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].

In vitro PtdIns 3-kinase assays [5]
C3H tumor, brain, liver and kidney were harvested from control and Wortmannin-treated mice. The tissues were homogenized using a polytron homogenizer in three volumes of lysis bu€er (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1 mM MgCl2, 1 mM CaCl2, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl ¯uoride (PMSF), 2 lg ml)1 leupeptin and 2 lg ml)1 aprotinin) at 4 °C. Cells were washed with phosphate-bu€ered saline (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl pH 7.0) and incubated at 4 °C for 20 min in 3 ml lysis bu€er. Tissue homogenates and cell lysates were centrifuged at 16 000 g for 5 min and the supernatant diluted to a ®nal protein concentration of 0.01 to 0.1 lg protein ll )1 in 20 mM Tris-HCl, pH 8.0, 137 mM NaCl and 1 mM EDTA. Prior to mixing reaction components, PtdIns 3-kinase inhibitors were dried into the reaction tubes from stocks prepared in 10 ll dimethylsulfoxide. Incubations contained 30 ll of diluted crude, partially puri®ed or recombinant PtdIns 3-kinase preparations with 10 ll PtdIns or D-3-deoxy-PtdIns (0.5 mg ml)1 in 20 mM HEPES, pH 7.6) as substrate. Reactions were initiated by the addition of 10 lM [ 32P]-ATP (1 Ci/mmol). Samples were incubated at 37 °C for 45 min, quenched by the addition of 100 ll 1 N HCl and extracted with 400 ll of 1:1 chloroform/methanol then centrifuged at 2000 g for 1 min. For each sample, 25 ll of the lower organic phase was spotted onto the preabsorbent strip of an individual lane in a multichannel thin-layer chromatography Silica gel 60 plate . Plates were developed in 65% n-propyl alcohol/35% 2 M acetic acid. [ 32P]-Labeled product was quantitated by phosphorimager analysis. The absence of a hydroxyl group at the 3¢ position of PtdIns in 3¢-deoxy-PtdIns prevents 3¢-phosphorylation and allows the measurement of non-PtdIns 3-kinase activity [12]. PtdIns 3-kinase activity was determined by subtracting the activity detected in D-3-deoxy-PtdIns-containing samples from that in corresponding PtdIns-containing samples. Values expressed as PtdIns-kinase activity signify phosphorylations at all possible positions of PtdIns. Values expressed in PtdIns 3-kinase activity signify phosphorylation only at the 3¢-OH position of PtdIns. Percent inhibition of enzyme activity was calculated from the quotient of the mean treated value divided by the mean control value.

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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.

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The surface engagement of high affinity immunoglobulin E receptor (Fc epsilon RI) of rat basophilic leukemia 2H3 (RBL-2H3) cells induced histamine secretion and leukotriene release following activation of the tyrosine kinase Lyn together with phosphatidylinositol 3-kinase (PI3-kinase). Wortmannin inhibited the activity of partially purified PI3-kinase from calf thymus, as well as the PI3-kinase activity in anti-PI3-kinase p85 immunoprecipitates from RBL-2H3 cells, at a concentration as low as 1.0 nM and with IC50 values of 3.0 nM, but did not inhibit PI4-kinase activity. The inhibition of PI3-kinase by wortmannin was irreversible. Wortmannin inhibited both Fc epsilon RI-mediated histamine secretion and leukotriene release up to 80% with IC50 values of 2.0 and 3.0 nM, respectively. Wortmannin inhibited PI3-kinase activity in intact cells up to 80% with an IC50 value of 2.0 nM, which is almost equal to those for PI3-kinase in vitro and for histamine secretion and leukotriene release. With anti-wortmannin antibody, we have shown that wortmannin binds to the 110-kDa protein, but not to PI3-kinase 85-kDa regulatory subunit both in vitro and in whole cells. Furthermore, there was a positive correlation between the potencies of wortmannin derivatives as inhibitors of PI3-kinase and as inhibitors of histamine secretion. Wortmannin had no effect on the activation of the tyrosine kinase Lyn. These results suggest that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion following stimulation of Fc epsilon RI and that wortmannin blocks these responses through direct interaction with the catalytic subunit of this enzyme[1].

