cell activation

Next, we examined which proteins were inhibited by AZD7762 in FcεRI-mediated signaling pathway of mast cells. In RBL-2H3 cells, phosphorylations of Syk, LAT, and PCLγ1 were increased by antigen stimulation. However, AZD7762 inhibited their phosphorylations in a dose-dependent manner (Fig. 4A). In addition, the phosphorylations of Akt, Erk1/2, p38, and JNK, which are involved in the synthesis of pro-inflammatory cytokines, also suppressed by AZD7762 (Fig. 4A). In BMMC, a typical primary mast cell, AZD7762 also inhibited the phosphorylations of Syk and LAT in a dose-dependent manner (Fig. 4B). Based on these results, we argue that AZD7762 inhibits the activation of Syk and LAT pathway in antigen-stimulated mast cells.

⦁ Effect of AZD7762 on activation of Lyn and Fyn kinase in vitro

We found that AZD7762 inhibited phosphorylation of Syk by antigen in mast cells (Fig. 4). Syk is known to be phosphorylated and activated by Lyn and Fyn in mast cells [9, 17]. Therefore, we examined whether AZD7762 inhibits Lyn and Fyn kinase in vitro. As a result of our experiments, AZD7762 inhibited the activity of Lyn and Fyn in a

dose-dependent manner (Fig. 5). These results indicate that AZD7762 suppresses mast cell activation via the direct inhibition of Lyn and Fyn in mast cells.

⦁ Effect of AZD7762 on passive cutaneous anaphylaxis in mice

We used PCA mice to measure the inhibitory effect of AZD7762 on allergic responses in vivo. PCA is a typical acute allergic animal model that induces allergic reactions by stimulating mast cells with antigen in the ear or back skin [18, 19]. As a result of the PCA test, the amount of Evans blue in the ear, which is increased by an allergic reaction, was inhibited by AZD7762 (ED50, ~35.8 mg/kg). In particular, the amount of dye was remarkably reduced at the highest dose of AZD7762 (100 mg/kg) (Figs. 6A and 6B). In addition, the number of degranulation of mast cells in the ear tissue was also largely reduced at 100 mg/kg of AZD7762 (Fig. 6C). These results indicate that AZD7762 inhibits allergic responses in vivo.

⦁ Effect of AZD7762 on degranulation and Syk activation in human mast cells

We next examined whether AZD7762 inhibited degranulation from human mast cells using LAD2 cells. AZD7762 inhibited the degranulation from LAD2 cells by FcεRI stimulation in a dose-dependent manner (IC50, ~49.9 nM) (Fig. 7A). In addition, the activation of Syk, the downstream signaling protein of Lyn and Fyn, was also inhibited in a dose-dependent manner (Fig. 7B). These results suggest that AZD not only inhibits the IgE-mediated allergic response in animal model, but could also work in allergy patients.

⦁ Discussion

Asthma and allergic diseases are increasing worldwide and direct and indirect economic costs are estimated to be over $20.9 billion [20, 21]. There is general acceptance that mast cells are the critical effector cells in various allergic diseases. We previously found not only the roles of various proteins involved in the activation of IgE mediated mast cells [22–25] but also compounds that inhibit IgE-mediated mast cell activation [13, 15, 16, 26]. In this study, we demonstrate that AZD7762 inhibits mast cell-mediated allergic responses in vitro and in vivo through directly inhibiting activity of Lyn and Fyn.
In general, the development of new drugs is conducted through a series of processes; identifying drug candidates, preclinical experiments, and clinical trials. According to Clinical Development Success Rates 2006-2015 reported by Biotechnology Innovation Organization, only 9.6% passed all phases from phase I to final approval [27]. The passing rate at each stage was 63.2% for phase I, 30.7% for phase II, and 49.6% for phase III, respectively. Only 15.3% of drugs that passed phase I made it to the market, and the remaining 84.7% were stopped due to the absence of pharmacological effect of drugs or their toxicity in humans. It takes approximately 13-15 years from preclinical stage to phase III for drug development on average. And as the phases progress, the expenses increased exponentially. If a company invests a lot of time and money but does not commercialize them, the company would have a lot of difficulty managing the company [11, 27].

