TLR7/9

TLR7 and TLR9 can be effectively inhibited by the oral drug E6446. E6446 substantially less efficiently suppresses the stimulation of HEK:TLR4 cells by LPS endotoxin or the stimulation of HEK:TLR7 cells by R848. However, it effectively inhibits the DNA stimulation of HEK:TLR9 cells with an IC50 value of 10 nM. E6446 is not effective when IL-6 is produced by the TLR3 ligand polyinosine-cytosine, but it is when IL-6 is induced by CpG2216. E6446's capacity to inhibit TLR7 is ligand-dependent. While IL-6 produced by the short molecule imidazoquinoline ligand R-848 is rather poorly inhibited by E6446, it is potently inhibited by RNA. In vitro, E6446 suppresses TLR9-DNA interaction with an IC50 between 1 and 10 µM [1]. TLR9 activation by CpG ODN 2006 is specifically inhibited by E6446 (0.01-0.03 μM), while TLR7/8 activation by 2-8 μM imidazoquinoline compound R848 is blocked. In HEK-TLR9 cells stimulated with oligo 2006 and human PBMC stimulated with oligo 2216, respectively, E6446 lowers TLR4 activation by 50% at 30 μM and demonstrates an IC50 of 0.01 μM and 0.23 μM [2].

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Target Inhibition of Nucleic Acid-Sensing TLRs by E6446. [2]
To test the effect of E6446 on TLR activation, we stimulated HEK293 cells stably transfected with TLR4/MD2, TLR7, or TLR9 and the ELAM-1–luciferase reporter gene under NF-κB promoter with each of the corresponding ligands (LPS, R848, or CpG ODN 2006, respectively) in the presence of increasing concentrations of E6446. Measurement of ELAM-1–luciferase activity showed that E6446 specifically inhibited TLR9 activation with CpG ODN 2006, in the range of 0.01–0.03 μM. A 100-fold higher concentration (2–8 μM) of E6446 was required to inhibit TLR7/8 activated by the imidazoquinoline compound R848. Incubation of cells with even higher concentrations (30 μM) of E6446 was required to reduce 50% of TLR4 activation (Fig. 2A). Treatment of human peripheral blood mononuclear cells (PBMCs) with E6446 also diminished IL-6 production in response to the TLR9 (CpG ODN 2402) and TLR8 [single-stranded RNA (ssRNA)] agonists, in the range of 0.05 and 0.5, respectively. Approximately 5 μM was required to inhibit 50% of the response to R848, which also activates mouse TLR7 and human TLR8 (Fig. 2A, Lower). Chloroquine, a widely used antimalarial drug, has immunomodulatory properties, specially by interfering with endosomal TLR function, and has been used in the treatment of autoimmune disorders. We thus compared the effect of E6446 with that of hydroxychloroquine. When tested in HEK-TLR9 cells stimulated with oligo 2006, E6446 was eightfold more potent than hydroxychloroquine (mean IC50 over several experiments of 0.01 vs. 0.08 μM for hydroxychloroquine). Similarly, in human PBMCs stimulated with oligo 2216, the IC50 for E6446 is 0.23 μM, whereas for hydroxychloroquine, it is 1.2 μM.

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E6446 inhibits adipogenic differentiation and hepatic lipogenesis [3]
To evaluate the pharmacological effects of the top 5 candidate compounds, two classic cell models (adipogenic differentiation and hepatic steatosis models), which model the most critical processes of fatty liver formation, were selected in this study (Fig. 2A-B). As shown in Fig. 2C-E, all five candidate compounds significantly inhibited TG accumulation, decreased the number of lipid droplets and significantly downregulated the adipocyte differentiation marker genes Pparg, Fasn, Fabp4 and C/ebpα in OP cells. In addition, in the hepatic steatosis model, only E6446, not the four candidate compounds, suppressed the accumulation of TG content, the formation of lipid droplets and the expression of C/ebpα, Fasn, and Fabp4 (Fig. 2F-H). In addition, none of the compounds affected the SCD2 expression, and E6446 had the strongest inhibitory effect on SCD1 activity (Figs. S1A-C; S12A-B). These results indicated that E6446 was the most potent inhibitor of adipogenic differentiation and hepatic lipogenesis among the selective compounds.

