KIN1148

Interferon regulatory factor 3 (IRF3)

KIN1148 是一种 IRF3（RIG-I 样受体）途径的小分子激动剂，也是一种新型流感疫苗佐剂，可增强流感疫苗的功效。它诱导 PH5CH8 细胞中 IRF3 的剂量依赖性核转位以及 IRF3 响应启动子的特异性激活。 KIN1148 还可诱导 PH5CH8 细胞更大程度地诱导内源 IRF3 依赖性 ISG54 和 OASL 表达。 KIN1148。 KIN1148 诱导剂量依赖性 IRF3 核转位和 IRF3 响应启动子的特异性激活。使用次优剂量的单价大流行性流感裂解病毒 H1N1 A/California/07/2009 疫苗加 KIN1148 对小鼠进行初免加强免疫，可防止小鼠适应流感病毒 (A/California/04/2009) 的致命攻击并诱导来自肺和肺引流淋巴结的 T 细胞产生流感病毒特异性 IL-10 和 Th2 反应。使用疫苗加 KIN1148 的初免-加强免疫（而非单独初免）诱导的抗体能够抑制流感病毒血凝素并中和病毒感染性。尽管如此，使用疫苗加 KIN1148 进行单次免疫比单独使用疫苗提供了更强的保护，并减少了攻击后肺部的病毒载量。这些发现表明，保护作用至少部分是由细胞免疫成分介导的，并且 KIN1148 佐剂疫苗诱导 Th2 和免疫调节细胞因子可能特别有益于改善与流感病毒相关的免疫发病机制。 KIN1148 在 PH5CH8 细胞中诱导 IRF3 剂量依赖性核转位以及 IRF3 响应启动子的特异性激活。 KIN1148 还可诱导 PH5CH8 细胞更大程度地诱导内源 IRF3 依赖性 ISG54 和 OASL 表达。

使用次优剂量的单价大流行性流感裂解病毒 H1N1 A/California/07/2009 疫苗加 KIN1148 对小鼠进行初免加强免疫，可防止小鼠适应流感病毒 (A/California/04/2009) 的致命攻击，并诱导来自肺和肺引流淋巴结的 T 细胞产生流感病毒特异性 IL-10 和 Th2 反应。使用疫苗加 KIN1148 进行 Primeboost 免疫（而非单独初次免疫）可诱导能够抑制流感病毒血凝素并中和病毒感染性的抗体。尽管如此，与单独使用疫苗相比，使用疫苗加 KIN1148 进行单次免疫可提供更强的保护，并减少攻击后肺部的病毒载量

KIN1148 是一种 IRF3（RIG-I 样受体）途径的小分子激动剂，也是一种新型流感疫苗佐剂，可增强流感疫苗的功效。它诱导 PH5CH8 细胞中 IRF3 的剂量依赖性核转位以及 IRF3 响应启动子的特异性激活。 KIN1148 还可诱导 PH5CH8 细胞更大程度地诱导内源 IRF3 依赖性 ISG54 和 OASL 表达。

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分离疫苗特异性IgG ELISA试剂盒[1]
在每ml PBS中加入1 μg HA的裂解疫苗包被ELISA板。用5% BSA加0.1% Tween-20 （PBST）的PBS液阻断培养皿。将小鼠血清按指定的血清稀释倍数（1:1000/ 1:1000）一式两份加入板中，与抗原结合1.5小时。用1:5000的生物素化山羊抗小鼠IgG抗体检测小鼠抗体。然后以1:20 000的比例加入链亲和素- hrp，孵育30分钟。洗板，用TMB底物显影2分钟，用1 M H3PO4停止。在450nm处测量单孔光密度（OD）

KIN1148 在 PH5CH8 细胞中诱导 IRF3 剂量依赖性核转位以及 IRF3 响应启动子的特异性激活。 KIN1148 还可诱导 PH5CH8 细胞更大程度地诱导内源 IRF3 依赖性 ISG54 和 OASL 表达。

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血凝抑制（HAI）和病毒微中和（VMN） [1]
根据世卫组织指南，使用0.5%新鲜全火鸡血进行HAI测定。对于VMN检测，血清在56°C下热灭活45分钟，并在感染培养基中稀释两倍。将病毒在感染培养基中稀释后，以感染倍数为0.15加入稀释后的血清中，在37℃加湿培养箱中孵育90 min。将病毒和血清混合液转移到MDCK细胞上，37℃孵育6 h，冷冻甲醇-丙酮（1:1）固定细胞10 min，抽吸固定液，PBS洗涤细胞1次。用含BSA的10%马血清（1mg /ml）和0.1% Triton X-100阻断1小时。将异硫氰酸荧光素标记的小鼠流感核蛋白IgG（1:3000）和Hoechst染料（1:5000）加入1%马血清培养皿中，室温孵育2小时。使用ArrayScan HCS仪器定量检测感染细胞计数和细胞核染色。中和效价测定为血清最高稀释度的倒数，导致感染比阴性对照（无血清）减少50%。

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

111

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。