For this reason, multinational pharmaceutical companies are attempting to develop new drugs with other efficacy by using compounds that have already been marketed or that have been discontinued at phase II and III of clinical trials. This approach is called “drug repositioning”. For example, sildenafil, originally developed as a drug for angina, has been commercialized as a drug for erectile dysfunction [28]. Thalidomide was originally developed for hyperemesis treatment. However, thalidomide was discontinued because of a cause of fetal anomaly, but has also been reborn as a drug for multiple myeloma [29, 30]. In addition, there are some drugs successfully developed via drug repositioning, and multinational pharmaceutical companies are now investing 10- 50% of their R&D budget for drug repositioning [11]. Therefore, instead of using new compounds to measure the anti-allergic effect, we have also performed to screen anti- allergy effect of compounds which have passed the phase I clinical trial test.
AZD7762 is a drug candidate that has passed phase I in the U.S.A. as an anti-cancer drug. AZD7762, a thiophene carboxamide urea inhibited the growth of cancer cells by inhibiting checkpoint kinase 1 in cancer cells and increased the anti-cancer effect when specially treated with gemcitabine [12]. Inhibition of the G2-M checkpoint by AZD7762 resulted in apoptosis of various tumor cells, such as multiple myeloma cells [31], breast cancer cells [32], ovarian cancer cells [33], lung cancer cells [34], and urothelial cancer cells [35]. It was also reported that AZD7762 inhibited various kinases, including cyclin-dependent kinases, protein kinase C isoforms, p38, and Src-family kinases including Lyn and Fyn, albeit to a lower extent, in an in vitro kinase assay [12]. Of these kinases, Lyn and Fyn are essential kinases for activation of mast cells by
antigen [6, 9]. However, the inhibitory effect of AZD7762 on mast cells and allergic

response was totally unknown. In this study, we found that AZD7762 inhibited the degranulation by antigen in mast cells (Fig. 2A) and suppressed the allergic response in mice (Fig. 6). These results strongly suggest that it is possible to expand the usage of AZD7762 to treat allergic diseases in humans.
Antihistamines, corticosteroids, and anti-IgE drugs have been used to treat allergic diseases [3]. Although these drugs have immediate effects in a variety of allergic diseases, antihistamines cause drug resistance due to repeated administration, and corticosteroids can cause side effects by inducing hormone changes in the body [36]. Omalizumab, an anti-IgE drug that interferes with the binding of FcεRI and IgE in mast cells, has an excellent efficacy but has a disadvantage in that it is expensive to patients [37]. Therefore, we are studying drugs that target signaling molecules that are involved in the activation of IgE-mediated mast cells. We demonstrate that AZD7762 inhibits the degranulation of mast cells and thus blocks the secretion of inflammatory mediators (Figs. 2A and 3). Furthermore, in our experiment, AZD7762 inhibited the degranulation of mast cells in the ear tissue and consequently inhibited the PCA reaction (Fig. 6), suggesting that AZD7762 can suppress allergic symptoms in patients with allergy.
Activation of IgE mediated mast cells involves a variety of signaling proteins [6, 7, 38]. For example, Lyn, Fyn, Syk, LAT, Gab2, PLCγ, PI3K/Akt, and MAP kinases are signaling proteins activated in mast cells by antigen [38]. Among them, Lyn and Fyn are the very early signaling proteins and play a key role in the initiation of FcεRI signaling cascades in mast cells. The Lyn/ Syk/LAT axis is the pivotal signaling pathway in FcεRI signals of mast cells by antigen stimulation [39, 40]. In addition, the

complementary signaling pathway is Fyn/Gab2/PI3K axis for the optimal activation of mast cells [8, 9, 17]. When these two signaling pathways are fully activated, degranulation and production of cytokines/chemokines are effectively induced. We observed that AZD7762 directly inhibited the activity of Lyn and Fyn in vitro (Fig. 5), and further inhibited the activation of downstream signal proteins such as Syk, LAT, PLCγ, Akt, and MAP kinases (Fig. 4).
In experiments with LAD2 human mast cells, we further observed that AZD7762 inhibited degranulation by the FcεRI stimulation in a dose-dependent manner (Fig. 7A). AZD7762 also suppressed the activation of Syk of LAD2 cells by antigen as in BMMCs (Fig. 7B). These results suggest that AZD7762 could suppress allergic response in patients with allergy. Altogether in this study, our results demonstrate that AZD7762 suppresses IgE-mediated degranulation and the release of cytokines from mast cells by inhibiting the activity of Lyn and Fyn kinase in vitro and in vivo. Therefore, AZD7762, which is undergoing clinical trials as an anticancer drug, could also become a candidate drug for allergic diseases.

Acknowledgements

This research was supported by the National Research Foundation of Korea (NRF) grant (NRF-2016R1A2B3015840) and in part by Basic Research Laboratory Program (No. 2013R1A4A1069575) funded by the Korea government.

Conflict of interest

The authors have declared that there is no conflict of interest.

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Figure legend

Fig. 1. Molecular structure of 3-[(aminocarbonyl)amino]-5-(3-fluorophenyl)-N-(3S)-3- piperidinyl-2-thiophenecarboxamide, AZD7762.

Fig. 2. AZD7762 reversibly suppresses antigen-stimulated degranulation in mast cells.