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The inhibitory effect of wortmannin on leukemic cells and the possible mechanisms were examined. K562 cells were treated with wortmannin of various concentrations (3.125-100 nmol/L) for 0-72 h. MTT assay was used to evaluate the inhibitory effect of wortmannin on the growth of K562 cells. Cell apoptosis was detected by both Annexin-V FITC/PI double-labeled cytometry and transmission electron microscopy (TEM). The expression of p-Akt, T-p-Akt, NF-kappaBp65 and IKK-kappaB was determined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that wortmannin obviously inhibited growth and induced apoptosis of K562 cells in vitro in a time- and dose-dependent manner. The IC(50) value of wortmannin for 24 h was 25+/-0.14 nmol/L. Moreover, wortmannin induced K562 cells apoptosis in a dose-dependent manner. TEM revealed typical morphological changes of apoptosis in wortmannin-treated K562 cells, such as chromatin condensation, karyopyknosis, karyorhexis and apoptotic bodies. Additionally, several important intracellular protein kinases such as p-Akt, NF-kappaBp65 and IKK-kappaB experienced degradation of various degrees in a dose-dependent manner both at protein level and transcription level when cultured with wortmannin, but the expression of total Akt showed no change. It is concluded that wortmannin can inhibit the proliferation and induce apoptosis of K562 leukemia cells possibly by down-regulating the survival signaling pathways (PI3K/Akt and NF-kappaB channels)[4].

Antitumor activity studies [5]
Scid mice were implanted s.c. with a 0.25-mg 21-day release 17- estradiol pellet and 24 h later inoculated with the C3H and MCF-7 hormonedependent breast cancer cell lines. Additional 17-estradiol pellets were implanted every 21 days for the duration of the study. Murine mammary C3H tumor fragments were implanted s.c. in the right mammary fat pad of 20-g female C3H mice as previously described. MCF-7 cells (10~7 ) were injected s.c. into the hind quarters of the mice. Tumor burdens were assessed from two-dimensional measurements taken three times a week during the course of study. Tumor volumes were calculated with the formula: Tumor volume …ml† ˆ Tumor length …cm† ‰Tumor width …cm†Š2 =2 In studies where only ®nal masses were reported, tumors were excised and weighed after the completion of the study period. Wortmannin was administered to mice by oral gavage as a 0.1 mg ml)1 solution in 0.1% Tween 20 with 10% ethanol at daily doses of 1 or 1.5 mg kg)1 for up to 14 days. A

Solubilized in 0.4 mg/mL in DMSO, and diluted with 0.9% NaCl before use; 0.175, 0.35, and 0.7mg/kg;i.v.

Human pancreatic adenocarcinoma cells PK1 are injected both s.c. and orthotopically into SCID mice.

[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.

Wortmannin is an organic heteropentacyclic compound belonging to the δ-lactone, acetate, and cyclic ketone classes. It possesses diverse biological activities, including as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor, an anticoronavirus agent, an anti-aging agent, an autophagy inhibitor, a Penicillium metabolite, a radiosensitizer, and an antitumor agent. Wortmannin is a steroidal metabolite of Penicillium funiculosum and Talaromyces wortmannii. This drug is a nonspecific covalent phosphatidylinositol 3-kinase (PI3K) inhibitor. Wortmannin has been discovered in Talaromyces wortmannii, and relevant data exist. Wortmannin is a potent fungal metabolite isolated from Penicillium wortmannii that selectively inhibits phosphatidylinositol 3-kinase and affects signal transduction pathways. (NCI)
An androstenediene metabolite produced by Penicillium funiculosum, it inhibits phosphatidylinositol 3-kinase and suppresses allogeneic antigen-specific activation of T lymphocytes in human tumor cell lines. It is widely used in cell biology research and has broad therapeutic potential.

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Binding to the high-affinity immunoglobulin E receptor (FcεRI) on the surface of rat basophilic leukemia 2H3 (RBL-2H3) cells induces histamine secretion and leukotriene release upon activation by tyrosine kinase Lyn and phosphatidylinositol 3-kinase (PI3-kinase). Watermanin inhibits the activity of partially purified PI3-kinase from calf thymus and the PI3-kinase activity in RBL-2H3 cell anti-PI3-kinase p85 immunoprecipitate at concentrations as low as 1.0 nM, with an IC50 value of 3.0 nM, but does not inhibit PI4-kinase activity. The inhibition of PI3-kinase by Wottermanin is irreversible. Wottermanin exhibits an 80% inhibition rate against FcεRI-mediated histamine secretion and leukotriene release, with IC50 values of 2.0 nM and 3.0 nM, respectively. Wottermanin also shows an 80% inhibition rate against PI3-kinase activity in intact cells, with an IC50 value of 2.0 nM, almost identical to the IC50 values for PI3-kinase activity, histamine secretion, and leukotriene release in vitro. Using an anti-Wottermanin antibody, we confirmed that Wottermanin binds to a 110 kDa protein in vitro and in intact cells, but not to the 85 kDa regulatory subunit of PI3-kinase. Furthermore, the potency of Wottermanin derivatives as PI3-kinase inhibitors and histamine secretion inhibitors is positively correlated. Wottermanin has no effect on the activation of tyrosine kinase Lyn. These results indicate that PI3-kinase is involved in the signal transduction pathway of histamine secretion after FcεRI stimulation, and Wottermanin blocks these responses by directly interacting with the catalytic subunit of this enzyme. [1]

