Interferon regulatory factor 3 (IRF3)

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.

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

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).

KIN1148 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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Mice 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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5 mg/mL; i.m.

Mice

[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 essential to drive a protective immune response in cases where vaccine antigens are weakly immunogenic, where vaccine antigen is limited, or where an increase in potency is needed for a specific population, such as the elderly. To discover novel vaccine adjuvants, we used a high-throughput screen (HTS) designed to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway leading to interferon regulatory factor 3 (IRF3) activation. RLRs are a group of cytosolic pattern-recognition receptors that are essential for the recognition of viral nucleic acids during infection. Upon binding of viral nucleic acid ligands, the RLRs become activated and signal to transcription factors, including IRF3, to initiate an innate immune transcriptional program to control virus infection. Among our HTS hits were a series of benzothiazole compounds from which we designed the lead analog, 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.[1]

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We identified KIN1148 as 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, reactogenicity and delivery. KIN1148 is a lipophilic small molecule compound with a cLogP of 4.76 and limited solubility in aqueous solutions. The incorporation of KIN1148 liposomes increased the adjuvant activity of the compound over KIN1148 formulated in PBS (data not shown). This effect is likely mediated by the homogenous dispersion of KIN1148 liposomes and split vaccine and the ability to deliver a higher compound dose. In contrast to nucleic acid RIG-I ligands, the KIN1148 adjuvant system does not require transfection reagents or the integration into DNA vaccines for in vivo adjuvant activity.[1]
Prime-boost immunization with the KIN1148/split vaccine adjuvant system elicited protective antibodies and induced IL-10 and a Th2 response in the lung and lung-draining lymph nodes after viral challenge. Similarly, DMXAA, a potent IRF3-dependent inducer of type I IFN, also induces a Th2 response to a split influenza virus vaccine.[1]
Although Th2 immune responses, particularly eosinophilia, have the potential to elicit allergic responses, recent research into Th2 immunity at mucosal surfaces has revealed plurality within the Th2 response. Th2 immune responses may fall into two categories of outcome: anti-inflammatory (tissue protective or reparative) or pro-inflammatory (tissue damaging mast cell degranulation, asthma, allergy, and inflammation). The observations of influenza antigen-specific IL-10-producing T cell populations in lungs and lung-draining lymph nodes obtained from mice after challenge implies that immunization with vaccine plus KIN1148 promotes a balanced immune response that dampens influenza virus-induced immunopathology. Our results encourage the preclinical development of small-molecule RLR agonists as novel immunomodulatory adjuvants for vaccines against RNA viruses. In addition, the use of KIN1148-adjuvanted vaccines for pathogenic human and avian influenza viruses, where the excessive inflammatory responses caused by these viruses might be controlled by the induction of a tissue-protective Th2 regulatory immune response will be explored.

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.)