Interferon regulatory factor 3 (IRF3)
KIN1148 acts on interferon regulatory factor 3 (IRF3) as an agonist [1]

KIN1148 is a small-molecule agonist of the IRF3 (RIG-I-like receptor) pathway and a novel influenza vaccine adjuvant found to enhance flu vaccine efficacy. It induces the dose-dependent nuclear translocation of IRF3 in PH5CH8 cells and specific activation of IRF3- responsive promoters. KIN1148 also elicits greater induction of endogenous IRF3-dependent ISG54 and OASL expression by PH5CH8 cells. KIN1148. KIN1148 induced dose-dependent IRF3 nuclear translocation and specific activation of IRF3-responsive promoters. Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induced an influenza virus-specific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Prime-boost immunization with vaccine plus KIN1148, but not prime immunization alone, induced antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provided increased protection over vaccine alone and reduced viral load in the lungs after challenge. These findings suggest that protection was at least partially mediated by a cellular immune component and that the induction of Th2 and immunoregulatory cytokines by a KIN1148-adjuvanted vaccine may be particularly beneficial for ameliorating the immunopathogenesis that is associated with influenza viruses. KIN1148 induces the dose-dependent nuclear translocation of IRF3 in PH5CH8 cells and specific activation of IRF3- responsive promoters. KIN1148 also elicits greater induction of endogenous IRF3-dependent ISG54 and OASL expression by PH5CH8 cells.

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KIN1148 induced dose-dependent nuclear translocation of IRF3 in PH5CH8 cells, and was more potent than its precursor KIN1000 in activating the expression of IRF3-responsive genes (ISG54 and OASL) in PH5CH8 cells [1]
- KIN1148 induced the production of IP-10 in PMA-activated THP-1 cells [1]

Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protect against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induce an influenza virusspecific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Primeboost immunization with vaccine plus KIN1148, but not prime immunization alone, induce antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provide increased protection over vaccine alone and reduce viral load in the lungs after challenge

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Prime-boost immunization (two immunizations, 21 days apart) of C57BL/6N mice with a suboptimal dose of monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against lethal challenge (10× LD₅₀ of mouse-adapted A/California/04/2009 influenza virus) 21 days after boost, significantly improving survival rate and reducing weight loss compared with vaccine plus PBS/vehicle [1]
- Prime-boost immunization with vaccine plus KIN1148 induced influenza virus-specific IL-10 and Th2 responses in T cells derived from lung and lung-draining lymph nodes of challenged mice (collected on day 7 post-challenge) [1]
- Prime-boost immunization with vaccine plus KIN1148 induced higher IgG titers (14 days post boost), neutralizing antibody titers and hemagglutination inhibition (HAI) titers compared with vaccine plus PBS/vehicle; prime immunization alone with vaccine plus KIN1148 also provided increased protection over vaccine alone and reduced viral load in the lungs after challenge [1]
- Passive transfer of serum from mice immunized with vaccine plus KIN1148 (prime-boost immunization) protected recipient C57BL/6N mice against lethal H1N1 challenge (10× LD₅₀), while serum from prime-only immunization showed weaker protective effects [1]

KIN1148 is a small-molecule agonist of the IRF3 (RIG-I-like receptor) pathway and a novel influenza vaccine adjuvant found to enhance flu vaccine efficacy. It induces the dose-dependent nuclear translocation of IRF3 in PH5CH8 cells and specific activation of IRF3- responsive promoters. KIN1148 also elicits greater induction of endogenous IRF3-dependent ISG54 and OASL expression by PH5CH8 cells.

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Split vaccine-specific IgG ELISA [1]
ELISA plates were coated with the split vaccine at 1 μg of HA per ml of PBS. Plates were blocked with 5% BSA in PBS plus 0.1% Tween-20 (PBST). Mouse sera were added at the indicated sera dilutions (1:1000/ 1:10,000) in duplicate to plates, and allowed to bind to antigen for 1.5 h. Mouse antibodies were detected with biotinylated goat anti-Mouse IgG antibodies at 1:5000. Streptavidin-HRP was then added at 1:20,000 and incubated for 30 min. Plates were washed, developed for 2 min with TMB substrate, and stopped with 1 M H3PO4. Individual well optical density (OD) was measured at 450 nm.

