Wortmannin (SL-2052; KY-12420)

PI3K (IC50 = 3 nM); DNA-PK (IC50 = 16 nM); PLK3 (IC50 = 48 nM); ATM (IC50 = 150 nM); ATR (IC50 = 1.8 μM); MLCK (IC50 = 200 nM)

Wortmannin (0-100 nM; 24-72 hours) inhibits the proliferation of K562 cells in a time- and dose-dependent manner. The IC50 values at 24 hours, 48 hours, and 72 hours, respectively, are 25±0.10 nM, 12.5±0.08 nM, and 6.25±0.11 nM[4]. YAP cannot enter the nucleus because of Wortmannin[6].

Wortmannin (One group of eight Scid mice receives a daily oral gavage dose of Wortmannin 1 mg/kg for a total of 14 days. For the first five days, the second group of eight mice receives wortmannin 1.5 mg/kg, and then the dose is lowered to 1 mg/kg for the remainder of the treatment period) treatment significantly slows the growth of human MCF-7 breast cancer xenograft and murine C3H mammary tumor. In mice with established murine C3H mammary tumors, a dose of 1 mg/kg Wortmannin for 7 days reduced tumor burdens by 54% compared to controls. After 14 days of 1 mg/kg Wortmannin treatment starting one day after tumor implantation, the burdens of human MCF-7 breast cancer xenografts are reduced by 97% in comparison to controls[5].

MLCK activity is assayed with peptide substrate (KKRPQRATSNVFS-NH2) or myosin light chain. The peptide substrate (24 μM) is phosphorylated in a reaction mixture containing 25 mM Tris-HC1 (pH 7.5), 0.5 mg/mL bovine serum albumin, 4 mM MgCl2, 0.5 mM CaCl2, 2.6 nM calmodulin, 1.5 nM MLCK, and 400 μM ATP in a final volume of 0.25 mL. After a 10-min preincubation at 28 ºC without ATP, the reaction is started by addition of ATP at 28 ºC and terminated by the addition of 0.1 mL of 10% (v/v) acetic acid after 30 min. The peptide substrate (24 μM) is phosphorylated in a reaction mixture with a final volume of 0.25 mL and the following ingredients: 25 mM Tris-HC1 (pH 7.5), 0.5 mg/mL bovine serum albumin, 4 mM MgCl2, 0.5 mM CaCl2, 2.6 nM calmodulin, 1.5 nM MLCK, and 400 μM ATP. The reaction is started by adding ATP at 28 ºC after a 10-min preincubation at that temperature without ATP. It is finished off by adding 0.1 mL of 10% (v/v) acetic acid at that temperature after 30 minutes. The mixture is analyzed by high performance liquid chromatography: column, Unisil Pack 5C18 4.6 X 150 mm; solvent, 18% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid in water; flow rate, 1.0 mL/min; temperature, 40 ºC; detection, absorbance at 220 nm. High performance liquid chromatography is used to analyze the mixture: column, Unisil Pack 5C18, 4.6 X 150 mm; solvent, 18% (v/v) acetonitrile; and 0.1% (v/v) trifluoroacetic acid in water; flow rate, 1.0 mL/min; temperature, 40 oC; and detection, absorbance at 220 nm. The ratio of the peak areas of the phosphorylated form to those of the unphosphorylated form is used to calculate the percentage of the reaction. The specific activity measured under the aforementioned circumstances is 0.81 μmol/min/mg. Myosin light chain (108 μg/mL) is phosphorylated in a reaction mixture with 25 mM Tris-HC1 (pH 7.5), 0.5 mg/mL bovine serum albumin, 4 mM MgCl2, 0.5 mg/mL CaCl2, 4.2 nM calmodulin, 0.92 nM enzyme, and 10 μM[γ-32P]ATP (100-900 cpm/pmol). The reaction is initiated by the addition of [γ-32P]ATP at 30 ºC after a 3-min preincubation without ATP, and is terminated by the addition of 0.125 mL of trichloroacetic acid after 5 min. After being collected on a nitrocellulose membrane filter, the acid-precipitable materials are washed with four 1-mL aliquots of 5% (v/v) trichloroacetic acid. Using a Packard Tri-Carb liquid scintillation spectrometer Model 4530 and a toluene scintillation fluid, the radioactivity on the filter is measured. 1.23 μmol/min/mg is the specific activity as determined by the conditions.

Primary NK cells were pretreated with wortmannin (1 μM) for 1 h, washed twice with RPMI 1640, and then treated with IL-15 (10 ng/mL) for 24 h. DMSO was used as control.

[1]. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J Biol Chem. 1993 Dec 5;268(34):25846-56.

[2]. Autophagy inhibitors as a potential antiamoebic treatment for Acanthamoeba keratitis. Antimicrob Agents Chemother. 2015 Jul;59(7):4020-5.

[3]. Polo-like kinases inhibited by wortmannin. Labeling site and downstream effects. J Biol Chem. 2007 Jan 26;282(4):2505-11.

[4]. Wortmannin inhibits K562 leukemic cells by regulating PI3k/Akt channel in vitro. J Huazhong Univ Sci Technolog Med Sci. 2009 Aug;29(4):451-6.

[5]. Wortmannin inhibits the growth of mammary tumors despite the existence of a novel wortmannin-insensitive phosphatidylinositol-3-kinase. Cancer Chemother Pharmacol. 1999;44(6):491-7.

[6]. Wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalianpolo-like kinase. Chem Biol. 2005 Jan;12(1):99-107.

C23H24O8

428.43

428.14712

C, 64.48; H, 5.65; O, 29.88

19545-26-7

White to light yellow solid powder

CC(O[C@@H](C1=C2C(C3=C4C(C(O[C@H](COC)[C@]14C)=O)=CO3)=O)C[C@]5(C)C(CC[C@]52[H])=O)=O

QDLHCMPXEPAAMD-QAIWCSMKSA-N

InChI=1S/C23H24O8/c1-10(24)30-13-7-22(2)12(5-6-14(22)25)16-18(13)23(3)15(9-28-4)31-21(27)11-8-29-20(17(11)23)19(16)26/h8,12-13,15H,5-7,9H2,1-4H3/t12-,13+,15+,22-,23-/m0/s1

(1S,6bR,9aS,11R,11bR)-1-(methoxymethyl)-9a,11b-dimethyl-3,6,9-trioxo-3,6,6b,7,8,9,9a,10,11,11b-decahydro-1H-furo[4,3,2-de]indeno[4,5-h]isochromen-11-yl acetate.

SL 2052; SL2052; SL-2052; Wortmannin

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: ~85 mg/mL (198.4 mM)
Water: <1 mg/mL (slightly soluble or insoluble)
Ethanol: <1 mg/mL

Solubility in Formulation 1: 2.08 mg/mL (4.85 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.85 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 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.85 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.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 8mg/mL

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