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E6446 targets SCD1 to inhibit adipogenic differentiation and hepatic lipogenesis [3]
Previous studies have found that E6446 can inhibit TLR7 and TLR9 signaling. However, TLR7 and TLR9 are primarily expressed in the brain and lymphoid tissue, and are barely expressed in the liver and adipose tissue. Our results are consistent with these findings, TLR7 and TLR9 was not detected in OP9 and AML12 cells (Fig. S2). To investigate whether E6446 targets SCD1 during adipogenic differentiation and hepatic lipogenesis, SCD1 was over-expressed and knockdown in OP9 and AML12 cells. The efficiency of transfection was shown in Fig. S3. The inhibitory effects of E6446 on adipogenic differentiation, including SCD1 protein expression, TG content and adipocyte differentiation marker gene expression were reversed when SCD1 was overexpressed in OP9 cells (Fig. 3A-D). In addition, E6446 also promoted the expression of UCP1 in OP9 cells (Fig. S4). Similarly, compared with the E6446 treatment group, SCD1 overexpression combined with E6446 treatment significantly increased SCD1 protein expression, lipid droplets, TG content and lipogenesis gene expression in AML12 cells (Fig. 3E-H). Meanwhile, knocking down SCD1 alone significantly inhibited both adipogenic differentiation and hepatic lipogenesis, and E6446 treatment did not enhance the phenotypes caused by SCD1 silencing alone (Fig. S5). Collectively, these results indicate that E6446 targets SCD1 to inhibit adipogenic differentiation and hepatic lipogenesis.

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E6446 is more potent than A939572 in preventing adipogenic differentiation and hepatic lipogenesis [3]
Numerous studies have shown that A939572 is a potent SCD1 inhibitor, and many preclinical studies have used A939572 as a control (Stearoyl-CoA desaturase 1 is a novel molecular therapeutic target for clear cell renal cell carcinoma. Monounsaturated fatty acids generated via stearoyl CoA desaturase-1 are endogenous inhibitors of fatty acid amide hydrolase. Loss of stearoyl-CoA desaturase activity leads to free cholesterol synthesis through increased Xbp-1 splicing). The efficacy and safety of E6446 versus A939572 in the treatment of NAFLD is unclear. To address this question, we tried to reveal the advantages and disadvantages between them through a few simple classical experiments. LDH and CCK-8 assays indicated that E6446 significantly inhibited free fatty acid-induced lipotoxicity in hepatocytes while A939572 did not (Fig. 4A-D). In addition, E6446 and A939572 exhibited excellent SCD1 potency with IC50 values in the micromole range (E6446 IC50 = 0.98 μM; A939572 IC50 = 2.8 μM). Next, we investigated whether E6446 was more effective than A939572 in adipogenic differentiation and hepatic lipogenesis. As shown in Fig. 4E-G, we found that E6446 treatment was significantly better at decreasing the expression of the adipogenic differentiation marker genes Pparg, Fasn, Fabp4 and C/ebpα, and lipid droplets than A939572 treatment. Moreover, compared to A939572 treatment, E6446 intervention significantly inhibited the accumulation of TG content, the expression of Fasn, Fabp4 and C/ebpα, and the formation of lipid droplets (Fig. 4H-J). Using SPR assay, the kinetics/affinity of E6446 and A939572 with SCD1 were determined. The results showed that the KD value of E6446 was 4.61 μM, while the KD value of A939572 was 11.65 μM, indicating a strong interaction ability between E6446 and SCD1 (Fig. 4K). These results suggested that E6446 is safer and more effective than A939572 in NAFLD treatment.