KIN1148 induces the dose-dependent nuclear translocation of IRF3 in PH5CH8 cells and specific activation of IRF3- responsive promoters. KIN1148 also elicits greater induction of endogenous IRF3-dependent ISG54 and OASL expression by PH5CH8 cells.

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Hemagglutination inhibition (HAI) and virus microneutralization (VMN) [1]
HAI assays were performed using 0.5% fresh whole turkey blood according to WHO guidelines. For VMN assays, serum was heat-inactivated at 56 °C for 45 min and diluted twofold in infection media. Virus was diluted in infection media and added to diluted serum at a multiplicity of infection of 0.15 and incubated for 90 min in a humidified incubator at 37 °C. The virus and serum mixture was then transferred onto MDCK cells and incubated at 37 °C for 6 h. Cells were fixed with ice-cold methanol-acetone (1:1) for 10 min. Fixing solution was aspirated and cells were washed once with PBS. Plates were blocked for 1 h with 10% horse serum containing BSA (1 mg/ml) and 0.1% Triton X-100. Fluorescein isothiocyanate-labeled mouse IgG to influenza nucleoprotein (1:3000) and Hoechst dye (1:5000) were added to plates in 1% horse serum and incubated for 2 h at room temperature. Infected cell counts and nuclear staining were quantified using an ArrayScan HCS instrument. Neutralizing titers were determined as the reciprocal of highest dilution of serum resulting in 50% reduction in infection over negative control (no serum).

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IRF3 nuclear translocation assay: PH5CH8 cells were treated with KIN1148 at different doses, and the nuclear translocation of IRF3 was detected; experiments were performed in duplicates and repeated three times independently [1]
- IRF3-responsive gene expression assay: PH5CH8 cells were exposed to KIN1148 (or KIN1000 as control), and the expression levels of ISG54 and OASL (IRF3-dependent genes) were measured; experiments were performed in duplicates and repeated three times independently [1]
- IP-10 production assay: THP-1 cells were activated with PMA and then treated with KIN1148, and the production of IP-10 in cell culture supernatants was detected; experiments were performed in triplicates and repeated three times independently [1]
- T cell cytokine detection assay: T cells isolated from lung and lung-draining lymph nodes of challenged mice were stimulated with split vaccine (SV), CD4 epitope peptide (MA-CA/04 NP₂₆₀₋₂₈₃), CD8 epitope peptide (MA-CA/04 NP₃₆₆₋₃₇₄) or concanavalin A (Con A) for 18 h; cytokines (including IL-10) in culture supernatants were measured using multiplex ELISA; for lung-derived T cells, samples from individual mice were evaluated, while lymph node-derived cells were pooled by group, and stimulations were performed in triplicate [1]

5 mg/mL; i.m.
\n Mice \nKIN1148 was formulated in liposomes containing phosphatidylcholine, pegylated phosphatidylethanol and cholesterol in phosphate-buffered saline (PBS) by 6 h ultrasonication. The final liposomes prior to mixing with vaccine contained KIN1148 (5 mg/ml) and phospholipids (40 mg/ml) and were delivered intramuscularly (KIN1148 at 50 μg and phospholipid at 400 μg per dose). Blank liposomes were prepared without KIN1148 as vehicle control. KIN1148 liposome and blank liposome formulations were stable for at least 4 months at 4 °C and were used for in vivo experiments within a month after preparation.[1]

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\nMice were anesthetized with isoflurane and immunized intramuscularly with 50 μl (3.3 μg total protein corresponding to 0.6 μg HA with and 0.069 EU) of vaccine, split between two 25-μl injections, one in each gastrocnemius muscle. The suboptimal dose of vaccine (3.3 μg) was determined by vaccinating with descending amounts of split-vaccine alone until reaching suboptimal protection from lethal challenge (>25%). All vaccination studies were performed using the same dose and batch of the Virapur split vaccine. For prime and prime/boost immunization, mice were immunized either once (prime) or twice 21 days apart. Twenty-one days after the receiving the final vaccine dose, mice were challenged intranasally with 10× the LD50 (2500 plaque-forming units/mouse, lethal virus dose that causes the death of 50% of the naïve mouse population inoculated with virus) of mouse-adapted influenza virus. Mice were euthanized humanely by carbon dioxide asphyxiation if displaying weight loss ⩾30% of starting weight or when displaying symptoms of severe disease including hunched posture, irregular breathing, pillory erection, and reduced activity. For serology, mice were anesthetized with isoflurane and whole blood (200 μl) was collected retro-orbitally for the processing of serum. For cytokine profiling, T cells from lung and lung-draining lymph nodes were stimulated for 18 h with split vaccine (1 μg/ml), CD4 epitope peptide MA-CA/04 NP260–283 (5 μM), CD8 epitope peptide MA-CA/04 NP366–374 (5 μM), or concanavalin A (Con A 5 μg/ml). Cytokines in culture supernatants were then measured using a multiplex ELISA (Quansys). Cytokine experiments were performed in triplicate. Bone marrow-derived dendritic cells were added as antigen-presenting cells to the lymph node T cell assays.[1]

