GSK-3β (Ki = 38 nM); GSK-3β (Ki = 38 nM)

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].

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.

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.

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.

[1]. Bhat R, Xue Y, Berg S, 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 that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC50 = 104 +/- 27 nM), in an ATP-competitive manner (Ki = 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50 > 100 microM) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3beta protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by beta-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5.[1]

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Glycogen synthase kinase (GSK)-3 has recently been implicated in the pathogenesis of neurodegenerative diseases. Although the neuroprotective effects of GSK-3 inhibitors in Alzheimer's disease have been established, their effects on amyotrophic lateral sclerosis (ALS) have not been well defined. This study was undertaken to evaluate the effects of GSK-3 inhibition in the G93A-SOD1 mouse model of ALS. Groups of G93A-SOD1 mice were treated with varying concentrations of GSK-3 inhibitor VIII, a specific GSK-3 inhibitor that crosses the BBB, intraperitoneally 5 days a week after 60 days of age. The GSK-3 inhibitor VIII treatment significantly delayed the onset of symptoms and prolonged the life span of the animals, and inhibited the activity of GSK-3 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 pathogenic mechanisms of ALS and that inhibition of GSK-3 could be a potential therapeutic candidate for ALS.[2]

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.)