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Transcriptomics analysis implicates ATF3 as a potential downstream target [3]
To identify candidate molecules that might participate in E6446 mediated inhibition of adipogenic differentiation and lipogenesis, we performed RNA-seq in adipogenic differentiation and hepatic steatosis models after E6446 treatment. The analysis identified 506 upregulated and 167 downregulated genes in OP9 cells, and 238 upregulated and 167 downregulated genes in AML12 cells (Fig. 5A-B). Among the two upregulated gene sets, 31 genes overlapped (Fig. 5C). Subsequently, the co-upregulated genes were subjected to GO analysis, and the results indicated that the gene set was significantly enriched in adipogenesis genes (Fig. 5D). The top 3 genes among adipogenesis-related genes were Id3, Ccn2 and ATF3. To verify the RNA-seq results, we performed RT–qPCR and found that Id3, Ccn2 and ATF3 were markedly increased during adipogenic differentiation and hepatic steatosis (Fig. 5E-F). However, when SCD1 was overexpressed, the ATF3 mRNA level was significantly decreased in AML12 cells, while the Id3 and Ccn2 levels were not affected (Fig. 5G). Additionally, among the three genes, the ATF3 mRNA level decreased most obviously in OP9 cells after SCD1 transfection (Fig. 5H). Therefore, these results indicated that ATF3 may be the potential downstream target of E6446-mediated SCD1 inhibition in adipogenic differentiation and hepatic lipogenesis.

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E6446 inhibited adipogenic differentiation and hepatic lipogenesis through SCD1-ATF3 signaling [3]
The above results demonstrated that SCD1-ATF3 signaling might represent a key downstream signaling pathway for E6446 treatment. Thus, we investigated whether ATF3 silencing blocked the inhibitory effects of E6446 on adipogenic differentiation and hepatic lipogenesis. Our results revealed that ATF3 knockdown significantly prevented the E6446-induced inhibitory effects on adipogenic differentiation, including TG accumulation, adipocyte differentiation marker gene expression and lipid droplets formation (Fig. 6A-C). Similarly, ATF3 silencing also blocked the E6446 treatment-induced the inhibition of hepatic lipogenesis (Fig. 6D-F). These observations suggested that E6446 inhibited adipogenic differentiation and hepatic lipogenesis through SCD1-ATF3 signaling.

E6446 (20 mg/kg, oral) reduces the generation of IL-6 caused by CpG1668 nearly entirely and inhibits the creation of ANA (antinuclear antibodies) in mice at dosages of 20 and 60 mg/kg in a dose-dependent manner [1]. In mice, TLR9 signaling is dose-dependently inhibited by E6446 (20, 60 mg/kg, oral). E6446 (60, 120 mg/kg, oral) inhibits the activation of TLR7 and TLR9, decreases TLR reactivity during acute malaria, and protects LPS-induced septic shock and TLR hyperresponsiveness in rodent malaria [2].

