GSK-3β (IC50 = 30 nM)
Target: GSK-3β (IC50 = 30 nM for kinase inhibitory activity; KD = 126 ± 11 nM by SPR)[1]

Treatment of a monolayer of P19 cells with 1 μM TWS119 causes 30–40% cells to differentiate specifically into neuronal lineages based on counting of TuJ1 positive cells with correct neuronal morphology (up to 60% neuronal differentiation occurred through the standard EB formation protocol with concomitant TWS119 treatment). Surface plasmon resonance (SPR) measurements of TWS119's strong GSK-3-binding affinity (K D = 126 nM) and IC50 of 30 nM further support this conclusion. [1] WS119 is found to potently induces neuronal differentiation in both mouse embryonal carcinoma and ES cells. [2] TWS119 treatment towards hepatic stellate cells (HSC) leads to reduced b-catenin phosphorylation, induces nuclear translocation of b-catenin, elevates glutamine synthetase production, impedes synthesis of smooth muscle actin and Wnt5a, but promotes the expression of glial fibrillary acidic protein, Wnt10b, and paired-like homeodomain transcription factor 2c.[3] TWS119 causes a sharp up-regulation of the expression of Tcf7, Lef1, and other Wnt target genes like Jun, Ezd7 (encoding Frizzled-7), and Nlk (encoding Nemo-like kinase) in addition to triggering a rapid accumulation of -catenin (mean 6.8-fold increase by densitometry). TWS119 causes a dose-dependent reduction in T cell-specific killing and IFN-g release along with the maintenance of IL-2 production.[4] In polyclonally activated human T cells, treatment with TWS119 induces Wnt signaling, according to a recent study. Unlike control-activated T cells, which develop a CD45RO(+)CD62L(-) effector phenotype in a TWS119 dose-dependent manner, these T cells maintain a native CD45RA(+)CD62L(+) phenotype. Because cell division is prevented by TWS119-induced Wnt signaling, T cell expansion is reduced. Additionally, degranulation and IFN- production in response to T cell activation—which are both indicators of T cell effector function—are impaired. The inability of TWS119-treated T cells to use autocrine IL-2 for expansion may be the cause of the block in T cell division. This is because TWS119 treatment reduces the expression of the IL-2R. [5]

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In Vitro: TWS119 (1-5 μM) induced 30-40% of P19 cells to differentiate into neurons (TuJ1-positive) with characteristic neuronal morphology in monolayer culture; up to 60% neuronal differentiation was achieved with embryoid body formation protocol.
Treatment with TWS119 for 2 days followed by compound-free medium for 2 days increased neuronal percentage to 40-60%, suggesting extended exposure to early differentiation signals is unfavorable for late-stage neuronal maturation.
Immunofluorescence staining showed that TuJ1-positive cells expressed nestin (neural progenitor marker), neurofilament-M, Map2(a+b), NeuN, glutamate, and synapsin I after longer incubation (up to 2 weeks in B27-supplemented neurobasal medium).
In mouse ESCs (D3 cell line), TWS119 at 400 nM induced approximately 50-60% neuronal differentiation as indicated by TuJ1, Map2(a+b), and neurofilament-M staining.
Western blot analysis showed that TWS119 (3 μM, 24 h) increased β-catenin levels in P19 cells compared to inactive control TWS121.
TCF/LEF reporter assay showed that TWS119 caused activation of β-catenin-induced TCF/LEF reporter activity in a dose-dependent manner with an 11-fold increase at 10 μM after 36 h treatment.
Affinity chromatography with immobilized TWS119 specifically bound two protein bands at ~47 and 49 kDa identified as GSK-3β (confirmed by LC/MS and Western blot).[1]
TWS119 induces neuronal differentiation in both mouse embryonal carcinoma and ES cells by targeting GSK-3β.[2]

A cell population that expressed low levels of CD44 and high levels of CD62L on the cell surface when 30 mg/kg of TWS119 is administered.[4]