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Polo-like kinases play a crucial role in mitosis. We previously reported that Wortmannin effectively inhibits Polo-like kinase 1 (Plk1). In this study, we found that Wortmannin also strongly inhibits Polo-like kinase 3 (Plk3). To further characterize this inhibition, we identified the labeling sites of the tetramethylrhodamine-Wortmannin conjugate AX7503 on Plk1 and Plk3. We found that the labeling of AX7503 on Plk1 and Plk3 occurred at conserved ATP-binding site residues. Furthermore, we found that Wortmannin inhibited Plk3 activity in living cells at concentrations commonly used to inhibit Wortmannin targets such as phosphatidylinositol 3-kinase. Importantly, we found that the inhibition of Plk3 by Wortmannin led to a decrease in the phosphorylation level of p53 serine 20 induced by DNA damage, indicating that Wortmannin acts on a downstream target of Plk3. In summary, our findings suggest that Wottermanin can affect multiple functions of Plk3 in cell cycle progression and DNA damage checkpoints. Identifying the labeling sites of Plk1 and Plk3 using AX7503 may help design more effective cancer therapeutic compounds targeting Polo-like kinases, and may also help in the structural study of Plk domains. [3]

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Objective: Phosphatidylinositol (PtdIns) 3-kinase is an important mediator of many cellular functions. Research on PtdIns 3-kinase has been promoted due to the existence of Wottermanin, a potent and irreversible p110 PtdIns 3-kinase inhibitor. This study aimed to investigate the relationship between the cell growth inhibitory activity and antitumor activity of Wottermanin and the inhibition of phosphatidylinositol 3-kinase (PtdIns 3-Kase). Methods: PtdIns 3-Kase activity in cells and tumor tissues was detected, and the role of Wottermanin was studied. Results: Wottermanin inhibited the growth of mouse C3H and human MCF-7 breast tumors in vivo. However, the ability of Watmanin to inhibit C3H tumor growth was independent of the inhibition of tumor PtdIns 3-Kase activity. Watmanin-insensitive PtdIns 3-Kase activity was detected in C3H and MCF-7 cell culture lysates, solid tumors, and normal mouse tissue homogenates. In addition to resistance to Watmanin inhibition, total PtdIns 3-kinase activity in MCF-7 cell lysates was also resistant to five other known p110 PtdIns 3-kinase inhibitors. Partial purification of Watmanin-insensitive PtdIns 3-kinase from MCF-7 cell lysates showed that its activity was independent of the PtdIns 3-kinase p85 regulatory subunit. Conclusion: The results of this study indicate that despite the presence of a novel PtdIns 3-kinase that is insensitive to Wottermanin in mouse and human breast tissues, Wottermanin was still able to inhibit the growth of these tumors, suggesting that the antitumor activity of Wottermanin may be mediated by other targets. [5]

C23H24O8

428.43

428.147

C, 64.48; H, 5.65; O, 29.88

19545-26-7

312145

White to light yellow solid powder

1.4±0.1 g/cm3

615.6±55.0 °C at 760 mmHg

234-240ºC

326.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.591

0.81

0

8

4

31

921

5

O(C(C([H])([H])[H])=O)[C@]1([H])C([H])([H])[C@]2(C([H])([H])[H])C(C([H])([H])C([H])([H])[C@@]2([H])C2C(C3=C4C(=C([H])O3)C(=O)O[C@]([H])(C([H])([H])OC([H])([H])[H])[C@]4(C([H])([H])[H])C=21)=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; 19545-26-7; Wartmannin; KY 12420; Antibiotic SL-2052; SL-2052; MFCD00133927; NSC221019;

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.)