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 E6446 Prevents Hyperresponsiveness of TLRs and LPS-Induced Septic Shock in Rodent Malaria. [2]
Because of its ability to block TLR activation by nucleic acids, we decided to evaluate the effect of E6446 on malaria pathogenesis. We first evaluated the efficacy of E6446 treatment on Plasmodium chabaudi infection. Although not usually lethal, we have previously shown that this murine model causes a proinflammatory priming of TLR responses rendering mice extremely susceptible to low doses of LPS (19). Treatment with 60 mg·kg−1·d−1 of E6446 diminished TLR responsiveness during acute malaria (Fig. 3A). Furthermore, when given at a higher concentration (120 mg·kg−1·d−1), which was not toxic, E6446 protected infected mice from LPS-induced shock (Fig. 3B). Of note, mice treated with 120 mg·kg−1·d−1 of E6446 presented even higher resistance (86% survival) to LPS-induced shock than TLR9−/− mice (38% survival) (19) compared with vehicle-treated mice (<10% survival). Based on our in vitro experiments (Fig. 2), it is conceivable that, in vivo, 120 mg·kg−1·d−1 of E6446 was able to inhibit activation of both TLR7 and TLR9. If this is the case, these results suggest that along with TLR9, TLR7 and TLR8 (in humans) may be involved in priming and enhancing the responsiveness of TLRs, systemic inflammatory reaction, and pathogenesis of malaria. Importantly, the highest serum drug concentration reached in vivo at a dose of 120 mg/kg is around 1 μM, which is 30- to 50-fold less than the E6446 concentration required to block in vitro activation of TLR4 by LPS. Furthermore, treatment with E6446 (120 mg·kg−1·d−1) did not prevent the death of noninfected mice after challenge with a lethal dose of LPS (500 μg) reinforcing the specific effect of E6446 for nucleic acid-sensing TLRs but not for TLR4, which is activated by LPS (Fig. 2A).

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 E6446 Prevents ECM in Mice. [2]
We next tested if blockade of cell signaling through nucleic acid-sensing TLRs has any impact on PbA-induced ECM. C57BL/6 mice were orally treated with 120 mg·kg−1·d−1 of E6446 or vehicle from day −1 to day 12 postinfection with 1 × 105 PbA-infected red blood cells (iRBCs). As expected for this malaria model (29), most mice treated with vehicle succumbed to PbA-mediated ECM by day 6–12 postinfection (Fig. 4A, Upper). Although no change in parasitemia was observed (Fig. 4A, Lower), treatment with E6446 had a pronounced effect on enhancing survival after infection with PbA.
To address the compelling efficacy of E6446 on an already established disease, we tested whether treatment with this compound could prevent the development of PbA ECM before the onset of symptoms. Mice were infected with PbA and treated with 120 mg/kg of E6446 from day 3, 5, or 7 up to day 12 postinfection. Although the efficacy of E6446 in preventing ECM decreased, mice treated with this compound had a better survival rate than vehicle-treated mice (Fig. 4B). E6446 failed to protect mice when administered after 6 d postinfection at the onset of the cerebral syndrome.
We next evaluated the effect of E6446 treatment on the cytokine profile during PbA infection. Mice treated with E6446 produced significantly lower levels of proinflammatory cytokines (Fig. 4C, Upper). Furthermore, PbA-mediated priming of TLR responses was also diminished by E6446 therapy (Fig. 4C, Lower). Nucleic acid-sensing TLRs (e.g., TLR9, TLR7) are highly expressed on dendritic cells (DCs) and were shown to be activated by Plasmodium products that play a relevant role in malaria pathogenesis (18, 30, 31). We thus sought to identify the effect of E6446 on cytokines produced by myeloid CD11c+ MHCII+ DCs using a flow cytometry (FACS) assay for intracellular cytokine staining. We found that in mice infected with PbA, the production of TNF-α was significantly increased at day 6 postinfection compared with noninfected mice. TNF-α production was abolished in cells from PbA-infected mice treated with E6446 (Fig. 4D).
To confirm the involvement of endosomal TLRs in ECM pathogenesis, we infected “three-deficient” (3d) mice with PbA. The 3d mice have a single mutation in the UNC93B1 gene and are not responsive to TLR3, TLR7, or TLR9 agonists (32). Importantly, our results in 3d mice resemble the results obtained in mice treated with E6446. These mice showed impaired production of proinflammatory cytokines and were more resistant to PbA-induced ECM, although presenting with similar parasitemia (Fig. S1). Hence, these results further support the hypothesis that E6446 therapy protects against PbA-induced ECM by blocking activation of nucleic acid-sensing TLRs.

