Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKβ and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice.[1]
SR12343 displayed a similar profile to the 8K-NBD peptide, showing significant inhibition on COX-2, IL-6, and iNOS expression at a much lower concentration (50 μM) compared to NBD peptide (400 μM). [1]
Raw 264.7 cells were pretreated with indicated drugs for 30 min and then stimulated with 1 μg/ml of LPS for 2 h. Cells were then harvested for RNA extraction and qRT-PCR analysis. Drug concentrations used are as follows: IKKi VII (2 μM), 8K-NBD peptide (200 μM), SR12343 (50 μM), SR12460 (50 μM), SR12454 (50 μM), and SR11481 (50 μM). [1]

Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.[1]

NBD mimetics target the NEMO/IKKß interaction to inhibit NF-κB signaling
To determine whether the novel NBD mimetics target the NEMO-IKKβ interaction in vivo, co-immunoprecipitations were performed using extracts from Raw 264.7 macrophages . All 4 of the mimetics reduce the IKKβ-NEMO interaction as well as or better than the NBD peptide, with SR12343 being the most effective. SR12343 also reduced the association between NEMO and IKKβ in Raw 264.7 cells in a dose-dependent manner . No interaction between NEMO and TNF receptor-associated factor 2 (TRAF2) or IκBα was observed under these conditions. Previous studies suggested that the NBD peptide also inhibits the interaction of NEMO with IKKα . However, SR12343 had only a marginal effect on NEMO/IKKα binding only at the highest dose, suggesting that these inhibitors affect the NEMO/IKKα interaction with a much lower efficiency . To demonstrate that SR12343—which was the most effective mimetic in disrupting the NEMO/IKKβ interaction in vivo—affects the NEMO/IKKβ interaction directly, in vitro glutathione S-transferase (GST) pull-down assays were performed using recombinant GST-NEMO and FLAG-IKKβ. As shown in Fig 5C, SR12343 was able to disrupt the interaction between GST-NEMO and FLAG-IKKβ even at a dose of 12.5 μM. To demonstrate that the reduction of NF-κB-mediated transcription upon stimulation of TNF-α and LPS is not due to off-target effects, the activation of NF-κB signaling and mitogen-activated protein kinase (MAPK) pathways was examined by western blot analysis. The levels of phosphorylated IKK complex, IκBα, and p65 in response to TNF-α and LPS were all reduced by SR12343 . Consistently, the degradation of IκBα was partially reduced . However, there were no changes in the levels of phospho-c-Jun N-terminal kinase (p-JNK), p-p38MAPK, total JNK, and p38MAPK by treatment with SR12343 (150 μM) in response to TNF-α or LPS stimulation. These results suggest that the observed inhibitory effects of SR12343 are mediated directly through IKK/NF-κB but not due to off-target effects such as through the JNK or p38MAPK pathways. SR12343 treatment also had no effect on the activation of noncanonical NF-κB pathway by anti-lymphotoxin β receptor (LTβR) as shown by unaltered processing from p100 to p52 .[1]

Novel NBD mimetics suppress LPS-induced acute pulmonary inflammation in vivo
To determine the stability of the NBD mimetics in vivo, their levels were measured in the plasma of mice 2 h post i.p. injection with 10 mg/kg of each compound. The concentration of SR12460 was very high in plasma (>6.5 μg/mL; 20 μM), while levels in brain, muscle, spleen, and liver were lower. SR12343 and SR12454 had lower plasma concentrations, with SR12343 showing a higher level in liver and SR12454 showing higher concentrations in muscle and spleen. The level of SR11481 was undetectable in plasma and tissues . Because SR12454 and SR12460 share similar chemical structures and SR11481 demonstrates poor in vivo exposure, SR12460 and SR12343 were selected for further in vivo analysis. However, it is important to note that SR12343 was not as orally active as SR123460 .[1]
To determine their NF-κB inhibitory effects in vivo, SR12343 and SR12460 were tested in an acute model of LPS-induced systemic endotoxemia. C57BL/6J mice were pretreated with vehicle control, 8K-NBD peptide, or NBD mimetics at 10 mg/kg for 30 min, followed by LPS induction at 10 mg/kg . Lung and liver were harvested 2–4 h post treatment for qRT-PCR analysis of NF-κB target genes. SR12343 was able to significantly inhibit NF-κB transcriptional activity in lung, demonstrated by the inhibition of iNOS, IκBα, COX-2, and IL-6, while expression of TNF-α was unchanged . The NF-κB inhibitory effects of the compounds were less effective in liver compared to lung, demonstrated by significant inhibition of only COX-2 expression. Despite lower plasma and tissue concentrations of SR12343 compared to SR12460, its inhibition of NF-κB/IKK in liver and lung were greater than that of SR12460 . Similarly, acute treatment with SR12343 also affected the LPS-induced expression of NF-κB target genes at protein level. The extent of phosphorylation of IκBα and the levels of COX-2 were reduced in lung and liver tissues treated with SR12343 . Serum levels of IL-6 detected by ELISA increased significantly following LPS induction and were reduced by acute treatment with SR12343. These results are consistent with our RT-PCR analysis shown in Fig 6C. However, no significant differences in lung histopathology were observed. Finally, there was a slight reduction in the number of white blood cells and neutrophils in the SR12343-treated group. Taken together, the results demonstrate that SR12343 and SR12460 are effective at attenuating LPS-induced acute lung inflammation by suppressing NF-κB target gene expression.[1]
Novel NBD mimetics alleviate necrosis and muscle degeneration in mdx mice Because SR12343 and SR12460 both reduced LPS-induced NF-κB activation in vivo, they were further tested in mdx mice, a mouse model of DMD in which NF-κB is chronically activated. Mdx mice develop normally at birth and undergo a massive myonecrosis starting at 3 wk. Treatment of mdx mice with IKK/NF-κB inhibitors effectively reduces inflammation, blocks necrosis, and increases muscle regeneration. Initially, the effect of acute treatment of SR12343 on NF-κB DNA binding activity in tibialis anterior (TA) muscle in 9-wk-old mdx mice was examined by EMSA. Treatment with a single injection of 30 mg/kg of SR12343 resulted in a reduction in NF-κB DNA binding 2 h post injection . Subsequently, Mdx mice were chronically treated with vehicle, SR12343 (30 mg/kg), SR12460 (30 mg/kg), or 8K-NBD (10 mg/kg) starting from day 21, 3 times/wk for 4 wk, similar to the dosing regimen used with the NBD peptide. No significant weight loss was observed in chronically treated mdx mice . In addition, there was no increase in levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), or alkaline phosphatase (ALP) in chronically treated mdx mice, suggesting that treatment with SR12343 and SR12460 had no overt liver toxicity.[1]

[1]. Development of novel NEMO-binding domain mimetics for inhibiting IKK/NF-κB activation. PLoS Biol. 2018 Jun 11;16(6):e2004663.

[2]. New Trends in Aging Drug Discovery. Biomedicines. 2022 Aug 18;10(8):2006.

C15H15BRCLN3O

368.656101465225

367.008

2055101-86-3

124117414

Off-white to light yellow solid powder

3.8

2

3

6

21

332

0

PEOFCAIUELALCR-UHFFFAOYSA-N

InChI=1S/C15H15BrClN3O/c16-12-3-1-2-11(8-12)6-7-18-15(21)10-20-14-5-4-13(17)9-19-14/h1-5,8-9H,6-7,10H2,(H,18,21)(H,19,20)

N-(3-Bromophenethyl)-2-((5-chloropyridin-2-yl)amino)acetamide

SR 12343SR-12343 SR12343

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.)