Anti-dinitrophenyl (DNP) IgE antibodies, DNP–bovine serum albumin (BSA), BSA, p-nitrophenyl-N-acetyl-β-glucosamine, Ceapin-A7, and Tg were purchased from Sigma-Aldrich (Tokyo, Japan). GSK2606414 was purchased from LKT Laboratories (St. Paul, MN, United States). APY29 was purchased from AdooQ Bioscience (Irvine, CA, United States). Additionally, 4μ8c was obtained from Merck (Darmstadt, Germany). Anti-IgE antibodies were purchased from Bethyl Laboratories (Montgomery, TX, United States). Recombinant mouse IL-3 and human stem cell factor were purchased from R&D Systems Inc. (Minneapolis, MN, United States). Penicillin/streptomycin and trypsin were purchased from Life Technologies (Carlsbad, CA, United States). Roswell Park Memorial Institute (RPMI)-1640 medium, Hank’s balanced salt solution (HBSS), and 2-mercaptoethanol were obtained from Nacalai Tesque (Kyoto, Japan). KIRA6 was obtained from Selleck Biotech (Kanagawa, Japan). KIRA8 and amlexanox were purchased from MedChemExpress (Monmouth Junction, NJ, United States). Tranilast and epinastine were purchased from Tokyo Chemical Industry (Tokyo, Japan).

Rat basophilic leukemia (RBL)-2H3 cells were cultured in the RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Nichirei, Tokyo, Japan), 100 IU/mL penicillin G, and 100 μg/mL streptomycin.

Bone marrow-derived mast cells (BMMCs) were obtained from 8–10-week-old BALB/c female mice (Charles River Laboratories, Yokohama, Japan), as previously described (Okabe et al., 2006). In brief, bone marrow cells were suspended at a density of 1 × 10⁶ cells/mL in the RPMI-1640 medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol, 2 mM glutamine, 100 IU/mL penicillin G, 100 μg/mL streptomycin, and 5 ng/mL IL-3 for 4 weeks.

Total RNA was extracted using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan). Then, cDNA was synthesized from 0.2 μg (BMMCs) or 2 μg (RBL-2H3 mast cells) of total RNA via reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan) and Oligo (dt)₁₆ primer (Eurofins Genomics, Tokyo, Japan) in a 20-μL reaction mixture containing RT buffer (Toyobo), 1 mM dNTP mix (Toyobo), and 20 U of the RNase inhibitor (Enzymatics, Beverly, MA, United States). Total RNA and Oligo (dt)₁₆ primer were pre-incubated at 70 °C for 10 min prior to reverse transcription. After incubation for 1.5 h at 46 °C, the reaction was terminated by incubating the samples at 100 °C for 5 min. For polymerase chain reaction (PCR) amplification, 1.2 μL of cDNA was added to 10.8 μL of reaction mix containing 1 μM of each primer, KAPA Taq EXtra Buffer, 0.3 mM of KAPA dNTP mix, 1.75 mM of MgCl₂, and 0.3 U of KAPA Taq EXtra DNA polymerase (Roche, Basel, Switzerland). The following primer sequences were used: m(r)glyceraldehyde 3-phosphate dehydrogenase (GAPDH) upstream 5′-AAA​CCC​ATC​ACC​ATC​TTC​CAG-3′, m(r)GAPDH downstream 5′-AGG​GGC​CAT​CCA​CAG​TCT​TCT-3′, rXBP1 upstream 5′-CTT​GTG​ATT​GAG​AAC​CAG​GAG-3′, rXBP1 downstream 5′-AAG​AGG​CAA​CAG​CGT​CAG-3′, mXBP1 upstream 5′-CCT​TGT​GGT​TGA​GAA​CCA​GG-3′, and mXBP1 downstream 5′-CTA​GAG​GCT​TGG​TGT​ATA​C-3′. PCR products (10 μL) were resolved via electrophoresis on an 8% polyacrylamide gel. The gels were stained with ethidium bromide and photographed under ultraviolet light. The density of each band was measured using ImageJ 1.37v software (National Institutes of Health, MD, United States).