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 Symptoms of ECM Are Attenuated by E6446 Therapy. [2]
Brain seizures with severe debilitation are strong indicators of PbA ECM. We therefore performed neurological examinations in each mouse during acute PbA infection. We show that mice treated with E6446 attained a better clinical outcome; symptoms were attenuated compared with vehicle-treated mice (Fig. 5A). Proinflammatory cytokines, such as TNF-α and IL-1β, have been shown to affect blood–brain barrier (BBB) permeability, leading to leak and infiltration of mononuclear cells contributing to ECM (33). Because of its inhibitory effect on cytokine production in mouse cells (Fig. 2B) and in malaria infection (Figs. 3B and 4C), we next tested the effect of E6446 on the integrity of the BBB during PbA infection. We assessed cerebral vascular leakage after i.v. injection of 1% Evans blue in mice with end-stage ECM. Mice treated with vehicle displayed a distinct vascular leakage, shown by a blue staining of the brain, that was absent in E6446-treated mice (Fig. 5B). The morphological correlate of the vascular leak was investigated in brain tissue sections. Consistent with the macroscopic observation, brain vessels of infected mice treated with vehicle displayed typical microvascular damage with sequestration of iRBCs, including the presence of hemozoin, mononuclear cell adhesion on the endothelium, and hemorrhage into the parenchyma, which was much less frequent in E6446-treated mice. In general, PbA-infected mice treated with E6446 presented significantly lower numbers of brain intravascular inflammatory foci (Fig. 5C).

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 E6446 improves HFD-induced NAFLD [3]
We next investigated the effect of E6446 on HFD-induced NAFLD in vivo. C57BL/6J mice were fed a HFD for 12 weeks and gavaged with E6446 or vehicle three times per week. HFD feeding for 18 weeks resulted in higher body weight, liver weight, and WAT weight than the normal control diet (NCD) group, while E6446 significantly reduced liver weight, WAT weight, and fat/body ratio (Fig. 7A-D). HFD-mice also showed more severe insulin resistance, as indicated by glucose tolerance tests (GTTs). E6446 significantly improved glucose tolerance (Fig. 7E). HFD-fed mice showed a significant increase in hepatic and serum TC, TG, LDL, ALT and AST compared the NCD group, while E6446 treatment significantly reduced these biochemical indices (Fig. 7F-G). H&E and Oil Red O staining revealed more lipid droplet in HFD-fed mice than in NCD mice, while E6446 treatment significantly decreased lipid droplets accumulation (Fig. 7H). These findings revealed that E6446 exhibited robust efficacy against HFD-induced hepatic steatosis.

Surface plasmon resonance (SPR) analysis [3]
SPR experiments were performed using Biacore X100. HBS-EP buffer as used as the working buffer, and the SCD1 recombinant protein was diluted to a final concentration of 20 μg/mL. A mixture of NHS and EDC (1:1, v/v) was injected into the instrument to activate the CM5 sensor. Then, the 20 μg/mL SCD1 recombinant protein was injected to immobilize it onto the CM5 chip through amino coupling. Subsequently, a 1 M ethanolamine hydrochloride (pH 8.5) was injected for 7 min to block and activate the chip surface. E6446 (100, 50, 25, 12.5, 6.25, 3.125 μmol/L) and A939572 (100, 50, 25, 12.5, 6.25, 3.125 μmol/L) were then injected using HBS-EP buffer and passed over the immobilized SCD1 chip sensor surface. The binding kinetics of E6446 and SCD1 were calculated based on the fitted data from the analysis software, with time as the abscissa and response values as the ordinate.

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 Inhibition of TLR9 and TLR7 Signaling by Small Molecule Ligands [1]
HEK293 cells expressing cloned human TLR9 and an NF-κB:luciferase reporter (HEK:TLR9 cells) were used to screen a compound library for small molecules that could suppress induction of NF-κB by stimulatory DNA (CpG2006). AT791 and E6446 (Fig. 1A) potently suppressed DNA stimulation of HEK:TLR9 cells, with IC50 values of 40 and 10 nM, respectively, but were significantly less effective at suppressing LPS endotoxin stimulation of HEK:TLR4 cells (Table 1) or R848 stimulation of HEK:TLR7 cells.