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Enzyme Assay: Affinity chromatography: P19 cells were lysed with PY buffer containing 0.4% Nonidet P-40. Protein extracts (300 μg) were added to affinity supports (AS-101, AS-102, AS-119, AS-113, AS-121) derived from active or inactive TWS analogs. After agitation at 4°C for 1 h, supports were washed three times with bead buffer. For competition assay, bead buffer containing 50 μM TWS119 was used. Bound proteins were eluted with SDS sample buffer, separated by 10% Tris-glycine SDS-PAGE, and visualized by silver staining or Western blot with anti-GSK-3β antibody. Two bands at ~47 and 49 kDa specifically bound to active analog-derived supports, and binding was blocked by free TWS119.[1]
Surface plasmon resonance (SPR): A GST-GSK-3β fusion protein was immobilized on S系列 CM5 biosensor chips via EDC coupling. TWS119 was injected for 90 s at increasing concentrations (0, 2, 4, 8, 16, 32, 62.5, 125, 250, 500, and 1,000 nM), and dissociation was followed for 300 s. A 1:1 binding model was assumed for determination of kinetic and thermodynamic binding constants. The average affinity (KD) for TWS119 binding to GSK-3β was determined as 126 ± 11 nM from four independent experiments.[1]
GSK-3β kinase inhibitory activity: TWS119 showed potent inhibition with IC50 = 30 nM (method details not fully described in the provided text).[1]

All rats are divided into four groups at random as follows: Sham group rats undergo the same surgical procedure, but the filament is not inserted, and they are given 1 mL of dimethyl sulfoxide (1% DMSO in saline); After MCAO, the rats in the vehicle group receive 1 mL of DMSO. At 4 hours after MCAO, the rats in the rtPA group receive rtPA (10 mg/kg, Actilyse®). At 4 hours after MCAO, the rats in the rtPA+TWS119 group receive intraperitoneal TWS119 (30 mg/kg, dissolved in 1 mL 1% DMSO).

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Cell Assay: P19 EC cells (clone P19Ta1Luc-17 stably transfected with pTα1-Luc reporter, containing 1.1 kb of the rat Tα1 tubulin 5'-flanking region) were plated in white 384-well plates at 2,000 cells per well in MEMα with 5% FBS. Compounds including TWS119 were added at 5 μM final concentration 12 h after plating. After 4 days, cells were lysed and luciferase activity measured. Primary hits were confirmed by direct immunostaining with βIII-tubulin/TuJ1 antibody and observation of neuronal morphology.[1]
Immunocytochemistry: Cells were fixed with 4% paraformaldehyde in PBS for 20 min. Primary antibodies used: βIII-tubulin (TuJ1) mouse monoclonal (1:500), rabbit polyclonal (1:2,000); microtubule-associated protein Map2(a+b) mouse monoclonal (1:1,000); neurofilament M mouse monoclonal (1:1,000); NeuN mouse monoclonal (1:100); nestin mouse monoclonal (1:1,000); synapsin I rabbit polyclonal (1:1,000); glutamate rabbit polyclonal (1:300). Secondary antibodies were Cy2- or Cy3-conjugated anti-rabbit or anti-mouse (1:500). Cells were imaged with a Nikon Eclipse TE2000 microscope at 200× magnification.[1]
TCF/LEF reporter assay: P19 cells in 100-mm dish were cotransfected with 6 μg of pTOPFASH (containing four consensus LEF-1/TCF-1 binding sites, a minimal promoter, and firefly luciferase reporter) and 3 μg of Renilla luciferase control reporter using FuGENE6. After 24 h, cells were trypsinized and replated into 96-well plates and treated with TWS119 (or RA in Petri dishes) in MEMα with 5% FBS 12 h post-plating. Thirty-six hours later, cells were lysed and luciferase activity assayed. Firefly luciferase activity was normalized to Renilla activity. TWS119 at 10 μM caused an 11-fold increase after 36 h.[1]
Phenotypic characterization: P19 cells were treated with 1-5 μM TWS119 for 2 days and cultured in compound-free MEMα supplemented with 2% FBS for an additional 2-14 days (B27-supplemented neurobasal medium for >10 days) before fixing and immunostaining. TuJ1-positive cells with neuronal morphology (round soma, asymmetric multiple processes) were observed. Most TuJ1-negative cells stained positive for nestin. No GFAP (glial marker) or MF20 (muscle marker) positive cells were detected.[1]
Mouse ESC differentiation: Undifferentiated D3 ESCs were cultured on gelatin-coated dishes with ESC growth medium containing 15% serum replacement and LIF. Cells were treated with 400 nM TWS119 under similar conditions, and neuronal differentiation (50-60%) was confirmed by immunostaining for TuJ1, Map2(a+b), and neurofilament-M.[1]