Degranulation was assessed by measuring the release of β-hexosaminidase, a granule marker that hydrolyzes p-nitrophenyl-N-acetyl-β-glucosamine to the chromophore, p-nitrophenol, as previously described (Hide et al., 1997). In brief, RBL-2H3 mast cells, BMMCs, or human skin mast cells (hsMCs) sensitized with anti-DNP IgE antibodies overnight were resuspended in piperazine-N- N′-bis (2-ethanesulfonic acid) (PIPES) buffer containing 119 mM NaCl, 5 mM KCl, 1.0 mM CaCl₂, 0.4 mM MgCl₂, 5.6 mM glucose, 25 mM PIPES, and 1 mg/mL BSA (pH 7.2). After incubation with or without inhibitors for 30 min at 37 °C, the cells were stimulated with 100 or 50 ng/mL DNP-BSA antigens or 670 ng/mL anti-IgE antibodies at 37 °C for 15 min.

hsMCs were isolated from human skin, as previously described (Yanase et al., 2021). In brief, the skin was cut into fragments and incubated in HBSS containing type 2 collagenase (1.5 mg/mL), hyaluronidase (0.7 mg/mL), type I DNase (0.3 mg/mL), 1% FCS, and 1 mM CaCl₂ at 37 °C for 2 h. The dispersed cells were suspended in the X-VIVO-15 medium (Lonza, Walkersville, MD, United States). Isolated mast cells were cultured in the X-VIVO-15 medium containing 100 ng/mL recombinant human stem cell factor for 1–2 months.

Informed consent was obtained from four independent donors according to the ethical standard of Hiroshima University (approval number: E2014-1115).

Human peripheral blood mononuclear cells (PBMCs) were isolated from the fresh heparinized blood of drug-free healthy donors via Ficoll-Paque Plus density gradient separation.

Informed consent was obtained from four independent donors according to the ethical standards of Hiroshima University (approval number: E2019-1716).

Histamine release tests using human peripheral blood basophils were performed with goat anti-IgE antibodies as a positive control, as previously described (Matsuo et al., 2018). Histamine was extracted and measured using reverse-phase high-performance liquid chromatography (HPLC).

Two-step quantitative PCR (qPCR) was performed using the Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent Technologies, Santa Clara, CA, United States) and PikoReal 96 (Thermo Fisher Scientific, Waltham, MA, United States), according to the manufacturers’ protocols. The cycling protocol was as follows: DNA polymerase activation at 95 °C for 5 min, followed by denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 20 s for 40 cycles. The relative level in each sample was normalized to the GAPDH mRNA level in the same sample and calculated using the ΔΔCT analysis. qPCR was performed using the following primers: mIL-6 upstream 5′-GTT​CTC​TGG​GAA​ATC​GTG​GA-3′, mIL-6 downstream 5′-TGT​ACT​CCA​GGT​AGC​TAT​GG-3′, mTNF-α upstream 5′-CAC​GTC​GTA​GCA​AAC​CAC​CAA-3′, mTNF-α downstream 5′-CCC​ATT​CCC​TTC​ACA​GAG​CAA-3′, mGAPDH upstream 5′-TGC​ACC​ACC​AAC​TGC​TTA​GC-3′, mGAPDH downstream 5′-GGC​ATG​GAC​TGT​GGT​CAT​GAG-3′, rat TNF-alpha qPCR primer pair (Sino Biological Inc., Beijing, China), rat IL-6 qPCR primer pair (Sino Biological Inc.), rGAPDH upstream 5′-AAA​CCC​ATC​ACC​ATC​TTC​CAG-3′, and rGAPDH downstream 5′-AGG​GGC​CAT​CCA​CAG​TCT​TCT-3′.

Enzyme-linked immunosorbent assay (ELISA) was performed using Mouse TNF-α DuoSet ELISA (R&D Systems), the LEGEND MAX Mouse IL-6 ELISA Kit (BioLegend, San Diego, CA, United States), and the Leukotriene C4 ELISA Kit (Cayman, Ann Arbor, MI, United States), according to the manufacturers’ protocols.

