Activating Transcription Factor 6 (ATF6) [2]
AA147 primarily targets activating transcription factor 6 (ATF6) and also activates nuclear factor erythroid 2-related factor 2 (NRF2) . ATF6 pathway: AA147 undergoes metabolic activation to generate a reactive electrophile that selectively modifies ER proteins (including multiple protein disulfide isomerases), thereby activating ATF6 signaling . NRF2 pathway: In neuron-derived cells, AA147 promotes NRF2 activation by covalently modifying KEAP1, leading to upregulation of antioxidant response genes .

Reactive oxygen species (ROS)-related damage is lessened by AA147 (20-0.078 μM (halved); 6 or 16 hours) in HT22 cells, preventing oxidative toxicity caused by valley induction [1]. AA147 (10 μM; 16 h) in HT22 AA147 (10 μM; 16 h) covalently alters KEAP1 in HT22 cells to encourage NRF2 activation [1]. AA147 recognizes ATF6 activation and upregulates phosphorylated cofilin in BPAEC (5, 10, 15 μM; 4, 8, 16, 24, 48 hours)[2]. In BPAEC, AA147 (10 μM; 24 hours) lessens the breakdown of the endothelium barrier caused by LPS [2].

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AA147 selectively induces ATF6 activation independently of endoplasmic reticulum (ER) stress in Bovine Pulmonary Artery Endothelial Cells (BPAEC). [2]
- Treatment of BPAEC with AA147 (10 μM) for various time points (4, 8, 16, 24, and 48H) significantly increased the activation of ATF6 (cATF6). [2]
- AA147 treatment (10 μM) in BPAEC upregulated the phosphorylation of Cofilin (pCofilin). [2]
- Exposure of BPAEC to increasing concentrations of AA147 (5, 10, 15 μM) for 24H resulted in a dose-dependent increase in both ATF6 activation and Cofilin phosphorylation. [2]
- AA147 (10 μM) pre-treatment (24H) of BPAEC counteracted LPS (1 μg/ml, 1H)-induced suppression of ATF6 activation. [2]
- In LPS-treated BPAEC, AA147 (10 μM) inhibited the activation of Cofilin and Myosin Light Chain 2 (MLC2), as indicated by reduced phosphorylation levels. [2]
- AA147 (10 μM) reduced LPS-induced VE-Cadherin phosphorylation, a key event in endothelial barrier disruption. [2]
- In a FITC-Dextran paracellular permeability assay using BPAEC monolayers grown on trans-well inserts, treatment with AA147 (10 μM, 24H) significantly opposed the increase in permeability caused by LPS (10 μg/ml, 2H). [2]
- Electric cell-substrate impedance sensing (ECIS) measurements on confluent BPAEC monolayers treated with AA147 (5 μM, 10 μM) showed a gradual increase in transendothelial electrical resistance (TEER) values, indicating decreased permeability and enhanced barrier integrity. [2]

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AA147 exhibits protective effects against various cellular insults in vitro. HT22 cells (neuronal model): AA147 (0.078-20 μM, 6 or 16 hours pre-incubation) dose-dependently protects HT22 cells from glutamate-induced oxidative toxicity, reducing reactive oxygen species-associated damage . At 10 μM for 16 hours, AA147 covalently modifies KEAP1 to promote NRF2 activation, significantly upregulating antioxidant genes including prolactin and glutathione transferase . BPAEC cells (endothelial model): AA147 (5, 10, 15 μM; 4-48 hours) induces ATF6 activation and upregulates phosphorylated cofilin . At 10 μM for 24 hours, AA147 reduces LPS-induced endothelial barrier disruption . AA28 analog: Replacement of the AA147 amide linker with a carbamate linker yields AA28, which activates ATF6 with an EC₅₀ of 2.9 μM in HEK293T cells (AA147: 1.1 μM) and induces comparable expression of the ATF6 target gene BiP .

By activating ATF6, AA147 (intrathecal injection; single anesthetic for three days) can stimulate distal motor neuron expression, cut motor neuron neuroprotection, and rebalance XBP1 expression in severe SMA specimens [3].

