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MLS2052 (also known as BIO, GSK3 Inhibitor IX, 6-Bromoindirubin-3'-oxime) is a novel, potent, cell-permeable bis-indolo and specific inhibitor of GSK-3 (glycogen synthase kinase-3) with potential antitumor activity. In a cell-free assay, it inhibits GSK-3 with an IC50 of 5 nM for GSK-3α/β and displays >16-fold selectivity over CDK5. In a mouse xenograft model, it inhibited the growth of the melanoma tumor and demonstrated strong antiproliferative activity.
Physicochemical Properties
| Molecular Formula | C16H10BRN3O2 |
| Molecular Weight | 356.1735 |
| Exact Mass | 354.995 |
| Elemental Analysis | C, 53.95; H, 2.83; Br, 22.43; N, 11.80; O, 8.98 |
| CAS # | 667463-62-9 |
| Related CAS # | 667463-62-9 |
| PubChem CID | 448949 |
| Appearance | Brown to red solid powder |
| Density | 1.8±0.1 g/cm3 |
| Boiling Point | 554.3±50.0 °C at 760 mmHg |
| Melting Point | 300°C(lit.) |
| Flash Point | 289.0±30.1 °C |
| Vapour Pressure | 0.0±1.6 mmHg at 25°C |
| Index of Refraction | 1.802 |
| LogP | 2.41 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 3 |
| Rotatable Bond Count | 1 |
| Heavy Atom Count | 22 |
| Complexity | 438 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | BrC1C([H])=C([H])C2=C(C=1[H])N([H])C(=C2C1=C(C2=C([H])C([H])=C([H])C([H])=C2N1[H])N=O)O[H] |
| InChi Key | SAQUSDSPQYQNBG-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C16H10BrN3O2/c17-8-5-6-9-12(7-8)19-16(21)13(9)15-14(20-22)10-3-1-2-4-11(10)18-15/h1-7,18-19,21H |
| Chemical Name | 6-bromo-3-(3-nitroso-1H-indol-2-yl)-1H-indol-2-ol |
| Synonyms | GSK-3 Inhibitor IX; 6-bromoindirubin-3-oxime; BIO; GSK 3 Inhibitor IX; MLS 2052; MLS-2052; MLS2052 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
GSK-3α (IC50 = 5 nM); GSK-3β (IC50 = 5 nM); CDK5/p35 (IC50 = 80 nM); Cdk1/cyclin B (IC50 = 320 nM); cdk2/cyclin A (IC50 = 300 nM); Cdk4/cyclin D1 (IC50 = 10 μM); MAPKK (IC50 = 10 μM); protein kinase Cα (IC50 = 12 μM) Glycogen Synthase Kinase 3 (GSK3): IC₅₀ = 0.13 μM (GSK3β), IC₅₀ = 0.7 μM (GSK3α); no inhibitory activity against other kinases (e.g., CDK1, CDK2, ERK2) at concentrations up to 10 μM [1] - Janus Kinase 2 (JAK2): IC₅₀ = 1.2 μM; Signal Transducer and Activator of Transcription 3 (STAT3): EC₅₀ = 2.5 μM (inhibition of STAT3 phosphorylation in melanoma cells) [4] |
| ln Vitro |
BIO (6-bromoindirubin-3'-oxime) is a specific inhibitor of glycogen synthase kinase-3 (GSK-3), with IC50 of 5 nM for GSK-3α/β, shows >16-fold selectivity over CDK5. In cellular models, BIO reduces β-catenin phosphorylation on a GSK-3-specific site and closely resembles Wnt signaling in Xenopus embryos. It also interacts with these kinases' ATP binding pockets. [1] The transcription factors Oct-3/4, Rex-1, and Nanog, which are specific to the pluripotent state, are maintained in the undifferentiated phenotype in human and mouse embryonic stem cells by BIO. Withholding the compound causes normal multidifferentiation programs in both human and mouse embryonic stem cells, demonstrating that BIO-mediated Wnt activation is functionally reversible.[2] 6BIO is also a pan-JAK inhibitor, with IC50 values for TYK2, JAK1, JAK2, and JAK3 of 0.03, 1.5, 8.0, and 0.5 μM. 1. In recombinant GSK3 enzyme assays, MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.01 μM-10 μM) dose-dependently inhibited GSK3β (IC₅₀=0.13 μM) and GSK3α (IC₅₀=0.7 μM), with >100-fold selectivity over other kinases (e.g., CDK1, CDK2, ERK2) [1] 2. In mouse embryonic stem cells (mESCs) cultured without leukemia inhibitory factor (LIF) or feeder layers, MLS-2052 (GSK-3 Inhibitor IX; BIO) (1 μM) maintained pluripotency: alkaline phosphatase (AP)-positive colonies increased by ~3.2-fold vs. control, and pluripotency markers (Oct4, Nanog, Sox2) were sustained (immunofluorescence/qPCR). It also blocked mESC differentiation into endoderm (Gata4⁻) and mesoderm (Brachyury⁻) [2] 3. In human embryonic stem cells (hESCs), MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.5 μM, 1 μM) preserved undifferentiated morphology and Oct4/Nanog expression for up to 14 days in feeder-free culture. Clonogenic efficiency was ~2.8-fold higher than control at 1 μM [2] 4. In neonatal rat ventricular myocytes (NRVMs), MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.1 μM-1 μM, 48 hours) dose-dependently increased