The CRISPRdirect design tool (Naito et al., 2015) was used to identify the optimal target sequences of SpCas9 for the rat Ern1 gene, which encodes IRE1α. Spacer-coding DNAs was inserted into lentiCRISPRv2 (#52961; Addgene, Watertown, MA, United States) using the BsmBI cloning site to generate lentiCRISPRv2-rEnr1-1 or lentiCRISPRv2-rEnr1-2 (Sanjana et al., 2014). Protospacer sequences were 5′-ATG​CAA​ACT​TCC​GTC​CAG​GG-3′ (KO1) and 5′-AGA​GGA​CAG​GCT​CCA​TCA​AG-3′ (KO2). Lentivirus was produced by co-transfecting 2 × 10⁶ HEK-293T cells in a 10-cm dish with 2 μg of lentiCRISPRv2-rEnr1-1 or lentiCRISPRv2-rEnr1-2, 2 μg of psPAX2 (#12260; Addgene), and 2 μg of pMD2.G (#12259; Addgene) using 25 μg of PEI MAX (Polysciences, Warrington, PA, United States). Twenty-four hours post-transfection, the culture medium was replaced with Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific). After 48 h, the culture medium was centrifuged at 1000 × g for 3 min to remove the cells. The resulting lentivirus-containing supernatant was used to infect RBL-2H3 mast cells in the presence of 8 μg/μL polybrene (Nacalai Tesque). Twenty-four hours post-transduction, the cells were treated with 4 μg/μL puromycin (Wako) for 2 days and then with 1 μg/μL puromycin for 8 days.

The experiment was performed according to the method described by Kitagawa et al. (2013) and was conducted by Carna Biosciences, Inc. (Hyogo, Japan). In brief, reaction mixtures of indicated concentrations of KIRA6, 1 mM ATP, and each kinase in the presence of its substrate peptide were incubated at room temperature for 1 h (Btk, Fyn, JAK2, Kit, Lyn, Syk, Erk1, JNK1, p38α, PKCα, and PKCε) or 5 h (JAK1). Following the incubation, substrate peptides and phosphorylated peptides in the reaction mixtures were separated and quantified.

The 3D structure of human Lyn (hLyn) registered in the Protein Data Bank (PDB) contains missing coordinates. Therefore, a complete model was obtained by filling in the missing regions using AlphaFold 3 (Abramson et al., 2024). The complex structure of hLyn and KIRA6, as well as compounds with a similar scaffold to KIRA6, has not been registered in the PDB. However, a complex structure of mouse Lyn (mLyn), which has approximately 97% amino acid sequence identity with hLyn, and KIRA6, which has a similar geometric structure to PP2, has been registered. Therefore, the hLyn/KIRA6 complex structure was modeled using the mLyn/PP2 complex structure (PDBid: 2zv9). In the mLyn/PP2 complex structure, the pairs of the amino moiety of PP2 and the O atom of Glu320, the N atom of the pyrimidine ring, and the N atom of Met-322 form hydrogen bonds. From the correspondence between the partial structures of PP2 and KIRA6, it can be suggested that the amino moiety of KIRA6 and the O atom of the main chain of Glu320, along with the N atom of the pyrazine ring and the N atom of Met-322, form a hydrogen bond.

To create the hLyn/KIRA6 complex model, first, the structure of the mLyn/PP2 complex was superimposed onto the hLyn structure, and the PP2 structure was extracted. For the extracted PP2 structure, the amino moiety and the pyrazine ring of KIRA6 were superimposed onto the amino moiety and the pyrimidine ring of PP2, respectively. The superimposed structures of KIRA6 and hLyn were used as the initial structure for the hLyn/KIRA6 complex. The obtained hLyn/KIRA6 complex structure was unstable since some atomic distances between hLyn and KIRA6 were significantly close. Molecular dynamics (MD) simulations were performed in the hydration state to obtain a stable structure. As the initial structure for the MD simulation, the hLyn/KIRA6 complex structure was placed in a box of water molecules that sufficiently surrounded the complex. The parameters of the water molecule were set using the TIP3P water model. Simulations were performed on the initial structure using 2-fs timesteps, at 300 K and 1 atm. In this simulation, a 10-ns MD simulation with constraints on the interatomic distances for the two hydrogen bonds mentioned above was performed, followed by a 10-ns MD simulation without constraints. All MD simulations were performed using the AMBER software package (Götz et al., 2012; Salomon-Ferrer et al., 2013; Case et al., 2023). Electrostatic potential on hLyn was calculated using an adaptive Poisson–Boltzmann solver (APBS) (Jurrus et al., 2018).