In Vitro Assays. [2]
E6446 was assayed for suppression of freshly isolated human PBMCs or BALB/c mouse spleen IL-6 production in response to stimulation by oligonucleotide 1668, R848, or ssRNA. The compound was added to dissociated cells [5 × 105 per well in complete RPMI/10% (vol/vol)] before addition of stimulants. Cells were stimulated for 72 h, and supernatant was removed for ELISA analysis of IL-6.

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Cytokine Analysis. [2]
Cytokines were assessed in the sera or in supernatants of cultured splenocytes from C57BL/6 mice infected i.p. with 105 P. chabaudi or P. berghei iRBCs treated or not treated with E6446. Each experiment was repeated two to three times. Each animal was analyzed individually. Spleens were aseptically removed and macerated through a nylon mesh over the course of the experimental infection. After RBC lysis, splenocytes were resuspended in RPMI, 10% (vol/vol) FCS, and 1% gentamicin at a density of 2.5 × 106 cells per milliliter. Splenocytes were subsequently cultured in 48-well plates for 48 h in a final volume of 1 mL in the presence of Con A, higly purified LPS, or CpG ODN. Measurements of cytokines were performed using commercially available ELISA kits. Serum cytokines from P. chabaudi-infected mice were quantified with the CBA inflammation kit.

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Intracellular Staining of Cytokines. [2]
At day 6 postinfection with 105 PbA iRBCs, spleens from mice treated or not treated with E6446 were harvested and CD11c+ splenic DCs were isolated using CD11c microbeads according to the manufacturer's instructions. Cells (5 × 105) were Fc-blocked with 2.4G2 mAb and labeled with efluor-conjugated anti-CD11c, Cy5-conjugated anti-mouse MHCII, and phycoerythrin (PE)-conjugated anti-mouse CD40, CD86, and CD80 mAbs. A nonrelated IgG mAb was used as a control for staining specificity. For intracellular analysis of cytokines, cells were fixed and permeabilized with BD Cytofix/Cytoperm and stained for intracellular TNF-α, IL-12 p40/p70 levels using PE-conjugated anti-mouse mAbs. Data were analyzed using Flowjo software.

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Cell culture [3]
Mouse OP9 cells were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2. Mouse AML12 cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/nutrient mixture F-12 supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2. The medium was replaced every 3 days if not otherwise stated. To induce adipogenic differentiation, 100% confluent OP9 preadipocytes were stimulated with 1 μM rosiglitazone in DMEM containing 5% FBS for 15 days. Adipogenic differentiation was induced by 1 μM rosiglitazone in DMEM containing 5% FBS with or without 10 μM E6446 for 15 days when OP9 cells grown to 90% confluence. To establish hepatic steatosis model, mouse AML12 cells were stimulated with palmitic acid (PA; 0.125 mM) and oleic acid (OA; 0.25 mM) with or without 10 μM E6446 at the indicated concentrations for 48 h.

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Lactate dehydrogenase (LDH) assay [3]
AML12 or OP9 cells were seeded into 96-well plates and treated with 0, 10, 2, 0.4, 0.08 μM E6446 or A939572 with palmitic acid (PA; 0.125 mM) and oleic acid (OA; 0.25 mM) for 48 h. LDH levels in AML12 cells were measured using commercial kits.

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CCK8 assay [3]
AML12 or OP9 cells were seeded into 96-well plates and treated with 0, 10, 2, 0.4, 0.08 μM E6446 or A939572 with palmitic acid (PA; 0.125 mM) and oleic acid (OA; 0.25 mM) for 48 h. Next, 10 μL of CCK8 solution was added and incubated at 37 °C with 5% CO2 for 2 h. The absorbance at 450 nm was measured using a microplate reader.

