JNK
AEG3482 directly binds HSP90 (as shown by affinity pull‑down; no IC50/Ki reported) and, through this interaction, promotes HSF1‑dependent transcription of HSP70 and HSP25. The resulting accumulation of HSP70 then binds and inhibits JNK activity. [1]

AEG3482 (0.3-30 μM; 2 d) inhibits nerve growth factor (NGF) withdrawal-induced death in SCG neurons, with an EC50 of ∼20 μM[1].
AEG3482 (1-80 μM; 40 h) inhibits PC12 cells' p75NTR- and NRAGE-mediated apoptosis in a dose-dependent manner[1].
AEG3482 (10-40 μM; 30 h) inhibits p75NTR- and NRAGE-mediated JNK activation in PC12 cells[1].

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AEG3482 inhibited NGF withdrawal‑induced death of rat superior cervical ganglion (SCG) sympathetic neurons with an EC50 of approximately 20 μM (cell viability measured by MTS assay after 2 days). [1]
- In PC12TTA cells overexpressing p75NTR or NRAGE (which causes JNK‑dependent apoptosis), AEG3482 reduced cell death in a dose‑dependent manner (LDH release assay). At 40 μM, it reduced p75NTR‑ or NRAGE‑induced cell death by >90%; at the highest concentration tested (80 μM) it slightly increased death in LacZ control cells. [1]
- AEG3482 attenuated NRAGE‑ or p75NTR‑induced JNK activation: immunoblots showed decreased phosphorylation of the JNK target c‑Jun and decreased caspase‑3 cleavage. Effects were detectable at 10 μM and virtually complete at 40 μM. [1]
- AEG3482 inhibited paclitaxel‑induced apoptosis (10 and 50 μM) and low‑dose cisplatin‑induced apoptosis (10 μM) but did not protect against high‑dose cisplatin (50 μM) or doxorubicin (JNK‑independent)‑induced death. [1]
- Treatment with AEG3482 for 18 h caused a large increase in cellular HSP70 and HSP25 protein levels in PC12 cells (immunoblot). Semi‑quantitative rtPCR showed increased HSP25 and HSP70 mRNA but no effect on HSP90 or actin mRNA. [1]
- In PC12TTA cells infected with LacZ or p75NTR, AEG3482 dose‑dependently increased HSP70 and HSP25 but not HSP40. NRAGE infection alone raised HSP70, and AEG3482 further increased it. [1]
- In wild‑type mouse embryonic fibroblasts (MEFs), AEG3482 induced HSP70 promoter activity (luciferase reporter) and increased endogenous HSP70 and HSP25 proteins; these effects were absent in HSF1‑null MEFs. [1]
- AEG3482‑conjugated Sepharose beads bound purified HSP90; binding was competed by excess free AEG3482 (immunoblot). [1]
- Analogs: AEG19940 (similar structure) induced HSP70 production, blocked p75NTR‑/NRAGE‑induced death, and reduced c‑Jun phosphorylation. AEG33691 and AEG33733 did not induce HSP70 and had no effect in these assays. [1]
- RNAi knockdown of HSP70 in PC12TTA cells partially attenuated the ability of AEG3482 (or AEG19940) to block c‑Jun phosphorylation. Using a GST‑c‑Jun reporter to assess JNK activity specifically in transfected cells, HSP70 siRNA almost completely abolished AEG19940‑mediated inhibition of NRAGE‑induced GST‑c‑Jun phosphorylation. [1]
- Unlike geldanamycin, AEG3482 did not block HSP90 binding to γ‑ATP‑Sepharose but rather increased association. AEG3482 enhanced phosphorylated Akt levels in PC12 cells, whereas geldanamycin reduced total and phosphorylated Akt. [1]

HSP90 pull‑down assay: AEG3482 was chemically crosslinked to Sepharose beads to generate an affinity resin. Purified HSP90 protein was incubated with the beads for 2 h, then beads were washed extensively. Bound proteins were eluted and analyzed by SDS‑PAGE and immunoblotting using anti‑HSP90 antibody. Control beads (without AEG3482) and competition with excess free AEG3482 (80 μM) were included. AEG3482 beads bound HSP90, while control beads did not, and binding was reduced by free compound. [1]
- γ‑ATP‑Sepharose binding assay: Purified HSP90 (1 μg) was preincubated with ATP, geldanamycin (1 μM), or increasing concentrations of AEG3482 in incubation buffer (10 mM Tris pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM DTT, 20 mM Na2MoO4, 0.01% NP‑40) for 10 min at room temperature. Then 25 μl of γ‑ATP‑Sepharose beads were added and incubated for 30 min at 30 °C. Beads were washed with ice‑cold buffer, bound proteins were eluted, and HSP90 content determined by immunoblotting. Geldanamycin blocked HSP90 binding to γ‑ATP‑Sepharose, whereas AEG3482 did not decrease binding but increased it. [1]
- In vitro JNK activity assay (mentioned but not detailed): AEG3482 had no effect on JNK activity in vitro (data not shown). [1]