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\nLethal influenza challenge model (prime-boost immunization): Groups of 10 C57BL/6N mice were immunized twice (21 days apart) with a suboptimal dose of monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 (vaccine plus PBS/vehicle as controls); 21 days after the boost immunization, mice were challenged with 10× the LD₅₀ of mouse-adapted influenza virus A/California/04/2009, and survival rate and body weight changes were monitored for 14 days post-challenge; the experiment was repeated twice independently [1]
\n- Humoral response assay: C57BL/6N mice were immunized twice (21 days apart) with vaccine plus KIN1148 (vaccine plus PBS/vehicle as controls); mice were bled at day 18 post prime and day 35 (14 days post boost), and serum IgG titers, neutralizing antibody titers and HAI titers were determined; n=15 for IgG titer detection, n=8 for neutralizing antibody and HAI titer detection; the experiment was repeated three times independently [1]
\n- Passive transfer assay: Immune sera were collected from mice immunized once (prime) or twice (prime-boost, 21 days apart) with vaccine plus KIN1148 (vaccine plus vehicle as control); groups of 6 C57BL/6N recipient mice received 200 μl of pooled immune sera via intraperitoneal injection on day -2 and day -1 before challenge; recipients were then challenged with 10× LD₅₀ of mouse-adapted A/California/04/2009 influenza virus and observed for 14 days post-challenge [1]
\n- Lethal influenza challenge model (prime immunization alone): C57BL/6N mice were immunized once with vaccine plus KIN1148 (vaccine plus PBS/vehicle as controls); after challenge with 10× LD₅₀ of influenza virus, survival rate was monitored (10 mice per group), lung viral load was measured 3 days post challenge (5 mice per group), and T cell cytokine responses in lung and lung-draining lymph nodes were detected 7 days post challenge (5 mice per group); the experiment was repeated at least twice independently [1]
\n- T cell cytokine response assay (in vivo): C57BL/6N mice (n=4 for vehicle group, n=8 for KIN1148 group) were immunized twice (21 days apart) with vaccine plus vehicle/KIN1148, then challenged with 10× LD₅₀ of influenza virus; on day 7 post challenge, T cells were isolated from lung and lung-draining lymph nodes, stimulated with specific antigens/Con A for 18 h, and cytokines in supernatants were measured by multiplex ELISA [1]

[1]. A small-molecule IRF3 agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response. Vaccine. 2017 Apr 4;35(15):1964-1971.

Vaccine adjuvants are crucial for inducing protective immune responses in cases where vaccine antigens have weak immunogenicity, limited antigen quantities, or where enhanced vaccine efficacy is needed in specific populations (e.g., the elderly). To discover novel vaccine adjuvants, we employed a high-throughput screening (HTS) approach to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway, which activates interferon regulatory factor 3 (IRF3). RLRs are cytoplasmic pattern recognition receptors crucial for recognizing viral nucleic acids during viral infection. Upon ligand binding to viral nucleic acids, RLRs are activated and signal transcription factors, including IRF3, to initiate a lead immune transcription program to control viral infection. Our HTS screening identified a series of benzothiazole compounds from which we designed the lead compound KIN1148. KIN1148 dose-dependently induces IRF3 nuclear translocation and specifically activates the IRF3 reactive promoter. A primary-boost immunization of mice with a suboptimal dose of a monovalent H1N1 influenza virus split vaccine (A/California/07/2009) in combination with KIN1148 effectively resisted lethal challenge with mouse adaptive influenza virus (A/California/04/2009) and induced influenza virus-specific IL-10 and Th2 responses in T cells derived from the lungs and pulmonary draining lymph nodes. The primary-boost immunization with the vaccine in combination with KIN1148 (rather than primary immunization alone) induced antibodies that inhibited influenza virus hemagglutinin and neutralized viral infectivity. However, a single immunization with the vaccine in combination with KIN1148 provided greater protection than the vaccine alone and reduced viral load in the lungs after challenge. These results suggest that the protective effect is at least partially mediated by cellular immune components, and that the induction of Th2 cells and immunomodulatory cytokines by the KIN1148 adjuvant vaccine may be particularly beneficial in improving influenza virus-related immunopathological processes. [1]