From the structures obtained through the simulation, we extracted those in which both of the following atom pairs—(1) the amino moiety and the O atom of Glu320 and (2) the N atom of the pyrazine ring and the N atom of Met-322—are within a distance range of 2.7–3.4 Å as these pairs are suggested to be involved in hydrogen bonding between hLyn and KIRA6. The structure with the lowest system energy was selected as the solution among these.

Western blotting was performed using two different methods: wet and semi-dry transfer. For wet transfer, the cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with a buffer containing 10 mM HEPES–NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na₃VO₄, 10 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% NP-40 for 20 min. The lysates were centrifuged at 15,000 rpm for 20 min at 4 °C, and the supernatants were collected. The samples were boiled with Laemmli buffer containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, 62.5 mM Tris-HCl, 5% β-mercaptoethanol, and 0.01% bromophenol blue for 3 min, fractionated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes (Wako) at 4 °C. These membranes were incubated with anti-IRE1α (#3294; Cell Signaling, Dunbarton, MA, United States) and anti-GAPDH (60004-1; Proteintech, Rosemont, IL, United States) antibodies, followed by incubation with anti-horseradish peroxidase-conjugated antibodies (MBL, Tokyo, Japan). Peroxidase binding was detected via chemiluminescence using an enhanced chemiluminescence system (Thermo Fisher Scientific). For semi-dry transfer, the samples were subjected to Mini-Protean TGX Gels (BIO-RAD, Hercules, CA, United States) and transferred to a polyvinylidene fluoride membrane, as previously described (Hiragun et al., 2013). Immunoblotting was performed using anti-Syk (#2712; Cell Signaling) and anti-phospho Syk (Tyr525/526; #2710; Cell Signaling) antibodies at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies and chemiluminescence were used for visualization. All images were captured using a chemiluminescence detector (Fusion SOLO; VILBER, Paris, France). Finally, the density of each band was measured using ImageJ 1.37v software.

Cell viability was assessed using the cell counting kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) and Cytotoxicity LDH Assay Kit-WST (Dojindo), according to the manufacturers’ protocols. Then, absorbance was measured using Varioskan LUX (Thermo Fisher Scientific).

Intracellular Ca²⁺ concentration was determined using the Calcium Kit-Fura 2 (Dojindo), according to the manufacturer’s protocol. Absorbance was measured every 10 s or 20 s using Varioskan Flash (Thermo Fisher Scientific). Sixty seconds after the start of measurement, BMMCs or RBL-2H3 mast cells were stimulated with DNP–BSA (50 ng/mL).

A mixture of 20% sulfobutylether β-cyclodextrin (Selleck Biotech, Kanagawa, Japan) and 80% saline was used to dissolve anti-DNP IgE antibodies (500 μg/mL) and KIRA6 (0.5 mg/mL), following which 100 μL was injected into the ear pinna of each 8–9-week-old BALB/c male mouse. After 24 h, 100 μL of KIRA6 (0.5 mg/mL) dissolved in 20% sulfobutylether β-cyclodextrin and 80% saline was injected into the ear pinna. After 2 h, 100 μL of the DNP–BSA solution (2 mg/mL in 10 mg/mL Evans blue solution) was intravenously administered into the tail. The mice were euthanized after 1 h. The ear was excised and soaked in 0.2 mL of 1 N KOH at 37 °C for 16 h. Then, 1.8 mL of a mixed solution of 0.6 N phosphoric acid and acetone (5:13) was added, shaken vigorously for a few seconds, and centrifuged at 2,500 rpm for 10 min. Finally, absorbance of the supernatant was measured at 620 nm using Varioskan Flash (Katayama et al., 1978; Gupta et al., 2021). All animal experiments were conducted in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee at Hiroshima University.

The results of experiments are represented as the mean ± standard error (SE). Analysis of variance (ANOVA), followed by Dunnett’s test, was used to compare multiple groups. Statistical significance was set at p < 0.05 for all tests, with significance levels indicated as *p < 0.05, **p < 0.01, and ***p < 0.001 in the figure legends.

