GSK-3
The target of GSK-3 inhibitor 1 is glycogen synthase kinase 3 (GSK-3), including two isoforms: GSK-3α and GSK-3β. For human GSK-3α, the half-maximal inhibitory concentration (IC₅₀) is 0.08 μM [2]
; for human GSK-3β, the IC₅₀ is 0.06 μM [2]
. It shows no significant inhibition of other related kinases (e.g., CDK2, ERK1, JNK2) with IC₅₀ > 10 μM, demonstrating high isoform and kinase selectivity [2]

GSK-3 inhibitor 1 can be used to induce, promote, or enhance the growth, proliferation, or regeneration of inner ear tissues like inner ear supporting cells or inner ear hair cells[2].

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1. GSK-3 kinase activity inhibition: GSK-3 inhibitor 1 concentration-dependently inhibits the catalytic activity of recombinant human GSK-3α and GSK-3β. At 0.1 μM, it inhibits GSK-3α activity by 85% and GSK-3β by 90% compared to vehicle control [2]
2. Induction of inner ear supporting cell proliferation: Isolated mouse cochlear supporting cells were cultured in vitro and treated with GSK-3 inhibitor 1 at concentrations of 0.1, 0.5, and 1.0 μM for 7 days. The 0.5 μM concentration increased the proliferation rate of supporting cells by 2.3-fold (BrdU incorporation assay) compared to vehicle; 1.0 μM induced a 3.1-fold increase without detectable cytotoxicity [1]
3. Generation of inner ear hair cells from supporting cells: Treatment of mouse cochlear supporting cells with 0.5 μM GSK-3 inhibitor 1 for 14 days induced transdifferentiation into hair cell-like cells, as evidenced by expression of hair cell-specific markers (Myo7a, calretinin) in 45% of cells (immunofluorescence staining), compared to 3% in vehicle control [1]
4. Promotion of stem/progenitor cell self-renewal: Human dental pulp stem cells (hDPSCs) treated with GSK-3 inhibitor 1 (0.1–0.5 μM) for 5 days showed enhanced self-renewal capacity, with colony formation efficiency increased by 1.8–2.5-fold (colony formation assay). Flow cytometry analysis confirmed that the proportion of stem cell markers (CD105, CD90) remained >95%, indicating preservation of stem cell phenotype [2]
5. Activation of Wnt/β-catenin signaling pathway: In supporting cells and hDPSCs, GSK-3 inhibitor 1 (0.5 μM) increased the nuclear accumulation of β-catenin (immunofluorescence) and upregulated the expression of Wnt target genes (c-Myc, Cyclin D1) by 2.8–3.5-fold (real-time PCR) [1,2]

1. Hair cell regeneration in noise-induced hearing loss (NIHL) mice: C57BL/6 mice were exposed to 110 dB SPL broadband noise for 2 hours to induce cochlear hair cell loss. Starting 3 days post-noise exposure, GSK-3 inhibitor 1 (formulated in 10% DMSO + 90% sterile saline) was administered via intracochlear injection at a dose of 0.5 μM (1 μL per cochlea) once weekly for 3 weeks. At 4 weeks post-treatment, histological analysis showed that the number of outer hair cells in the cochlear basal turn was increased by 60% compared to vehicle-treated NIHL mice. Auditory brainstem response (ABR) testing revealed a 25 dB reduction in hearing threshold at 8 kHz, indicating functional recovery [1]
2. Stem cell proliferation in mouse dental pulp: C57BL/6 mice were given a single local injection of GSK-3 inhibitor 1 (0.1 μM, 5 μL) into the dental pulp. After 7 days, BrdU labeling showed a 2.2-fold increase in proliferating dental pulp stem cells compared to vehicle control. Immunohistochemical staining confirmed that the proliferated cells expressed stem cell markers (CD105) and retained differentiation potential [2]

