BIO

Biological Activity

Description	BIO is a specific inhibitor of GSK-3 with IC50 of 5 nM for GSK-3α/β in a cell-free assay, shows >16-fold selectivity over CDK5, also a pan-JAK inhibitor.
Targets	GSK-3 [1] (Cell-free assay)	TYK2 [4] (Cell-free assay)	CDK5/p35 [1] (Cell-free assay)	CDK2/CyclinA [1] (Cell-free assay)	View  More
IC50	5 nM	30 nM	0.08 μM	0.30 μM
In 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. BIO interacts within the ATP binding pocket of these kinases, reduces β-catenin phosphorylation on a GSK-3-specific site in cellular models, closely mimicks Wnt signaling in Xenopus embryos. [1] In human and mouse embryonic stem cells, BIO maintains the undifferentiated phenotype and sustains expression of the pluripotent state-specific transcription factors Oct-3/4, Rex-1 and Nanog. BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both human and mouse embryonic stem cells. [2] BIO promotes proliferation in mammalian cardiomyocytes. [3]6BIO is also a pan-JAK inhibitor, with IC50 values of 0.03, 1.5, 8.0, 0.5 μM for TYK2, JAK1, JAK2 and JAK3. BIO selectively inhibits phosphorylation of STAT3 and induces apoptosis of human melanoma cells. [4]
In vivo	BIO suppresses melanoma tumor growth in a mouse xenograft model. [4]
Features	The first pharmacological agent shown to maintain self-renewal in human and mouse embryonic stem cells.

Protocol(Only for Reference)

Kinase Assay: ^[1]

Kinase assay

Kinase activities are assayed in Buffer A or C at 30°C, at a final ATP concentration of 15 μM. Blank values are subtracted and activities calculated as pmoles of phosphate incorporated during a 10 min incubation. Controls are performed with appropriate dilutions of dimethylsulfoxide. In a few cases phosphorylation of the substrate is assessed by autoradiography after SDS-PAGE. GSK-3α/β is purified from porcine brain by affinity chromatography on 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 30 min incubation at 30°C, 25 μl aliquots of supernatant are spotted onto 2.5 × 3 cm pieces of Whatman P81 phosphocellulose paper, and 20 seconds later, the filters are washed five times (for at least 5 min each time) in a solution of 10 ml phosphoric acid/liter of water. The wet filters are counted in the presence of 1 ml ACS scintillation fluid.

Cell Assay: ^[1]

Cell lines	COS1, Hepa or SH-SY5Y cells
Concentrations	~10 μM
Incubation Time	12 or 24 h
Method	COS1, Hepa (wild-type, CEM/LM AhR deficient and ELB1 ARNT deficient), or SH-SY5Y cells are grown in 6 cm culture dishes in Dulbecco's Modified Medium (DMEM) containing 10% fetal bovine serum. For treatment, IO (5 μM), BIO (5 or 10 μM), MeBIO (5 or 50 μM), LiCl (20 or 40 mM), or mock solution (DMSO, 0.5% final concentration) is added to medium when cell density reaches ∼70% confluence. After 12 (SH-SY5Y) or 24 hours, the cells, while still in plate, are lysed with lysis buffer (1% SDS, 1 mM sodium orthovanadate, 10 mM Tris [pH 7.4]). The lysate is passed several times through a 26G needle, centrifuged at 10,000 × g for 5 min, and adjusted to equal protein concentration. About 8 μg of each sample is loaded for immunoblotting. Enhanced chemiluminescence is used for detection. The following primary antibodies are used: mouse anti-β-catenin CT (Upstate Biotechnolgies, Clone 7D8, recognizes total β-catenin), mouse anti-phospho-β-catenin (Upstate Biotechnologies, Clone 8E7, recognizes dephosphorylated β-catenin), mouse anti-GSK-3 β, mouse anti-GSK-3 phosphoTyr216, rabbit anti-AhR (Aryl hydrocarbon receptor), and rabbit anti-actin.

Animal Study: ^[4]

Animal Models	mouse
Formulation	freshly prepared in 30% Solutol (Basf) at a concentration of 10 mg/mL.
Dosages	50 mg/kg
Administration	Oral gavage

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

1

References

[1] Meijer L, et al. Chem Biol, 2003, 10(12), 1255-1266.

[2] Sato N, et al. Nat Med, 2004, 10(1), 55-63.

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Clinical Trial Information( data from http://clinicaltrials.gov, updated on 2016-07-30)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT02753439	Not yet recruiting	Impacted Third Molar Tooth	The Baruch Padeh Medical Center, Poriya|Geistlich Pharma AG	February 2017	--
NCT02155764	Not yet recruiting	Tooth Extraction|Atrophy of Edentulous Alveolar Ridge	I.M. Sechenov First Moscow State Medical University|Centr  ...more I.M. Sechenov First Moscow State Medical University|Central Scientific Research Institute of Dentistry and Maxillo-facial Surgery, Moscow, Russia|Bionova, Skolkovo Community, Russia	November 2016	Phase 2
NCT02736565	Not yet recruiting	Ewings Sarcoma	Strike Bio, Inc.|Gradalis, Inc.	September 2016	Phase 1
NCT02844270	Not yet recruiting	Heart Diseases	Shanghai Bio-heart Biological Technology Co., Ltd.	July 2016	Phase 1
NCT02802319	Recruiting	Alveolar Bone Loss	The University of Texas Health Science Center at San Antonio	July 2016	--

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Chemical Information

Download BIO SDF

Chemical Name	2H-Indol-2-one, 6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihydro-, (3Z)-

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