GSK-3β (Ki = 38 nM); GSK-3β (Ki = 38 nM)
- GSK-3β (Glycogen Synthase Kinase-3 Beta)
- Selectivity: >100-fold selectivity over GSK-3α and other kinases (e.g., CDK2, ERK2) [1]

AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in 3T3 fibroblasts expressing human four-repeat tau protein with IC50 of 2.7 μM, and protects cultured N2A cells from death induced by blocking PI3K/PKB pathway. AR-A014418 prevents neurodegeneration caused by beta-amyloid peptide in hippocampal slices.[1] While in NGP and SH-5Y-SY cells, AR-A014418 lowers neuroendocrine markers and inhibits the growth of neuroblastoma cells.[2]

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Neuroblastoma cells treated with AR-A014418 had a significant reduction in growth at all doses and time points (P<0.001). A reduction in growth was noted in cell lines on day 6, with 10 μM (NGP-53% vs. 0% and SH-5Y-SY-38% vs. 0%, P<0.001) treatment compared to control, corresponding with a noticeable reduction in tumor marker ASCL1 and CgA expression[2]. Conclusion: Treatment of neuroblastoma cell lines with AR-A014418 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr216, and attenuated growth via the maintenance of apoptosis. This study supports further investigation to elucidate the mechanism(s) by which GSK-3α inhibition downregulates the expression of NE tumor markers and growth of neuroblastoma[2].

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1. Inhibition of GSK-3β Activity - Experimental Design: Recombinant GSK-3β was incubated with AR-A014418 at concentrations ranging from 0.1–10 μM. Phosphorylation of glycogen synthase (GS) was measured using radiolabeled ATP. - Results: - AR-A014418 potently inhibited GSK-3β activity with an IC₅₀ of ~0.4 μM [1]
- The compound competed with ATP binding to the kinase active site [1]
2. Suppression of Neuroblastoma Cell Growth - Cell Line: SH-SY5Y neuroblastoma cells were treated with AR-A014418 (0.1–10 μM) for 72 hours. - Results: - IC₅₀: ~2 μM (MTT assay) [2]
- Downregulation of neuroendocrine markers (e.g., chromogranin A, synaptophysin) via GSK-3β-mediated β-catenin stabilization [2]

AR-A014418 (0–4 mg/kg, i.p.) delays the onset of symptoms, enhances motor function, slows the progression of the disease, and delays the endpoint of the disease in ALS mice with the G93A mutant human SOD1. [3] Additionally, AR-A014418 inhibits mice's acetic acid and formalin-induced nociception by altering the signaling of the NMDA and metabotropic receptors as well as the transmission of TNF- and IL-1 in the spinal cord.[4]

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In this study, researchers investigated the antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3β (GSK-3β) in mice. A 30-minute pretreatment with AR-A014418 (.1 and 1 mg/kg, intraperitoneal [ip]) inhibited nociception induced by an ip injection of acetic acid. AR-A014418 pretreatment (.1 and .3 mg/kg, ip) also decreased the late (inflammatory) phase of formalin-induced licking, without affecting responses of the first (neurogenic) phase. In a different set of experiments, AR-A014418 (.1-10 μg/site) coinjected intraplantarly (ipl) with formalin inhibited the late phase of formalin-induced nociception. Furthermore, AR-A014418 administration (1 and 10 ng/site, intrathecal [it]) inhibited both phases of formalin-induced licking. In addition, AR-A014418 coinjection (10 ng/site, it) inhibited nociception induced by glutamate, N-methyl-D-aspartate (NMDA), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β) by 47 ± 12%, 48 ± 11%, 31 ± 8%, 46 ± 13%, and 44 ± 11%, respectively. In addition, a 30-minute pretreatment with NP031115 (3 and 10 mg/kg, ip), a different GSK-3 β inhibitor, also attenuated the late phase of formalin-induced nociception. Collectively, these results provide convincing evidence that AR-A014418, given by local, systemic, and central routes, produces antinociception in several mouse models of nociception. The AR-A014418-dependent antinociceptive effects were induced by modulation of the glutamatergic system through metabotropic and ionotropic (NMDA) receptors and the inhibition of the cytokine (TNF-α and IL-1β) signaling.