All rats are divided into four groups at random as follows: Sham group rats undergo the same surgical procedure, but the filament is not inserted, and they are given 1 mL of dimethyl sulfoxide (1% DMSO in saline); After MCAO, the rats in the vehicle group receive 1 mL of DMSO. At 4 hours after MCAO, the rats in the rtPA group receive rtPA (10 mg/kg, Actilyse®). At 4 hours after MCAO, the rats in the rtPA+TWS119 group receive intraperitoneal TWS119 (30 mg/kg, dissolved in 1 mL 1% DMSO).

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[1]. Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7632-7.

[2]. Nat Biotechnol. 2004 Jul;22(7):833-40.

3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol is a member of the pyrrole class of compounds.

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Additional Info: TWS119 was identified from a screen of kinase-directed combinatorial libraries using a neuronal differentiation-specific luciferase reporter (pTα1-Luc) in P19 EC cells. The compound acts by inhibiting GSK-3β, leading to stabilization and nuclear translocation of β-catenin, which interacts with TCF/LEF DNA binding proteins to regulate transcription. This mechanism is involved in the Wnt signaling pathway. GSK-3β is a multifunctional serine/threonine kinase active in embryonic development, cell fate determination, transcriptional control, metabolism, oncogenesis, and neurological diseases. TWS119 can induce neurogenesis without embryoid body formation or retinoic acid treatment, suggesting a novel mechanism on early processes determining cell fate. The compound may promote neural induction or survival of neuronal progenitors via canonical Wnt signaling or other novel mechanisms.[1]
TWS119 is an example of a small molecule identified from cell-based phenotypic screens that can be used to selectively control stem cell proliferation and differentiation. It induces neuronal differentiation in murine ESCs and may provide insights into molecular mechanisms controlling stem cell fate, potentially useful for in vivo stem cell biology and therapy.[2]

C18H14N4O2

318.3294

318.111

C, 67.92; H, 4.43; N, 17.60; O, 10.05

601514-19-6

TWS119 TFA;1507095-58-0

9549289

Off-white to yellow solid powder

1.4±0.1 g/cm3

646.0±55.0 °C at 760 mmHg

344.5±31.5 °C

0.0±2.0 mmHg at 25°C

1.753

3.54

3

5

3

24

424

0

O(C1=C([H])C([H])=C([H])C(=C1[H])O[H])C1C2C([H])=C(C3C([H])=C([H])C([H])=C(C=3[H])N([H])[H])N([H])C=2N=C([H])N=1

VPVLEBIVXZSOMQ-UHFFFAOYSA-N

InChI=1S/C18H14N4O2/c19-12-4-1-3-11(7-12)16-9-15-17(22-16)20-10-21-18(15)24-14-6-2-5-13(23)8-14/h1-10,23H,19H2,(H,20,21,22)

3-((6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenol

TWS-119; TWS 119; TWS119

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: ~64 mg/mL (201.0 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.85 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 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.85 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 25.0 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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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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