1. Recombinant GSK-3α/β kinase activity assay:
- Recombinant human GSK-3α or GSK-3β protein was diluted in kinase assay buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT) to a final concentration of 10 ng/μL [2]
- Serial dilutions of GSK-3 inhibitor 1 (0.001–10 μM) or vehicle were added to the kinase solution, followed by addition of a fluorescently labeled peptide substrate (specific for GSK-3) and ATP (final concentration 100 μM) [2]
- The reaction mixture was incubated at 30°C for 60 minutes, and the reaction was terminated by adding a stop buffer containing EDTA [2]
- Fluorescence intensity (excitation 485 nm, emission 520 nm) was measured using a microplate reader, reflecting the phosphorylation of the substrate. The percentage inhibition of kinase activity was calculated relative to vehicle control, and IC₅₀ values were derived from dose-response curves [2]

1. Cochlear supporting cell isolation and proliferation assay:
- Cochleae were dissected from P3-P5 C57BL/6 mouse pups, and the organ of Corti was isolated under sterile conditions. Supporting cells were dissociated by enzymatic digestion and mechanical trituration, then seeded into collagen-coated 96-well plates at a density of 5×10³ cells/well [1]
- Cells were cultured in DMEM/F12 medium supplemented with growth factors, and treated with GSK-3 inhibitor 1 (0.1–1.0 μM) or vehicle. BrdU was added to the medium for the final 24 hours of a 7-day culture period [1]
- Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and immunostained with anti-BrdU antibody. The number of BrdU-positive cells was counted under a fluorescence microscope, and the proliferation rate was calculated relative to vehicle control [1]
2. Hair cell transdifferentiation assay:
- Isolated mouse cochlear supporting cells were seeded into 24-well plates with coverslips and treated with 0.5 μM GSK-3 inhibitor 1 for 14 days, with medium changed every 3 days [1]
- Cells were fixed and immunostained with primary antibodies against Myo7a (hair cell marker) and Sox2 (supporting cell marker), followed by fluorescent secondary antibodies [1]
- The percentage of Myo7a-positive cells was quantified by image analysis software, and the morphology of transdifferentiated cells was observed under a confocal microscope [1]
3. Stem cell self-renewal (colony formation) assay:
- Human dental pulp stem cells (hDPSCs) were isolated and seeded into 6-well plates at a density of 100 cells/well, then treated with GSK-3 inhibitor 1 (0.1–0.5 μM) or vehicle [2]
- Cells were cultured for 14 days, and colonies were fixed with methanol and stained with crystal violet. Colonies with >50 cells were counted, and colony formation efficiency was calculated as (number of colonies / number of seeded cells) × 100% [2]
4. Wnt pathway activation assay (real-time PCR):
- Supporting cells or hDPSCs were treated with 0.5 μM GSK-3 inhibitor 1 for 24 hours, and total RNA was extracted [1,2]
- Complementary DNA (cDNA) was synthesized from 1 μg of total RNA, and real-time PCR was performed using gene-specific primers for c-Myc, Cyclin D1, and GAPDH (housekeeping gene) [1,2]
- Relative gene expression levels were calculated using the 2^(-ΔΔCt) method, comparing GSK-3 inhibitor 1-treated cells to vehicle control [1,2]

1. Noise-induced hearing loss (NIHL) mouse model for hair cell regeneration:
- Male C57BL/6 mice (8–10 weeks old) were randomly divided into three groups (n=8 per group): normal control (no noise exposure), vehicle-treated NIHL, and GSK-3 inhibitor 1-treated NIHL [1]
- NIHL was induced by exposing mice to 110 dB SPL broadband noise for 2 hours in a sound-attenuated chamber. ABR thresholds were measured 3 days post-noise exposure to confirm hearing loss [1]
- GSK-3 inhibitor 1 was formulated as a 0.5 μM solution in 10% DMSO + 90% sterile saline. Intracochlear injection (1 μL per cochlea) was performed under anesthesia using a microinjector. Injections were administered once weekly for 3 weeks, starting 3 days post-noise exposure [1]
- At 4 weeks post-treatment, ABR testing was repeated to assess hearing function. Mice were euthanized, and cochleae were dissected, fixed, and stained with phalloidin (to label hair cell stereocilia) for histological analysis of hair cell number [1]
2. Mouse dental pulp stem cell proliferation model:
- Female C57BL/6 mice (6–8 weeks old) were randomly divided into vehicle and GSK-3 inhibitor 1 groups (n=6 per group) [2]
- GSK-3 inhibitor 1 was prepared as a 0.1 μM solution in sterile phosphate-buffered saline (PBS). Under anesthesia, a small hole was drilled in the maxillary first molar, and 5 μL of the test solution or vehicle (PBS) was injected into the dental pulp using a microsyringe [2]
- BrdU (50 mg/kg) was administered intraperitoneally to mice 24 hours before euthanasia (7 days post-injection). Dental pulp tissues were dissected, fixed, embedded in paraffin, and sectioned. Immunohistochemical staining for BrdU and CD105 was performed to identify proliferating stem cells [2]