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Daily intrathecal injection of AA147 from postnatal day 7 (P7) to P10 in severe SMA-like mice resulted in increased Xbp1u mRNA expression in the spinal cord, confirming ATF6 activation in vivo. [3]
- This treatment reactivated the IRE1α/XBP1 pathway in the spinal cord of severe SMA-like mice, as evidenced by increased mRNA levels of Xbp1s and its target Bip/Grp78. Importantly, Chop mRNA was not increased, suggesting that apoptosis was not induced. [3]
- In vivo ATF6 activation by AA147 led to a significant increase in exon-7 containing SMN transcripts (E7-E8) in the spinal cord, without increasing total SMN mRNA (E2a-E2b), confirming the promotion of SMN exon-7 inclusion. This was confirmed at the protein level, with increased SMN protein expression in the spinal cord of AA147-treated mice. [3]
- AA147 treatment exerted a neuroprotective effect on spinal motor neurons (MNs). While vehicle-treated severe SMA-like mice showed a 20% loss of ChAT-positive MNs in the ventral spinal cord compared to controls, the number of spinal MNs in AA147-treated SMA mice was no longer statistically different from healthy controls. [3]
- The neuroprotection provided by AA147 was associated with improved motor behavior. AA147-treated severe SMA-like mice showed a significantly increased grip time at P10 and a progressive increase in exploratory activity in an open field test from P8 to P11, compared to vehicle-treated SMA mice. [3]
- While vehicle-treated severe SMA-like mice exhibited a severe body weight reduction from P8 until death around P10, the AA147-treated severe SMA-like mice maintained their weight. Furthermore, 30% of AA147-treated mice survived longer than the 10-day mean lifespan of the vehicle-treated group. [3]

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AA147 demonstrates efficacy in various animal models. Spinal muscular atrophy model: AA147 (intrathecal injection; single dose for three days) stimulates motor neuron expression and provides neuroprotection in severe SMA models by activating ATF6 . Ischemic stroke model: Pharmacokinetic analysis shows AA147 can cross the blood-brain barrier. Post-stroke treatment with AA147 improves long-term functional recovery in both young and aged mice after permanent stroke . Multiple sclerosis model (EAE): AA147 ameliorates clinical symptoms of EAE, reduces ER stress, oligodendrocyte loss, and demyelination. It also suppresses CNS T cells without altering peripheral immune responses. In oligodendrocytes, AA147 promotes nuclear translocation of ATF6 but not NRF2; in microglia, it enhances both ATF6 and NRF2 target gene expression . Spinal cord injury model: In a mouse spinal cord injury model, AA147 promotes neuronal survival, alleviates neuronal apoptosis, enhances all three arms of the unfolded protein response, and reduces ROS accumulation in neurons by upregulating catalase expression, while also promoting motor function recovery . Retinal study: In mouse retina, AA147 induces ATF6-regulated gene expression without causing retinal cell death, compromising vision, or triggering ER stress . Myocardial ischemia/reperfusion injury model: AA147 protects the heart against ischemia/reperfusion injury in an ATF6-dependent manner .

AA147 acts through metabolic activation to generate reactive electrophiles that covalently modify ER proteins such as KEAP1 and protein disulfide isomerases .

cell viability assay [1]
Cell Types: HT22 cell
Tested Concentrations: 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 20 μM
Incubation Duration: 6 or 16 h (pre-incubation)
Experimental Results: demonstrated dose-dependent increases in the viability of glutamate-treated HT22 cells when pretreated with AA147 for 6 or 16 h prior to the glutamate challenge (addition concurrently with the glutamate challenge did not improve the viability of glutamate-treated cells). diminished ROS accumulation in cells when pre-incubation of 16 h.

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Cell viability assay[1]
Cell Types: HT22 Cell
Tested Concentrations: 10 μM
Incubation Duration: 16 hrs (hours)
Experimental Results: The expression of genes related to antioxidant activity was Dramatically increased in the neuronal model, including prolactin and glutathione transferase. NRF2 is activated through a mechanism involving metabolic activation and covalent KEAP1 protein modification.

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Cell viability assay[2]
Cell Types: BPAEC
Tested Concentrations: 5, 10 μM
Incubation Duration: 135 hrs (hours)
Experimental Results: ATF6 activation reduces cell permeabi

