TG003 (TG-003; TG 003) is a novel, potent, selective and ATP-competitive Cdc2-like kinase (Clk) inhibitor with potential anticancer activity. Its IC50 values for Clk1/2/4 inhibition are 20 nM, 200 nM, and 15 nM, respectively. TG003 exhibits no inhibitory action towards PKC, SRPK1, SRPK2, or Clk3.

Physicochemical Properties

Molecular Formula	C13H15NO2S
Molecular Weight	249.33
Exact Mass	249.082
Elemental Analysis	C, 62.62; H, 6.06; N, 5.62; O, 12.83; S, 12.86
CAS #	719277-26-6
Related CAS #	300801-52-9;719277-26-6
PubChem CID	1893668
Appearance	white solid powder
LogP	2.381
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	17
Complexity	329
Defined Atom Stereocenter Count	0
SMILES	CC(/C=C1SC2=CC=C(OC)C=C2N\1CC)=O
InChi Key	BGVLELSCIHASRV-QPEQYQDCSA-N
InChi Code	InChI=1S/C13H15NO2S/c1-4-14-11-8-10(16-3)5-6-12(11)17-13(14)7-9(2)15/h5-8H,4H2,1-3H3/b13-7-
Chemical Name	(1Z)-1-(3-ethyl-5-methoxy-1,3-benzothiazol-2-ylidene)propan-2-one
Synonyms	TG003; TG-003; TG 003
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	CLK1 (IC50 = 20 nM); CLK2 (IC50 = 200 nM); CLK4 (IC50 = 15 nM)
ln Vitro	TG003, exhibits the strongest effect on Clk1/Sty and Clk4 (IC50, 15–20 nM) and lesser on Clk2 (200 nM). TG003 suppresses Clk-mediated phosphorylation to prevent SF2/ASF-dependent splicing of β-globin pre-mRNA in vitro. In mammalian cells, it inhibits the phosphorylation of serine/arginine-rich proteins, the dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing[1]. Through modification of TP53 intron 9 alternative splicing, the small drug TG003 increases the endogenous expression of the protein isoforms p53β and p53β[2].
ln Vivo	TG003 (10 μM) corrects the embryonic defects caused by overactive Clk activity in Xenopus.
Enzyme Assay	In a reaction mixture containing 200 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 8 mM dithiothreitol, 4 mM EGTA, 1–20 μM ATP, 1 μCi of [γ–32P]ATP, 1 μg of synthetic peptide of SF2/ASF RS domain, and 0.1–1 μg of purified kinases in a final volume of 40 μL are used to measure the kinase activity of Clks and SRPKs. Regardless of the concentration of the inhibitor, the final concentration of Me2SO is adjusted to 1%. Half of the reaction mixture is spotted on P81 phosphocellulose membrane after it is incubated for 10 minutes at 30 °C for mammalian recombinant proteins and 25 °C for Xenopus recombinant proteins. The incubation time and substrate and kinase concentrations are among the parameters of the kinase assay that are optimized to preserve linearity. For at least fifteen minutes, the membrane is cleaned with either a 5% phosphoric acid solution or a 5% trichloroacetic solution. A liquid scintillation counter is utilized to quantify the radioactivity [1].
Cell Assay	Plate 2x105 HeLa or 1.5x105 COS-7 cells resuspended in 2 mL of medium on 6-well plates; in some wells, add 2 μL of 10 mM TG003 dissolved in Me2SO (final concentration at 10 mM) or 2 μL of Me2SO. Trypsinized cells are counted every 24 hours for three days, after which the density is determined. After fixing the cells in 1 mL of ice-cold 70% ethanol, they are next cleaned with PBS, incubated for 20 minutes at 37 °C in 1 mL of PBS containing 50 μg/mL propidium iodide and 1 μg/mL DNase-free RNase A, and then subjected to cell cycle analysis[1].
Animal Protocol	Intrathecal injection of either TG003 (1-100 pM) or IC261 (0.1-1 nM) dose-dependently decreases mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Xenopus
References	[1]. Manipulation of alternative splicing by a newly developed inhibitor of Clks. J Biol Chem. 2004 Jun 4;279(23):24246-54. [2]. Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response. Cell Death Differ. 2014 Sep;21(9):1377-87. [3]. Alleviation of behavioral hypersensitivity in mouse models of inflammatory pain with two structurally different casein kinase 1 (CK1) inhibitors. Mol Pain. 2014 Mar 10;10:17.

Solubility Data

Solubility (In Vitro)

DMSO: ~6 mg/mL (~24.1 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.34 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (8.34 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (8.34 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.0107 mL	20.0537 mL	40.1075 mL
	5 mM	0.8021 mL	4.0107 mL	8.0215 mL
	10 mM	0.4011 mL	2.0054 mL	4.0107 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.