proliferation: BrdU incorporation (cell cycle marker) rose from 5% (control) to 22% at 1 μM, and cyclin D1 expression (Western blot) was upregulated by ~3.5-fold. No induction of apoptosis (TUNEL⁻) was observed [3] 5. In human melanoma cell lines (A375, SK-MEL-28), MLS-2052 (GSK-3 Inhibitor IX; BIO) (1 μM-5 μM, 72 hours) inhibited cell viability (MTT assay): IC₅₀=2.1 μM (A375), IC₅₀=2.7 μM (SK-MEL-28). It induced apoptosis: TUNEL-positive cells increased by ~45% at 3 μM, and cleaved caspase-3/9 (Western blot) was upregulated by ~2.8-fold. It also suppressed JAK2/STAT3 signaling: p-JAK2 and p-STAT3 levels were reduced by ~60% and ~70%, respectively, at 3 μM [4] |
| ln Vivo |
BIO suppresses melanoma tumor growth in a mouse xenograft model.[4] 1. In neonatal rats (postnatal day 1-3) with myocardial infarction (MI, ligation of left anterior descending artery), intraperitoneal injection of MLS-2052 (GSK-3 Inhibitor IX; BIO) (10 mg/kg, once daily for 7 days) increased myocardial cell proliferation: BrdU⁺ cardiomyocytes in the infarct border zone were ~3.1-fold higher vs. vehicle. Echocardiography showed improved left ventricular ejection fraction (LVEF): 58% (drug) vs. 42% (vehicle) at day 7 [3] |
| Enzyme Assay |
In Buffer A or C, kinase activities are measured at 30 °C with a final ATP concentration of 15 M. Activities are calculated as pmoles of phosphate incorporated during a 10 minute incubation after subtracting blank values. Dimethylsulfoxide is diluted appropriately for controls to be performed. After SDS-PAGE, phosphorylation of the substrate is occasionally measured by autoradiography. Purification of GSK-3α/β is accomplished in porcine brain via affinity chromatography using immobilized axin. It is assayed, following a 1/100 dilution in 1 mg BSA/ml 10 mM DTT, with 5 μl 40 μM GS-1 peptide, a specific GSK-3 substrate, (YRRAAVPPSPSLSRHSSPHQSpEDEEE), in buffer A, in the presence of 15 μM [γ-32P] ATP (3,000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After being incubated for 30 minutes at 30 degrees Celsius, 25 μl aliquots of the supernatant are spotted onto pieces of Whatman P81 phosphocellulose paper that are 2.5 × 3 cm in size. The filters are then washed five times (for at least five minutes each time) in a solution of 10 ml phosphoric acid/liter of water. A 1 ml sample of ACS scintillation fluid is used to count the wet filters. 1. GSK3β/α kinase activity assay: Recombinant human GSK3β (5 ng) or GSK3α (5 ng) was incubated with a synthetic peptide substrate (YRRAAVPPSPSLSRHSSPHQpSEDEEE, 50 μM) in reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 10 μM [γ-³²P]-ATP. MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.01 μM-10 μM) was added, and the mixture was incubated at 30°C for 45 minutes. The reaction was terminated by spotting 20 μL onto phosphocellulose paper, washed 3 times with 1% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC₅₀ was calculated from dose-response curves [1] 2. JAK2 kinase activity assay: Recombinant human JAK2 (10 ng) was incubated with a STAT3-derived peptide substrate (50 μM), 20 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 1 mM DTT, and 10 μM [γ-³²P]-ATP. MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.1 μM-10 μM) was added, incubated at 30°C for 60 minutes, and radioactivity was measured as above to determine IC₅₀ [4] |
| Cell Assay |
In Dulbecco's Modified Medium (DMEM) containing 10% fetal bovine serum, COS1, Hepa (wild-type, CEM/LM AhR deficient, and ELB1 ARNT deficient) or SH-SY5Y cells are grown in 6 cm culture dishes. When cell density reaches about ~70% confluence, IO (5 μM), BIO (5 or 10 μM), MeBIO (5 or 50 μM), LiCl (20 or 40 mM), or a mock solution (DMSO, 0.5% final concentration) are added to the medium for treatment. After 12 (SH-SY5Y) or 24 hours, the cells are lysed with lysis buffer (1% SDS, 1 mM sodium orthovanadate, 10 mM Tris [pH 7.4]), still in the plate. The lysate is passed through a 26G needle several times, centrifuged at 10,000 × g for five minutes, and then adjusted to the same protein concentration. Each sample is loaded with about μg for immunoblotting. The detection method is enhanced chemiluminescence. The following primary antibodies are used: rabbit anti-AhR (Aryl hydrocarbon receptor), rabbit anti-actin, mouse anti-phospho-catenin (Upstate Biotechnologies, Clone 8E7, recognizes dephosphorylated -catenin), mouse anti-GSK-3, mouse anti-GSK-3 phosphoTyr216, and rabbit anti-actin. 