E6446
目录号 : GC33900
E6446 is an inhibitor of Toll-like receptor (TLR) 7 and 9 signaling in a variety of human and mouse cell types and inhibits DNA-TLR9 interaction in vitro.
Cas No.:1219925-73-1
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Cell experiment: | E6446 is assayed for the suppression of BALB/c mouse spleen interleukin-6 (IL-6) production in response to stimulation by oligonucleotide CpG1668. E6446 is added to dissociated splenocytes (5 × 105 per well in complete RPMI/10% fetal bovine serum in a 96-well plate) before addition of TLR agonists. Cells are stimulated for 72 hours, and supernatants are removed for ELISA analysis of IL-6. Mouse bone marrow-derived dendritic cells (BMDCs) are generated by culturing BALB/c marrow cells in RPMI containing 100 ng/mL Flt3 ligand for 7 days. Cells (1 × 105) in 50 μL are assayed for IL-6 production after overnight or 24-hour stimulation with various TLR ligands. For studies using human peripheral blood mononuclear cells, Ficoll-separated mononuclear cells are isolated from healthy volunteer donors, washed, and plated with stimulatory oligonucleotide CpG2216 in complete RPMI for 72 hours. Interferon in supernatant is quantified by ELISA[1]. |
Animal experiment: | Mice[1]MRL/lpr mice are dosed orally five times a week with 20 or 60 mg/kg E6446 or 60 mg/kg hydroxychloroquine beginning at 5 weeks of age. Cytoxan is administered at 50 mg/kg i.p. every 10 days. A serum sample is taken immediately before the beginning of treatment to monitor changes in autoreactive antibodies. Subsequently, serum samples are collected approximately monthly and analyzed for anti-dsDNA by ELISA after 1:500 dilution. Body weights and urine samples are taken at the same interval, and proteinuria is assessed by ChemStrips. Anti-nuclear antibodies (ANA) are assessed using commercially available HEp2 slide kits, with serum diluted to 1:100 in kit buffer. ANA scores are read blinded[1]. |
References: [1]. Lamphier M, et al. Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo. Mol Pharmacol. 2014 Mar;85(3):429-40. | |
E6446 is an inhibitor of Toll-like receptor (TLR) 7 and 9 signaling in a variety of human and mouse cell types and inhibits DNA-TLR9 interaction in vitro.
[1] Marc Lamphier, et al. Mol Pharmacol. 2014 Mar;85(3):429-40.
| Cas No. | 1219925-73-1 | SDF | |
| Canonical SMILES | C1(C2=CC=C(OCCCN3CCCC3)C=C2)=NC4=CC=C(OCCCN5CCCC5)C=C4O1 | ||
| 分子式 | C27H35N3O3 | 分子量 | 449.59 |
| 溶解度 | Soluble in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.2242 mL | 11.1212 mL | 22.2425 mL |
| 5 mM | 0.4448 mL | 2.2242 mL | 4.4485 mL |
| 10 mM | 0.2224 mL | 1.1121 mL | 2.2242 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
mRNA transfection by a Xentry-protamine cell-penetrating peptide is enhanced by TLR antagonist E6446
PLoS One 2018 Jul 30;13(7):e0201464.PMID:30059522DOI:10.1371/journal.pone.0201464.
Messenger RNA (mRNA) transfection is a developing field that has applications in research and gene therapy. Potentially, mRNA transfection can be mediated efficiently by cell-penetrating peptides (CPPs) as they may be modified to target specific tissues. However, whilst CPPs are well-documented to transfect oligonucleotides and plasmids, mRNA transfection by CPPs has barely been explored. Here we report that peptides, including a truncated form of protamine and the same peptide fused to the CPP Xentry (Xentry-protamine; XP), can transfect mRNAs encoding reporter genes into human cells. Further, this transfection is enhanced by the anti-malarial chloroquine (CQ) and the toll-like receptor antagonist E6446 (6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole), with E6446 being >5-fold more potent than CQ at enhancing this transfection. Finally, E6446 facilitated the transfection by XP of mRNA encoding the cystic fibrosis transmembrane regulator, the protein mutated in cystic fibrosis. As such, these findings introduce E6446 as a novel transfection enhancer and may be of practical relevance to researchers seeking to improve the mRNA transfection efficiency of their preferred CPP.
Administration of a TLR9 Inhibitor Attenuates the Development and Progression of Heart Failure in Mice
JACC Basic Transl Sci 2019 May 22;4(3):348-363.PMID:31312759DOI:10.1016/j.jacbts.2019.01.002.