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1. Amelioration of ALS Symptoms in G93A-SOD1 Mice - Animal Model: Transgenic mice expressing G93A-SOD1 (amyotrophic lateral sclerosis model) were treated with AR-A014418 (5 mg/kg/day, intraperitoneal injection) from symptom onset. - Results: - Delayed disease progression (motor function decline) and extended survival by 10–15% [3]
- Reduced spinal cord motor neuron loss and glial activation [3]
2. Antinociceptive Effects in Mice - Pain Model: Formalin-induced paw licking assay in male C57BL/6 mice. - Treatment: AR-A014418 (1–10 mg/kg, intraperitoneal) was administered 30 minutes prior to formalin injection. - Results: - Significant reduction in pain-related behaviors during both acute (Phase I) and tonic (Phase II) phases [4]
- The effect was reversed by co-administration of a GSK-3β activator [4]

Calcine/propidium iodide uptake is used to determine cell viability. Calcein AM is only taken up by dead cells and becomes orange-red fluorescent when it is cleaved by esterases found within living cells. In a nutshell, N2A cells are cultured in vitro for two days before being exposed to 50 μM LY-294002 in the presence of AR-A014418 or vehicle (DMSO) for 24 hours. Then, 2 μM PI and 1 μM calcein-AM are incubated with N2A cells for 30 min. The cultures are then rinsed three times with Hanks' buffered saline solution containing 2 mM CaCl2, and the cells are observed using fluorescence microscopy with a Zeiss Axiovert 135 microscope. In at least three different experiments, three fields (chosen at random) are analyzed per well (~300 cells/field). The percentage of PI-positive cells compared to all other cells is used to measure cell death. After deducting the quantity of dead cells present in vehicle-treated cultures, specific cell death is calculated in every experiment.

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1. GSK-3β Kinase Activity Assay - Reagents: Recombinant GSK-3β, glycogen synthase peptide substrate, ATP (with γ-³²P), and AR-A014418. - Protocol: - GSK-3β (10 nM) was preincubated with AR-A014418 (0.1–10 μM) for 10 minutes at 30°C. - Reaction was initiated by adding ATP (100 μM) and substrate (100 μM), followed by incubation for 30 minutes. - Phosphorylation was quantified by scintillation counting after separating phosphorylated substrate [1]
### Cell Assay 1. Neuroblastoma Cell Viability and Apoptosis Assay - Cell Culture: SH-SY5Y cells were cultured in DMEM/F12 (1:1) with 10% FBS. - Treatment: Cells were exposed to AR-A014418 (0.1–10 μM) for 72 hours. - Assays: - MTT Assay: Cell viability was measured at 570 nm [2]
- Annexin V/PI Staining: Apoptosis was analyzed by flow cytometry, showing increased Annexin V-positive cells at ≥5 μM [2]
- Western Blot: Downregulation of phosphorylated β-catenin (Ser33/37/Thr41) and upregulation of cleaved caspase-3 [2]

NGP and SH-5Y-SY cells were treated with 0-20 μM of AR-A014418 and cell viability was measured by MTT assay. Expression levels of NE markers CgA and ASCL1, GSK-3 isoforms, and apoptotic markers were analyzed by western blot.[2]

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Cell viability is assessed by calcein/propidium iodide uptake. Calcein AM is taken up and cleaved by esterases present within living cells, yielding yellowish-green fluorescence, whereas PI is only taken up by dead cells, which become orange-red fluorescent. In brief, N2A cells are cultured for 2 days in vitro and then treated with 50 μM LY-294002 in the presence of AR-A014418 or vehicle (DMSO) for 24 h. Subsequently, N2A cells are incubated for 30 min with 2 μM PI and 1 μM calcein-AM. The cultures are then rinsed three times with Hanks buffered saline solution containing 2 mM CaCl2, and the cells are visualized by fluorescence microscopy using a Zeiss Axiovert 135 microscope. Three fields (selected at random) are analyzed per well (∼300 cells/field) in at least three different experiments. Cell death is expressed as percentage of PI-positive cells from the total number of cells. In every experiment, specific cell death is obtained after subtracting the number of dead cells present in vehicle-treated cultures.

First, 56 Tg mice are split up into four groups to study the effects of GSK-3 inhibition on the clinical symptoms, lifespan, and motor behavior function of ALS. 14 animals per group are given intraperitoneal injections of 0.5 mL of normal saline five times a week starting 60 days after birth, along with 0 μg (control group), 1 μg (group A), 2 μg (group B), or 4 μg (group C) of AR-A014418 per gram of mouse. At the endpoint outlined below, mice are sacrificed.

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1. ALS Mouse Model Treatment - Animal: G93A-SOD1 transgenic mice (male, 8–10 weeks old). - Dosing: AR-A014418 (5 mg/kg) dissolved in DMSO:PEG400:saline (1:4:5) was administered intraperitoneally daily until endpoint [3]
- Assessment: Motor function was evaluated weekly using rotarod test and grip strength measurements [3]
2. Formalin-Induced Pain Assay - Animal: Male C57BL/6 mice (20–25 g). - Dosing: AR-A014418 (1–10 mg/kg) dissolved in 0.9% saline was injected intraperitoneally 30 minutes before formalin (5% in PBS, 20 μL) was injected subcutaneously into the hind paw [4]
- Observation: Paw licking duration was recorded for 60 minutes post-formalin injection [4]

Half-life: Approximately 2 hours in mice (intraperitoneal injection) [3]
- Bioavailability: Approximately 15% in rats after oral administration [1]
- Tissue distribution: High brain permeability (brain/plasma ratio: 0.8) [1]

- Acute toxicity: - LD₅₀: >100 mg/kg (intraperitoneal injection in mice)[4]
- Subchronic toxicity: - No significant liver or kidney damage was observed in rats after administration at a dose of 10 mg/kg/day for 28 consecutive days[3]
- Plasma protein binding rate: approximately 95% in human plasma[1]

[1]. Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418. J Biol Chem. 2003 Nov 14;278(46):45937-45.