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We discovered that KIN1148 is a small molecule agonist of the RLR pathway that activates IRF3. Unlike nucleic acid RLR agonists, our small molecule agonist can be optimized for efficacy, reactivity, and delivery. KIN1148 is a lipophilic small molecule compound with a cLogP value of 4.76 and limited solubility in aqueous solution. The addition of KIN1148 liposomes to KIN1148 improved the adjuvant activity of the compound compared to KIN1148 formulated in PBS (data not shown). This effect may be mediated by the uniform dispersion of KIN1148 liposomes and the split vaccine, as well as the ability to deliver higher doses of the compound. Unlike nucleic acid RIG-I ligands, the KIN1148 adjuvant system does not require transfection reagents or integration into DNA vaccines to exert adjuvant activity in vivo. [1]
After primary-boost immunization using the KIN1148/split vaccine adjuvant system, protective antibodies, IL-10, and Th2 responses were induced in the lungs and pulmonary draining lymph nodes after viral challenge. Similarly, DMXAA (a potent IRF3-dependent type I interferon inducer) can also induce a Th2 response to a split influenza virus vaccine. [1] Although Th2 immune responses, particularly eosinophilia, can potentially trigger allergic reactions, recent studies on mucosal surface Th2 immunity have shown diversity within Th2 responses. Th2 immune responses can be categorized into two types: anti-inflammatory responses (tissue protection or repair) or pro-inflammatory responses (tissue damage, mast cell degranulation, asthma, allergies, and inflammation). In mice infected with influenza virus, we observed influenza antigen-specific IL-10-producing T cell populations in their lungs and pulmonary draining lymph nodes, suggesting that vaccine-assisted KIN1148 immunization can promote a balanced immune response, thereby alleviating the immunopathology caused by influenza virus. Our findings support the use of small molecule RLR agonists as novel immunomodulatory adjuvants in the preclinical development of RNA virus vaccines. In addition, the use of KIN1148 adjuvanted vaccines against pathogenic human and avian influenza viruses will be explored in order to control the excessive inflammatory response caused by these viruses by inducing a tissue-protective Th2 regulatory immune response. KIN1148 is a small molecule IRF3 agonist derived from a series of benzothiazole compounds identified by high-throughput screening (HTS). It targets the RIG-I-like receptor (RLR) pathway, thereby activating IRF3 [1]. KIN1148 acts as an adjuvant for influenza vaccines by regulating antiviral immune responses. The body's protection against influenza virus attack is at least partially mediated by cellular immune components, and the Th2 cells and immunomodulatory cytokines induced by KIN1148 adjuvanted vaccines may alleviate influenza-related immunopathology [1]. RLR is a cytoplasmic pattern recognition receptor that is essential for recognizing viral nucleic acids. After activation, RLR signals transcription factors such as IRF3 to initiate an innate immune response against viral infection [1].

C19H11N3OS2

361.44

361.034

C, 63.14; H, 3.07; N, 11.63; O, 4.43; S, 17.74

1428729-56-9

71549151

Light yellow to yellow solid powder

5.3

1

5

2

25

519

0

YAISOECYKYATLL-UHFFFAOYSA-N

InChI=1S/C19H11N3OS2/c23-18(13-6-5-11-3-1-2-4-12(11)9-13)22-19-21-14-7-8-15-16(17(14)25-19)20-10-24-15/h1-10H,(H,21,22,23)

N-(benzo[1,2-d:3,4-d']bis(thiazole)-2-yl)-2-naphthamide

KIN 1148; KIN1148; 1428729-56-9; N-Benzo[1,2-d; KIN-1148; N-(benzo[1,2-d:3,4-d']bis(thiazole)-2-yl)-2-naphthamide; N-([1,3]thiazolo[5,4-e][1,3]benzothiazol-2-yl)naphthalene-2-carboxamide; SCHEMBL14847549; KIN-1148;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 10.5~100 mg/mL ( 29.05~276.67 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)