Mitochondrial deoxyribonucleic acid, containing the unmethylated cytidine-phosphate-guanosine motif, stimulates Toll-like receptor 9 to induce inflammation and heart failure. A small chemical, E6446 [(6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole)], is a specific Toll-like receptor 9 inhibitor in cardiomyocytes. In this study, we showed that E6446 exerts beneficial effects for the prevention and treatment of pressure overload-induced heart failure in mice. When administered before the operation and chronically thereafter, E6446 prevented the development of left ventricular dilatation as well as cardiac dysfunction, fibrosis, and inflammation. Furthermore, when administered after the manifestation of cardiac dysfunction, E6446 slowed progression of cardiac remodeling. Thus, the inhibitor may be a novel therapeutic agent for treating patients with heart failure.
Chronic Inhibition of Toll-Like Receptor 9 Ameliorates Pulmonary Hypertension in Rats
J Am Heart Assoc 2021 Apr 6;10(7):e019247.PMID:33787285DOI:10.1161/JAHA.120.019247.
Background Recent accumulating evidence suggests that toll-like receptor 9 (TLR9) is involved in the pathogenesis of cardiovascular diseases. However, its role in pulmonary hypertension remains uncertain. We hypothesized that TLR9 is involved in the development of pulmonary hypertension. Methods and Results A rat model of monocrotaline-induced pulmonary hypertension was used to investigate the effects of TLR9 on hemodynamic parameters, vascular remodeling, and survival. Monocrotaline-exposed rats significantly showed increases in plasma levels of mitochondrial DNA markers, which are recognized by TLR9, TLR9 activation in the lung, and interleukin-6 mRNA level in the lung on day 14 after monocrotaline injection. Meanwhile, monocrotaline-exposed rats showed elevated right ventricular systolic pressure, total pulmonary vascular resistance index and vascular remodeling, together with macrophage accumulation on day 21. In the preventive protocol, administration (days -3 to 21 after monocrotaline injection) of selective (E6446) or nonselective TLR9 inhibitor (chloroquine) significantly ameliorated the elevations of right ventricular systolic pressure and total pulmonary vascular resistance index as well as vascular remodeling and macrophage accumulation on day 21. These inhibitors also significantly reduced NF-κB activation and interleukin-6 mRNA levels to a similar extent. In the short-term reversal protocol, E646 treatment (days 14-17 after monocrotaline injection) almost normalized NF-κB activation and interleukin-6 mRNA level, and reduced macrophage accumulation. In the prolonged reversal protocol, E6446 treatment (days 14-24 after monocrotaline injection) reversed total pulmonary vascular resistance index and vascular remodeling, and improved survival in monocrotaline-exposed rats. Conclusions TLR9 is involved in the development of pulmonary hypertension concomitant via activation of the NF-κB‒IL-6 pathway. Inhibition of TLR9 may be a novel therapeutic strategy for pulmonary hypertension.
Inhibition of TLR9-NF-κB-mediated sterile inflammation improves pressure overload-induced right ventricular dysfunction in rats
Cardiovasc Res 2019 Mar 1;115(3):658-668.PMID:30239623DOI:10.1093/cvr/cvy209.
Aims: Recent accumulating evidence suggests that sterile inflammation plays a crucial role in the progression of various cardiovascular diseases. However, its contribution to right ventricular (RV) dysfunction remains unknown. The aim of this study was to elucidate whether toll-like receptor 9 (TLR9)-NF-κB-mediated sterile inflammation plays a critical role in the pathogenesis of RV dysfunction. Methods and results: We performed main pulmonary artery banding (PAB) in rats to induce RV pressure overload and dysfunction. On Day 14 after PAB, the pressure overload impaired RV function as indicated by increased RV end-diastolic pressure concomitant with macrophage infiltration and fibrosis, as well as maximal activation of NF-κB and TLR9. Short-term administration (days 14-16 after PAB) of a specific TLR9 inhibitor, E6446, or an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) significantly attenuated NF-κB activation. Furthermore, long-term administration of E6446 (treatment: days 14-28) or PDTC (prevention: days -1 to 28; treatment: days 14 to 28) improved RV dysfunction associated with mitigated macrophage infiltration and fibrosis in right ventricle and decreased serum brain natriuretic peptide levels. Conclusion: Inhibition of TLR9-NF-κB pathway-mediated sterile inflammation improved PAB-induced RV dysfunction in rats. This pathway plays a major role in the progression of pressure overload-induced RV dysfunction and is potentially a novel therapeutic target for the disorder.
Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria
Proc Natl Acad Sci U S A 2011 Mar 1;108(9):3689-94.PMID:21303985DOI:10.1073/pnas.1015406108.
Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.