Sympathetic neuron survival assay (MTS): Rat SCG neurons initially maintained in 10 ng/ml NGF were withdrawn from NGF and cultured in media containing AEG3482 at indicated concentrations. After 2 days, cell viability was measured using an MTS assay according to the manufacturer’s instructions. Results were normalized to survival in 10 ng/ml NGF. [1]
- PC12 cell death assay (LDH): PC12TTA cells were infected with recombinant adenoviruses (AdLacZ, Adp75, or AdNRAGE) and concurrently treated with increasing concentrations of AEG3482 or vehicle for 40 h. Cell death was assessed using a lactate dehydrogenase (LDH) release assay following the manufacturer’s protocol. Untreated cells and cells treated with 1% Triton X‑100 were used to define the assay range. [1]
- Immunoblotting: Cells were lysed, normalized for protein content, and analyzed by SDS‑PAGE and immunoblotting with antibodies against phospho‑c‑Jun, total c‑Jun, cleaved caspase‑3, JNK, IκBα, NRAGE, p75NTR, HSP70, HSP25, HSP40, Akt, phospho‑Akt, and actin. [1]
- RT‑PCR: PC12 cells were treated with increasing concentrations of AEG3482 for 18 h. mRNA was isolated, cDNA generated with random hexamers, and PCR performed using primers specific for rat HSP70, HSP25, and actin. [1]
- Transcriptional reporter assay: MEFs (wild‑type or HSF1‑null) were transfected with pGL3B‑HSP70 (HSP70 promoter‑luciferase) or empty vector. The next day, AEG3482 (40 μM) was added, and cells were harvested 48 h after transfection. Luciferase activity was measured using a commercial assay system. [1]
- GST‑c‑Jun reporter assay: A cDNA encoding amino acids 2‑79 of human c‑Jun was cloned into a mammalian GST expression vector. PC12TTA cells were transfected with this construct together with control siRNA or HSP70‑specific siRNA. After 24 h, cells were infected with AdNRAGE or AdLacZ and treated with AEG19940 or vehicle. After 30 h, cells were lysed, GST‑c‑Jun was recovered using glutathione‑conjugated beads (1 h at 4 °C), and phosphorylation was assessed by immunoblotting with anti‑phospho‑c‑Jun (Ser63) antibody. Equivalent pull‑down was confirmed with anti‑c‑Jun and anti‑GST antibodies. [1]
- RNAi transfection: PC12TTA cells were transfected with a “SmartPool” of HSP70‑specific small interfering RNAs or control RNA using Lipofectamine 2000. After 24 h, cells were infected and treated as indicated. [1]

At the highest concentration tested (80 μM), AEG3482 exerted a slight toxic effect in PC12TTA cells infected with the control LacZ adenovirus (as measured by LDH release assay). [1]

[1]. AEG3482 is an antiapoptotic compound that inhibits Jun kinase activity and cell death through induced expression of heat shock protein 70. Chem Biol. 2006 Feb;13(2):213-23.

6-Phenylaceto-2-imidazo[2,1-b][1,3,4]thiadiazole sulfonamide is a member of the imidazolium class of compounds.

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Background and mechanism: AEG3482 is the first compound shown to inhibit JNK activation through a mechanism involving induced production of HSP70. It binds HSP90, causing release of HSF1, which then transactivates HSP70 and HSP25 genes. Accumulated HSP70 directly binds JNK (likely at or near the docking groove) and inhibits its activity, thereby blocking apoptosis. This action is independent of HSP70’s chaperone function. [1]
- Comparison with geldanamycin: Unlike geldanamycin, AEG3482 does not occupy the ATP binding pocket of HSP90 (it does not block γ‑ATP‑Sepharose binding), does not reduce levels or phosphorylation of the HSP90 client protein Akt, and may enhance Akt phosphorylation. The compound is proposed to interact with the peptide‑binding domain of HSP90, facilitating HSF1 release while retaining HSP90 chaperone activity. [1]
- Therapeutic potential: The authors anticipate that AEG3482 and its analogs will serve as useful tools for basic research and have therapeutic potential for the treatment of acute and chronic neurological disorders. [1]

C10H8N4O2S2

280.32612

280.008

C, 42.85; H, 2.88; N, 19.99; O, 11.41; S, 22.87

63735-71-7

63735-71-7

698112

White to light yellow solid powder

1.8±0.1 g/cm3

1.835

1.32

1

6

2

18

405

0

C1=CC=C(C=C1)C2=CN3C(=N2)SC(=N3)S(=O)(=O)N

MQUYTXDAVCOCMX-UHFFFAOYSA-N

InChI=1S/C10H8N4O2S2/c11-18(15,16)10-13-14-6-8(12-9(14)17-10)7-4-2-1-3-5-7/h1-6H,(H2,11,15,16)

6-phenylimidazo[2,1-b][1,3,4]thiadiazole-2-sulfonamide

AEG-3482; AEG 3482; AEG3482

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: ~250 mg/mL (~891.8 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.42 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 20.8 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: ≥ 2.08 mg/mL (7.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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