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Cell Viability (MTT Assay): BPAEC were seeded in 96-well plates at a density of 10,000 cells per well. The cells were then treated with either vehicle (0.1% DMSO) or increasing concentrations of AA147 (1, 5, 10, 25, 50, 100, 200 μM) for 24 hours and 48 hours. After the treatment period, MTT (0.5 mg/ml) was added to each well and incubated for 3.5 hours. The formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm to assess cell viability. [2]
- Western Blotting for Protein Expression: BPAEC were cultured and treated with various conditions (e.g., AA147 alone or in combination with LPS). Proteins were isolated using RIPA buffer. Equal amounts of protein were separated by electrophoresis on 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk and incubated overnight at 4°C with primary antibodies against targets like cATF6, pCofilin, Cofilin, pMLC2, MLC2, pVE-cadherin, and VE-cadherin, with β-actin as a loading control. After incubation with appropriate HRP-conjugated secondary antibodies, protein signals were visualized using a ChemiDoc imaging system. Densitometric analysis of the immunoblots was performed using ImageJ software. [2]
- Paracellular Permeability (FITC-Dextran Assay): BPAEC were seeded onto trans-well inserts in 24-well culture plates at a density of 200,000 cells per insert and allowed to form monolayers. The cells were pre-treated with vehicle (0.1% DMSO) or AA147 (10 μM) for 24 hours. Subsequently, the cells were exposed to either vehicle (PBS) or LPS (10 μg/ml) for 2 hours. Following the LPS challenge, FITC-dextran (70 kDa, 1 mg/ml) was added to the inserts for 20 minutes. Media samples (100 μl) were collected from the receiver wells, and fluorescence intensity was measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively, using a microplate reader. [2]
- Transendothelial Electrical Resistance (ECIS): The barrier function of BPAEC monolayers was assessed using the electric cell-substrate impedance sensing (ECIS) system. Cells were grown on ECIS arrays until they reached a steady-state resistance of at least 800Ω. Confluent monolayers were then treated with vehicle (0.1% DMSO) or AA147 (5 μM, 10 μM), and the real-time changes in transendothelial electrical resistance (TEER), which inversely correlates with permeability, were monitored throughout the experiment. [2]

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HT22 Cell Viability Assay: HT22 cells are seeded in culture plates and pre-treated with various concentrations of AA147 (0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 20 μM) for 6 or 16 hours, followed by glutamate (5 mM) challenge for 24 hours. Cell viability is assessed by MTT assay. Results show that AA147 pre-treatment for 6 or 16 hours dose-dependently increases viability of glutamate-treated HT22 cells, whereas simultaneous addition of AA147 with glutamate provides no protective effect . Apoptosis Detection: HT22 cells pre-treated with AA147 for 6 or 16 hours are challenged with glutamate (5 mM) for 24 hours, then stained with Annexin V and/or propidium iodide. Apoptotic cell proportions are quantified by flow cytometry .

Mouse Model: The study used a severe SMA-like mouse model. [3]
- Drug Administration: AA147 was administered via intrathecal injection. Neonatal mice at postnatal day 7 (P7) were injected daily from P7 to P10. [3]
- Formulation/Dosage: The specific dose, vehicle, and formulation details for AA147 are not provided in the text. [3]
- Behavioral Analysis:
- Grip Test: The grip time of the mice was measured at P10. [3]
- Open Field Test: The spontaneous activity of the mice was analyzed in an open field. The total number of crossings and peripheral crossings during a 5-minute period were recorded from P8 to P11. [3]
- Tissue Collection and Analysis: Mice were sacrificed at P10. The lumbar spinal cord (L1-L5) was collected for molecular analysis (RT-qPCR, Western blot) and for immunohistochemistry. Motor neuron survival was assessed by counting ChAT-positive cells in the ventral horn of the lumbar spinal cord. [3]
- Survival and Weight Monitoring: Body weight was monitored daily, and the lifespan (survival) of the mice was recorded. [3]

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Mouse Model: The study used a severe SMA-like mouse model. [3]
- Drug Administration: AA147 was administered via intrathecal injection. Neonatal mice at postnatal day 7 (P7) were injected daily from P7 to P10. [3]
- Formulation/Dosage: The specific dose, vehicle, and formulation details for AA147 are not provided in the text. [3]
- Behavioral Analysis:
- Grip Test: The grip time of the mice was measured at P10. [3]
- Open Field Test: The spontaneous activity of the mice was analyzed in an open field. The total number of crossings and peripheral crossings during a 5-minute period were recorded from P8 to P11. [3]
- Tissue Collection and Analysis: Mice were sacrificed at P10. The lumbar spinal cord (L1-L5) was collected for molecular analysis (RT-qPCR, Western blot) and for immunohistochemistry. Motor neuron survival was assessed by counting ChAT-positive cells in the ventral horn of the lumbar spinal cord. [3]
- Survival and Weight Monitoring: Body weight was monitored daily, and the lifespan (survival) of the mice was recorded. [3]

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EAE Mouse Model (Multiple Sclerosis): The experimental autoimmune encephalomyelitis mouse model is used to evaluate AA147 efficacy. AA147 administration ameliorates clinical symptoms and reduces demyelination and oligodendrocyte loss . Spinal Cord Injury Model: A C57BL/6J mouse spinal cord injury model is established, and behavioral experiments are performed to assess AA147's effect on motor function recovery . Stroke Model: In a permanent stroke model, AA147 is administered post-stroke, and long-term functional recovery is evaluated through behavioral and histological assessments .