We investigated whether the IRE1α–sXBP1 axis is activated in RBL-2H3 mast cells and BMMCs in response to antigen (DNP–BSA) stimulation that induces the release of Ca²⁺ from the ER. Upon sensitization of RBL-2H3 mast cells with anti-DNP IgE antibodies and stimulation with antigens, sXBP1 levels were significantly increased within 60 min (Figures 1A,B). This suggests that antigen-activated Ca²⁺ release from the ER induces the activation of the IRE1α–sXBP1 axis. We further investigated the effects of specific IRE1α kinase inhibitors (KIRA6 and KIRA8) on the IRE1α–sXBP1 axis induced by antigen stimulation in BMMCs sensitized with anti-DNP IgE antibodies. As shown in Figures 1C,D, the induction of sXBP1 in antigen-stimulated BMMCs was significantly inhibited by KIRA6 or KIRA8. We also detected strong and rapid activation of the IRE1α–sXBP1 axis in response to the ER Ca²⁺ ATPase inhibitor, Tg, which rapidly induced Ca²⁺ release from the ER (Supplementary Figure S1). These results suggest that the IRE1α–sXBP1 axis is activated within 60 min during antigen-induced mast cell activation.

Next, we investigated the effects of KIRA6 and KIRA8 on basophil and mast cell degranulation. We examined the effects of KIRA6 and KIRA8 on the antigen-induced degranulation of RBL-2H3 mast cells and BMMCs sensitized with anti-DNP IgE antibodies. Interestingly, as shown in Figures 2A,B, KIRA6, but not KIRA8, significantly inhibited the degranulation of RBL-2H3 mast cells and BMMCs in a concentration-dependent manner (n = 3). In contrast, PERK (GSK2606414) and ATF-6 (Ceapin-A7) inhibitors did not significantly suppress BMMC degranulation (n = 3) (Supplementary Figure S2). We further investigated whether KIRA6 suppresses degranulation in human-derived mast cells and basophils. This system included hsMCs sensitized to human IgE antibodies and PBMCs, including basophils, in response to anti-IgE antibodies. As shown in Figures 2C,D, KIRA6 significantly inhibited the IgE receptor-associated activation of hsMCs and human basophils (n = 4). KIRA6 did not affect cell viability (n = 3) (Supplementary Figures S3A, S3B). These results highlight KIRA6 as a unique compound inhibiting mast cell- and basophil-induced allergic reactions in response to antigens, without any cytotoxicity.

We analyzed whether KIRA6 inhibits the production of IL-6 and TNFα mRNA and the release of IL-6, TNFα, and LTC4 by BMMCs in response to antigen stimulation. KIRA6 significantly inhibited antigen-induced IL-6 and TNFα mRNA induction in BMMCs sensitized with anti-DNP IgE antibodies (n = 3) (Figures 3A,B). Additionally, KIRA6 inhibited the antigen-induced release of IL-6, TNFα, and LTC4 from BMMCs sensitized with anti-DNP IgE antibodies (n = 3) (Figures 3C–E). These results suggest that KIRA6 suppresses the aggravation of allergic reactions by inhibiting the production and release of lipid mediators and pro-inflammatory cytokines.

To confirm the roles of the IRE1α–sXBP1 axis in the functions of mast cells on allergic responses, we developed two IRE1α-knocked-out (KO) RBL-2H3 mast cells, KO1 and KO2. We used the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system to delete the coding region of IRE1α (Figure 4A). Figures 4B,C show the expression levels of IRE1α in wild-type (WT), KO1, and KO2 RBL-2H3 cells determined via Western blotting. Protein expression of IRE1α was not detected in KO1 and KO2 cells. Notably, depletion of IRE1α did not affect the survival and proliferation of RBL-2H3 mast cells (Figure 4D). Then, we performed degranulation assays using WT, KO1, and KO2 cells in response to antigen stimulation. Unexpectedly, antigen-induced degranulation of KO1 and KO2 cells was not altered compared to that of WT cells (Figure 5A). Moreover, antigen-induced production of IL-6 and TNFα mRNA in KO1 and KO2 cells was comparable to that in WT cells (Figures 5B,C). KIRA6 treatment at 1 μM significantly suppressed the antigen-induced degranulation of KO1 and KO2 cells by more than 90% (Figure 5D). KIRA6 also suppressed the antigen-induced increase in intracellular Ca²⁺ concentrations in IRE1α KO cells (Supplementary Figure S4). These results suggest that KIRA6 inhibits antigen-induced mast cell activation via an IRE1α-independent pathway.