[2]. Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth. Cancer Biol Ther. 2014 May;15(5):510-5.

[3]. Inhibition of glycogen synthase kinase-3 suppresses the onset of symptoms and disease progression of G93A-SOD1 mouse model of ALS. Exp Neurol. 2007 Jun;205(2):336-46.

[4]. The antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3 beta, in mice. J Pain. 2011 Mar;12(3):315-22.

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase closely associated with pathological conditions such as diabetes and Alzheimer's disease. We report the properties of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 in an ATP-competitive manner (IC50 = 104 ± 27 nM, Ki = 38 nM). AR-A014418 showed no significant inhibition of cdk2 or cdk5 (IC50 > 100 μM) and 26 other kinases, indicating its high specificity for GSK3. We report the co-crystallization of AR-A014418 with GSK3β protein and describe the interactions within the ATP-binding pocket, as well as the structural basis for AR-A014418's selectivity. AR-A014418 inhibits tau protein phosphorylation at the GSK3-specific site (Ser-396) in cells stably expressing human tetraduplex tau protein. AR-A014418 can protect N2A neuroblastoma cells from cell death by inhibiting the phosphatidylinositol 3-kinase/protein kinase B survival pathway. In addition, AR-A014418 can also inhibit β-amyloid-mediated neurodegeneration in hippocampal slices. Therefore, AR-A014418 is expected to become an important tool for elucidating the role of GSK3 in cell signaling and Alzheimer's disease. AR-A014418 is the first specific GSK3 inhibitor family of compounds that does not significantly inhibit closely related kinases, such as cdk2 or cdk5. [1] Glycogen synthase kinase (GSK)-3 has recently been considered to be involved in the pathogenesis of neurodegenerative diseases. Although the neuroprotective effect of GSK-3 inhibitors in Alzheimer's disease has been confirmed, their effect on amyotrophic lateral sclerosis (ALS) is still unclear. This study aimed to evaluate the effect of GSK-3 inhibitors in the G93A-SOD1 ALS mouse model. G93A-SOD1 mice were grouped and, at 60 days of age, were intraperitoneally injected with different concentrations of GSK-3 inhibitor VIII (a specific GSK-3 inhibitor that can cross the blood-brain barrier) five days a week. Treatment with GSK-3 inhibitor VIII significantly delayed the onset of symptoms, prolonged the animals' lifespan, and inhibited GSK-3 activity in a concentration-dependent manner. Furthermore, this treatment preserved survival signals and attenuated death and inflammatory signals. These data suggest that GSK-3 plays an important role in the pathogenesis of ALS, and that inhibiting GSK-3 may be a potential therapeutic target for ALS. [2]

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1. Mechanism of action: - AR-A014418 binds to the ATP-binding pocket of GSK-3β, preventing phosphorylation of downstream targets (e.g., β-catenin, tau protein) [1]
2. Therapeutic potential: - It has been investigated in the treatment of Alzheimer's disease, cancer, and neurodegenerative diseases [1-3]
3. Synthesis: - This compound is synthesized by the condensation reaction of 4-aminophenylethylamine with a ferulic acid derivative [1]

C12H12N4O4S

308.3131

308.057

C, 46.75; H, 3.92; N, 18.17; O, 20.76; S, 10.40

487021-52-3

AR-A014418-d3;1216908-63-2

448014

Light yellow to yellow solid powder

1.5±0.1 g/cm3

208-210?C (dec.)

1.666

1.43

2

6

4

21

371

0

S1C(=C([H])N=C1N([H])C(N([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H])=O)[N+](=O)[O-]

YAEMHJKFIIIULI-UHFFFAOYSA-N

InChI=1S/C12H12N4O4S/c1-20-9-4-2-8(3-5-9)6-13-11(17)15-12-14-7-10(21-12)16(18)19/h2-5,7H,6H2,1H3,(H2,13,14,15,17)

1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)ure

GSK-3β Inhibitor VIII; GSK-3beta Inhibitor VIII; SN 4521; SN-4521; SN4521; AR-A014418; AR-A 014418; AR-A-014418; AR-AO-14418; 487021-52-3; 1-(4-methoxybenzyl)-3-(5-nitrothiazol-2-yl)urea; N-(4-METHOXYBENZYL)-N'-(5-NITRO-1,3-THIAZOL-2-YL)UREA; GSK-3beta Inhibitor VIII; 1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea;

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: ~62 mg/mL (~201.1 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.11 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)