AA147 is metabolically activated to a reactive electrophile (quinone methide AA147-QM or quinone-imine AA147-QI), a process likely involving ER-localized oxidases such as cytochrome P450. This metabolic activation is essential for its biological activity, as shown by the requirement of the 2-amino-p-cresol moiety and inhibition by the P450 inhibitor resveratrol. [1]
- An analogue with a tertiary amine on the linker amide (AA147N‑methyl), which disfavors oxidation to the quinone-imine, shows significantly less protection against glutamate toxicity and fails to activate luciferase reporters. [1]
Pharmacokinetic analysis demonstrates that AA147 can cross the blood-brain barrier, a property that supports its application in CNS disease research .

The effects of AA147 on cell viability were assessed using the MTT assay in BPAEC. Exposure to AA147 at concentrations between 1 and 50 μM for 24 hours, and between 1 and 25 μM for 48 hours, did not significantly affect cell viability. However, higher doses (e.g., 100-200 μM) were found to be toxic, causing a significant reduction in viable cells. [2]
Retinal study: In mouse retina, AA147 induces ATF6-regulated gene expression at doses that do not cause retinal cell death, compromise vision, or trigger ER stress .

[1]. Metabolically Activated Proteostasis Regulators Protect against Glutamate Toxicity by Activating NRF2. ACS Chem Biol. 2021 Dec 17;16(12):2852-2863.

[2]. Activating transcription factor 6 protects against endothelial barrier dysfunction. Cell Signal. 2022 Aug 4;99:110432.

[3]. Activating ATF6 in spinal muscular atrophy promotes SMN expression and motor neuron survival through the IRE1α-XBP1 pathway. Neuropathol Appl Neurobiol. 2022 Aug;48(5):e12816.

[4]. Regulators of the endoplasmic reticulum proteostasis network. WO2017117430A1.

AA147 (N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide) is characterized as a small molecule that selectively induces the activating transcription factor 6 (ATF6) signaling pathway independently of inducing endoplasmic reticulum (ER) stress. [2]
- The mechanism of action involves AA147 being metabolically converted to an active compound by cytochrome P450, an ER-resident enzyme, leading to the selective modification of ER proteins and subsequent ATF6 activation. [2]
- In the context of this study, the protective role of AA147-mediated ATF6 activation was investigated against Lipopolysaccharides (LPS)-induced endothelial barrier dysfunction. The findings demonstrate that AA147 supports endothelial barrier function by opposing LPS-induced activation of Cofilin and MLC2, as well as VE-Cadherin phosphorylation, thereby reducing hyperpermeability. This suggests that pharmacological activation of ATF6 could be a potential therapeutic strategy for diseases associated with vascular leak, such as sepsis and ARDS. [2]

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AA147 (CAS: 393121-74-9) is an endoplasmic reticulum proteostasis regulator, first discovered and reported by the team of Jeffery W. Kelly and R. Luke Wiseman at the Scripps Research Institute. This compound undergoes metabolic activation to generate a reactive electrophile that covalently modifies ER-resident proteins, thereby selectively activating the ATF6 signaling pathway. In neuron-derived cells, AA147 can also activate the NRF2 antioxidant response pathway. AA147 and its analog AA28 have demonstrated protective effects in various disease models, including ischemic stroke, multiple sclerosis, spinal cord injury, and myocardial ischemia/reperfusion injury.

C16H17NO2

255.311684370041

255.1259

C, 75.27; H, 6.71; N, 5.49; O, 12.53

393121-74-9

882909

Light brown to brown solid powder

3.3

2

2

4

19

287

0

O=C(CCC1C=CC=CC=1)NC1C(=CC=C(C)C=1)O

AWHLTHOHBAGPMY-UHFFFAOYSA-N

InChI=1S/C16H17NO2/c1-12-7-9-15(18)14(11-12)17-16(19)10-8-13-5-3-2-4-6-13/h2-7,9,11,18H,8,10H2,1H3,(H,17,19)

N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide

ATF6 activator 147; ATF6-activator 147; 393121-74-9; N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide; AA147; AA 147; ATF6-activator-147; ATF6 activator-147

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 : ~50 mg/mL (~195.84 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (19.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 5 mg/mL (19.58 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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 (Please use freshly prepared in vivo formulations for optimal results.)