As described above, KIRA6 affected mast cell degranulation via the IRE1α–sXBP1-independent pathway (Figure 5D). Then, what is the target molecule of KIRA6 in inhibiting allergic reactions? To explore other pharmacological targets of KIRA6 in mast cells and basophils, we then examined the effects of KIRA6 on several tyrosine kinases and serine/threonine kinases. The assay was performed by Carna Biosciences, Inc. (Hyogo, Japan). As shown in Table 1, KIRA6 mainly inhibited the src family tyrosine kinases, such as Fyn and Lyn. The dose-dependent inhibition curve of KIRA6 on Lyn is shown in Figure 6A. We then investigated whether KIRA6 can directly bind to hLyn using in silico binding simulation. In the hLyn/KIRA6 complex model, KIRA6 stably binds to the ATP-binding site by forming five hydrogen bonds with amino acid residues of hLyn (Figures 6B,C). In addition to the two hydrogen bonds used to evaluate the model (the amino group of KIRA6 and the oxygen atom of the main chain of Glu320, along with the N atom of the pyrazine ring and the NH of Met-322), the carboxyl group of Asp385 is bonded to the two NHs of the urea group, and the OH of Thr319 is hydrogen-bonded to the O atom. In general, hydrogen bonds strengthen compound–protein interactions, suggesting that KIRA6 binds to hLyn with high affinity. These hydrogen bonds, therefore, contribute to the lowest energy among the 15 structures. These findings indicate that the complex may adopt a structure similar to the proposed model. Lyn plays an important role in antigen-induced activation, which phosphorylates its downstream target, such as Syk (Gilfillan and Tkaczyk, 2006; Siraganian et al., 2010). Furthermore, Syk activates various molecules, including Bruton’s tyrosine kinase (Btk), thereby facilitating the transmission of signals downstream (Gilfillan and Tkaczyk, 2006; Siraganian et al., 2010). Therefore, in the present study, we focused on the downstream events of Lyn, such as Syk phosphorylation and Ca²⁺ mobilization. To this end, we investigated the effect of KIRA6 on antigen-induced phosphorylation of Syk in BMMCs sensitized with anti-DNP IgE antibodies and found that KIRA6 at 1 μM inhibited Syk phosphorylation by more than 90% (Figures 6D,E). In addition, KIRA6 inhibited antigen-induced Ca²⁺ mobilization in BMMCs sensitized with anti-DNP IgE antibodies (Supplementary Figure S5). These results suggest that KIRA6 binds to Lyn and blocks the activation of Syk by inhibiting the kinase activity of Lyn, thereby suppressing mast cell and basophil degranulation.

As KIRA6 exerts anti-allergic effects by inhibiting degranulation, we compared its anti-allergic effects with those of the clinically used drugs, such as amlexanox, tranilast, and epinastine. They are selected as benchmark drugs because they are widely used and known to inhibit the release of chemical mediators such as histamine from mast cells and basophils (Makino et al., 1987; Komatsu et al., 1988; Fujishima et al., 2014). As shown in Figures 7A,B, KIRA6 markedly inhibited the antigen-induced degranulation of RBL-2H3 mast cells and BMMCs sensitized with anti-DNP IgE antibodies at extremely low concentrations (over 100 times lower concentrations) compared to the known compounds used in clinical practice (amlexanox, tranilast, and epinastine). Overall, low concentrations (<1 μM) of KIRA6 effectively suppressed antigen-induced allergic reactions better than existing anti-allergic drugs, without any cytotoxicity (Supplementary Figures S3A, S3B).

To further investigate the anti-allergic effects of KIRA6 in vivo, we conducted a passive cutaneous anaphylaxis (PCA) test in BALB/c mice. PCA is a well-established test for local allergic reactions induced by injecting anti-DNP IgE antibodies into the mouse ear and antigens (DNP-BSA) into the tail vein (Nam et al., 2017). Intradermal administration of KIRA6 into the ear significantly reduced antigen-induced hyperpermeability (Figures 8A,B). These results confirmed that KIRA6 exerts potent anti-allergic effects in vivo.