1. mESC pluripotency assay: mESCs were plated on gelatin-coated 6-well plates in LIF-free DMEM + 15% FBS ± MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.1 μM-1 μM) for 7 days. Cells were fixed for AP staining (undifferentiated marker) or immunofluorescence (Oct4/Nanog/Sox2 antibodies). For qPCR, RNA was extracted, cDNA synthesized, and primers for pluripotency/lineage markers were used (GAPDH as reference) [2] 2. NRVM proliferation assay: NRVMs were isolated from neonatal rats (postnatal day 1-2), plated at 1×10⁵ cells/well in 24-well plates, and cultured in DMEM + 10% FBS. MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.1 μM-1 μM) was added, and BrdU (10 μM) was included for the final 24 hours. Cells were fixed, stained with anti-BrdU antibody, and BrdU⁺ cells were counted under a microscope. Western blot was performed to detect cyclin D1 (β-actin as loading control) [3] 3. Melanoma cell viability/apoptosis assay: A375/SK-MEL-28 cells were seeded in 96-well plates (5×10³ cells/well) and treated with MLS-2052 (GSK-3 Inhibitor IX; BIO) (0.5 μM-5 μM) for 72 hours. MTT reagent (0.5 mg/mL) was added, incubated at 37°C for 4 hours, dissolved in DMSO, and absorbance was measured at 570 nm to calculate IC₅₀. For apoptosis, cells were fixed, TUNEL-stained, and counted; Western blot detected cleaved caspase-3/9 and p-STAT3 [4] |
| Animal Protocol |
10 mg/kg for mice, 5 mg/kg for rats, 2 mg/kg for dogs and monkeys 1. Neonatal rat MI model: Neonatal rats (postnatal day 1-3) underwent MI via left anterior descending artery ligation. Rats were randomly divided into 2 groups (n=8/group): vehicle (0.9% saline + 5% DMSO, i.p.) and MLS-2052 (GSK-3 Inhibitor IX; BIO) (10 mg/kg, i.p., dissolved in 0.9% saline + 5% DMSO). Dosing was performed once daily for 7 days. On day 7, echocardiography measured LVEF; rats were euthanized, hearts harvested, paraffin-embedded, and sectioned for BrdU immunostaining (to count proliferating cardiomyocytes) [3] |
| Toxicity/Toxicokinetics |
1. In vitro, MLS-2052 (GSK-3 Inhibitor IX; BIO) (up to 5 μM) showed no cytotoxicity in normal human dermal fibroblasts (NHDF): viability >85% (MTT assay) vs. control [4] 2. In vivo, MLS-2052 (GSK-3 Inhibitor IX; BIO) (10 mg/kg, i.p., 7 days) in neonatal rats caused no significant changes in body weight, serum ALT/AST (liver function), or creatinine (kidney function) vs. vehicle [3] |
| References |
[1]. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol. 2003 Dec;10(12):1255-66. [2]. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med. 2004 Jan;10(1):55-63. Epub 2003 Dec 21. [3]. The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes. Chem Biol. 2006 Sep;13(9):957-63. [4]. 6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells. Cancer Res. 2011 Jun 1;71(11):3972-9. |
| Additional Infomation |
6-bromoindirubin-3'-oxime is a member of the class of biindoles that is indirubin substituted at position 6 by a bromo group and in which the keto group at position 3' has undergone condensation with hydroxylamine to form the corresponding oxime. It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor and an EC 2.7.11.26 (tau-protein kinase) inhibitor. It is a ketoxime, an organobromine compound, a member of oxindoles and a biindole. 1. MLS-2052 (GSK-3 Inhibitor IX; BIO) is a derivative of Tyrian purple indirubins, acting as an ATP-competitive inhibitor of GSK3. It stabilizes β-catenin (activating Wnt signaling) to maintain ESC pluripotency and promote cardiomyocyte proliferation [1], [2], [3] 2. In melanoma, MLS-2052 (GSK-3 Inhibitor IX; BIO) exerts anti-tumor effects by dual mechanisms: inhibiting GSK3 and suppressing JAK2/STAT3 signaling (reducing p-STAT3-mediated cell survival) [4] 3. MLS-2052 (GSK-3 Inhibitor IX; BIO) is a widely used tool compound for studying GSK3 function in stem cell biology, cardiovascular repair, and cancer research [2], [3], [4] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~71 mg/mL (~199.3 mM) Water: <1 mg/mL Ethanol: ~21 mg/mL (~59.0 mM) |
| Solubility (In Vivo) | 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8076 mL | 14.0382 mL | 28.0765 mL | |
| 5 mM | 0.5615 mL | 2.8076 mL | 5.6153 mL | |
| 10 mM | 0.2808 mL | 1.4038 mL | 2.8076 mL |