(A) DNP-specific IgE primed RBL-2H3 cells (2.0 × 105 cells/well) and BMMCs (3.0 × 105 cells/tube) were stimulated by 50 ng/ml antigen for 15 min with or without the pre- treatment of AZD7762 or PP2 for 30 min. Data are expressed as the mean ± s.e.m. from three independent experiments in triplicate. Statistical analysis was performed by Kruskal-Wallis test and Dunn’s post hoc for comparisons between groups. Significant differences against the antigen-stimulated controls without AZD7762 are indicated, **p
< 0.01 (n = 9). (B) RBL-2H3 cells and BMMCs were incubated with or without AZD7762 for 4 h and cell viability was determined by CCK-8. (C) RBL-2H3 cells were

pre-incubated with or without AZD7762 for 30 min, and then stimulated by antigen for

15 min after washing out 5 times. More details are described in the section for “Materials and methods”. The values indicate mean ± s.e.m. from three independent experiments in triplicate. Statistical significance was determined by Mann-Whitney U test. Significant differences are indicated, **p < 0.01 (n = 9). PP2, a Src-family kinase inhibitor.

Fig. 3. AZD7762 suppresses secretion of IL-4 and TNF-α from antigen-stimulated mast cells. IgE-primed RBL-2H3 cells were stimulated with 50 ng/ml antigen for 3 h after pre-incubating with or without AZD7762 for 30 min. And then, the supernatant was collected. The amount of released IL-4 and TNF-α was measured by using BDTM ELISA kit according to manufacturer’s protocol. Absorbance rate was measured with a microplate reader of 450 nm. The detailed information was described in the section “Materials and methods”. Data are expressed as the mean ± s.e.m. from three independent experiments in triplicate. Statistical analysis was performed by Kruskal- Wallis test and Dunn’s post hoc for comparisons between groups. Significant differences against the antigen-stimulated controls without AZD7762 are indicated, *p
< 0.05; **p < 0.01 (n = 9).

Fig. 4. AZD7762 suppresses phosphorylation of Syk and downstream signaling proteins in antigen-stimulated mast cells. (A) RBL-2H3 Cells and (B) BMMCs were primed with antigen-specific IgE overnight, and then incubated with or without AZD7762 for

30 min, subsequently they were stimulated with antigen for 10 min. The cell lysates were subjected to Western blotting analysis. More details were described in the “Materials and methods” section. Representative images and the band density for phosphorylated proteins from three independent experiments are shown. Densitometric analysis of phosphorylated proteins were performed using Multi Gauge Ver 3.0. The data for band density were presented as the mean ± s.e.m. from three independent experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Significant differences against the antigen- stimulated controls without AZD7762 are indicated, *p < 0.05; **p < 0.01 (n = 3).

Fig. 5. AZD7762 inhibits Lyn and Fyn kinase activity. The cells were lysed in NP-40 based lysis buffer. One mg of precleared whole cell lysate was used for immunoprecipitation of Lyn or Fyn as described in the “Materials and methods” section. The activity of Lyn and Fyn in vitro was measured using a Universal Tyrosine Kinase Assay Kit according to the manufacturer’s protocol. The values indicate mean ± s.e.m. from three independent experiments. Data are expressed as the mean ± s.e.m. from three independent experiments in triplicate. Statistical analysis was performed by Kruskal- Wallis test and Dunn’s post hoc for comparisons between groups. Significant differences against the value without AZD7762 are indicated, *p < 0.05 and **p < 0.01 (n = 9).

Fig. 6. AZD7762 inhibits IgE-mediated PCA reaction. IgE was intradermally injected into the ears of mice. After one day, AZD7762 (10, 30, 100 mg/kg) was orally administered 1 h prior to the challenge of antigen including Evans blue into the tail vein. The mice were put down after 1 h. More details are described in the “Materials and Methods” section. (A) Representative images and (B) values for the amount of Evans blue dye for PCA-induced mice ears are presented (n = 5). (C) The ratio of degranulated mast cells to total mast cells in the ear tissues is shown (3 sections per mouse, n = 15). Data are expressed as the mean ± s.e.m. of values obtained from 5 different mice. Statistical analysis was performed by Kruskal-Wallis test and Dunn’s post hoc for comparisons between groups. Significance differences between the antigen-stimulated control group without AZD7762 and other treatment groups are indicated, *p < 0.05 and
**p < 0.01.

Fig. 7. AZD7762 inhibits the activation of LAD2 human mast cells. (A-B) IgE-primed LAD2 cells were incubated with or without AZD7762 for 30 min. (A) The cells (2.0 × 105 cells/well) were subsequently stimulated with streptavidin (SA) for 30 min. The degree of degranulation of mast cells was expressed as % of β-hexosaminidase secreted out of total β-hexosaminidase (n = 9). (B) The cells (3.0 × 106 cells/tube) were stimulated with SA for 10 min. The cell lysates were subjected to Western blot analysis as described in the “Materials and Methods” section. Representative images and the band density for phosphorylated Syk from three independent experiments are shown (n
= 3). (A-B) The values indicate mean ± s.e.m. from three independent experiments. Statistical analysis was performed by Kruskal-Wallis test and Dunn’s post hoc for

comparisons between groups. Significant differences against the SA-stimulated controls without AZD7762 are indicated, *p < 0.05 and **p <0.01.

*Graphical Abstract (for review)

Graphical abstract