Drug and Treatment. [2]
The compound E6446-02 was dissolved in water, and its concentrations were adjusted so that the final dose in body weight (mg/kg) was given in 0.1 mL. Mice treated with vehicle (water) were used as a control group. The animals were treated through oral gavage once daily at the dose stated for several days postinfection. The drug was administered 24 h before infection.

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Experimental animals [3]
Male C57BL/6J mice (8 weeks old, weighing 21 ± 2 g) were housed at room temperature, 12-h light–dark cycle, with free access to food and water. The mice were divided into 3 experimental groups: normal control diet group (NCD; chow diet, 10% of calories derived from fat), high-fat diet (HFD, 60% of calories derived from fat) + Vehicle group, and HFD + E6446 group. After 12 weeks, mice were three times in a week oral gavage with Vehicle or 20 mg/kg E6446 for 6 weeks. The body weight was measured weekly during oral gavage experimental phases. Mice were sacrificed through isoflurane inhalation, and then blood, adipose, and liver samples were collected for further analysis.

[1]. Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo. Mol Pharmacol. 2014 Mar;85(3):429-40.

[2]. 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.

[3]. Identification of novel SCD1 inhibitor alleviates nonalcoholic fatty liver disease: critical role of liver-adipose axis. Cell Commun Signal. 2023 Sep 30;21(1):268.

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)-containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti-double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs. [1]

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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. [2]

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Due to the complexity and incomplete understanding of the crosstalk between liver and adipose tissue, especially the processes of hepatic lipogenesis and adipogenic differentiation, there are currently no effective drugs for the treatment of nonalcoholic fatty liver disease (NAFLD). Stearoyl-coenzyme A desaturase 1 (SCD1), which is abundantly expressed in liver and adipose tissue, may mediate the cross-talk between liver and adipose tissue. Thus, it is essential to develop specific SCD1 inhibitors that target the liver-adipose axis. Herein, we identified a novel SCD1 inhibitor, E6446, through a high-throughput virtual screen. E6646 significantly inhibited adipogenic differentiation and hepatic lipogenesis via SCD1-ATF3 signaling. The SPR results showed that E6446 had a strong interaction ability with SCD1 (KD:4.61 μM). Additionally, E6646 significantly decreased hepatic steatosis, hepatic lipid droplet accumulation and insulin resistance in high-fat diet (HFD)-fed mice. Taken together, our findings not only suggest that E6446 can serve as a new, safe and highly effective anti-NAFLD agent for future clinical use but also provide a molecular basis for the future development of SCD1 inhibitors that inhibit both adipogenic differentiation and hepatic lipogenesis. [3]

C27H35N3O3

449.595

449.268

C, 72.13; H, 7.85; N, 9.35; O, 10.68

1219925-73-1

E6446 dihydrochloride;1345675-25-3

45102599

Solid powder

5.1

0

6

11

33

559

0

O(C1=CC=C2C(=C1)OC(C1C=CC(=CC=1)OCCCN1CCCC1)=N2)CCCN1CCCC1

YMYJXFUPMPMETB-UHFFFAOYSA-N

InChI=1S/C27H35N3O3/c1-2-14-29(13-1)17-5-19-31-23-9-7-22(8-10-23)27-28-25-12-11-24(21-26(25)33-27)32-20-6-18-30-15-3-4-16-30/h7-12,21H,1-6,13-20H2

6-(3-pyrrolidin-1-ylpropoxy)-2-[4-(3-pyrrolidin-1-ylpropoxy)phenyl]-1,3-benzoxazole

E-6446; E 6446; 1219925-73-1; 6-(3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)benzo[d]oxazole; CHEMBL4065452; 6-(3-pyrrolidin-1-ylpropoxy)-2-[4-(3-pyrrolidin-1-ylpropoxy)phenyl]-1,3-benzoxazole; SCHEMBL12396763; E6446

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)