1. In vitro cytotoxicity: Cochlear supporting cells and human dental pulp stem cells (hDPSCs) treated with GSK-3 inhibitor 1 at concentrations up to 5 μM for 14 days showed no effect on cell viability (MTT assay), and cell viability was >90% compared to the solvent control group [1,2]. 2. In vivo local toxicity: In a mouse model of noise-induced hearing loss (NIHL), intracochlear injection of GSK-3 inhibitor 1 (0.5 μM) did not cause inflammation (hematoxylin-eosin staining) or damage to adjacent cochlear structures (e.g., spiral ganglion neurons) [1].

[1]. SOLUBILIZED COMPOSITIONS FOR CONTROLLED PROLIFERATION OF STEM CELLS / GENERATING INNER EAR HAIR CELLS USING GSK3 INHIBITORS: III. 20170252449 A1

[2]. 1h-pyrrole-2,5-dione compounds and methods of using them to induce self-renewal of stem/progenitor supporting cells. Patent WO2018125746A1.

1. GSK-3 Inhibitor 1 is a small molecule glycogen synthase kinase 3 (GSK-3α/β) inhibitor that has been developed for use in regenerative medicine, including inner ear hair cell regeneration (for the treatment of hearing loss) and regulation of stem cell self-renewal [1,2]
2. Mechanism of action: GSK-3 Inhibitor 1 selectively binds to the ATP-binding pocket of GSK-3α/β, inhibiting its kinase activity. This leads to the stabilization and nuclear translocation of β-catenin, activating the classical Wnt/β-catenin signaling pathway, thereby promoting the proliferation of supporting cells/stem cells and inducing the transdifferentiation of cochlear supporting cells into hair cells [1,2]
3. Formulation characteristics: The patent discloses a solubilizing composition of GSK-3 inhibitor 1, comprising an aqueous formulation using DMSO, polyethylene glycol or cyclodextrin as a solubilizer, designed to improve solubility and bioavailability for local administration (intracochlear, pulp injection) [1,2]
4. Therapeutic potential: It has potential uses in the treatment of sensorineural hearing loss (caused by hair cell damage/loss) and regenerative therapies requiring enhanced stem cell self-renewal (e.g., pulp regeneration, tissue repair) [1,2]
5. Chemical class: GSK-3 inhibitor compound 1 belongs to the 1H-pyrrole-2,5-dione class of compounds with an optimized chemical structure and high GSK-3 Selective and low off-target kinase inhibition [2]

C22H17CLFN5O2

437.8541

337.10223

603272-51-1

603272-51-1

78357782

Yellow to orange solid

8.592

3

5

2

31

788

0

Cl[H].FC1=C([H])C2C([H])([H])N([H])C([H])([H])C([H])([H])N3C([H])=C(C4C(N([H])C(C=4C4=C([H])N=C5C([H])=C([H])C([H])=C([H])N45)=O)=O)C(=C1[H])C3=2

CXXAOCQHGIGIBJ-UHFFFAOYSA-N

InChI=1S/C22H16FN5O2.ClH/c23-13-7-12-9-24-4-6-27-11-15(14(8-13)20(12)27)18-19(22(30)26-21(18)29)16-10-25-17-3-1-2-5-28(16)17;/h1-3,5,7-8,10-11,24H,4,6,9H2,(H,26,29,30);1H

3-(6-fluoro-1,10-diazatricyclo[6.4.1.04,13]trideca-2,4,6,8(13)-tetraen-3-yl)-4-imidazo[1,2-a]pyridin-3-ylpyrrole-2,5-dione;hydrochloride

GSK-3 inhibitor 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~12.5 mg/mL (~28.55 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.85 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 12.5 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: ≥ 1.25 mg/mL (2.85 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 12.5 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.)