Natural diterpenoid/fungal pytotoxin; 14-3-3 PPI/protein-protein interactions

Fusicoccin A can induce GCN1 renewal and neurite development (EC50=29 mM) by stabilizing the complex between 14-3-3 and the stress response regulator GCN1 [3]. Plants infected with Clostridin A experience wilt and water loss as a result of Clostridin A's stabilization of plasma membrane H+-ATPase's interaction with the 14-3-3 protein. Fusicoccin A inhibits human GBM cell line migration and proliferation in vitro, including a number of cell lines that show variable degrees of resistance to proapoptotic stimuli. Fusicoccin A's IC50 growth inhibitory concentration in Hs683 glioma cells is 83 µM, while it is 92 µM in U373-MG cells [4].

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fusicoccin A, a fungal metabolite from Fusicoccum amygdali, decreased the proliferation and migration of human GBM cell lines in vitro, including several cell lines that exhibit varying degrees of resistance to pro-apoptotic stimuli. The data demonstrate that fusicoccin A inhibits GBM cell proliferation by decreasing growth rates and increasing the duration of cell division and also decreases two-dimensional (measured by quantitative video microscopy) and three-dimensional (measured by Boyden chamber assays) migration. These effects of fusicoccin A treatment translated into structural changes in actin cytoskeletal organization and a loss of GBM cell adhesion. Therefore, fusicoccin A exerts cytostatic effects but low cytotoxic effects (as demonstrated by flow cytometry). These cytostatic effects can partly be explained by the fact that fusicoccin inhibits the activities of a dozen kinases, including focal adhesion kinase (FAK), that have been implicated in cell proliferation and migration. Overexpression of FAK, a nonreceptor protein tyrosine kinase, directly correlates with the invasive phenotype of aggressive human gliomas because FAK promotes cell proliferation and migration. Fusicoccin A led to the down-regulation of FAK tyrosine phosphorylation, which occurred in both normoxic and hypoxic GBM cell culture conditions. In conclusion, the current study identifies a novel compound that could be used as a chemical template for generating cytostatic compounds designed to combat GBM[4].

A Single Application of Fusicoccin-A (FC-A) Reduces Corticospinal Axon Die-Back after Dorsal Hemisection Spinal Cord Injury[3]
The damaged CNS is characterized by the presence of growth inhibitors including chondroitin sulfate proteoglycans (CSPGs) produced by reactive glia. fusicoccin-A (FC-A) significantly improved neurite outgrowth on aggrecan substrates, suggesting that it may be efficacious in improving growth after injury in vivo (Figure S6). We therefore sought to determine whether fusicoccin-A (FC-A) could improve axon growth after a dorsal hemisection spinal cord injury, which transects the entire corticospinal tract (CST). Mice received an immediate local application of FC-A in a rapidly polymerizing fibrin gel onto the injury site. CST axons were then anterogradely traced by injecting biotinylated dextran amine (BDA) into the motor cortex and the mice were analyzed on day 21 (Figure 7A). Surgeries and analysis were performed blinded to the experimental condition in two independent cohorts. Mice that received a single treatment of FC-A had a significant reduction in axonal die-back away from the injury site compared to control mice treated with vehicle-containing fibrin gel. While the cut axons of control mice retracted an average distance of ∼72 μm, the end-bulbs of FC-A-treated mice were within the lesion or in close proximity to the lesion (Figures 7B and 7C). These results indicate that transient exposure to FC-A is sufficient to diminish the collapse and retraction of severed axons in vivo.[3]

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Fusicoccin-A (FC-A) Stimulates Optic Nerve Regeneration[3]
Next, we determined whether Fusicoccin-A (FC-A) could stimulate CNS axon regeneration past the lesion with longer therapeutic exposure using the optic nerve crush (ONC) model and intravitreal injections. To visualize fusicoccin-A (FC-A) distribution after intravitreal injection, we generated an Alexa 488 FC-A conjugate. 488-FC-A was enriched in the retinal ganglion cell layer 1 day after injection, suggesting that it is readily taken up by retinal ganglion cells (RGCs) (Figure S7A). We also examined retinal GCN1 expression by western blot as a readout for activity. GCN1 was unchanged at 1 day post-injection, but significantly downregulated 3 days post-injection. Intriguingly, GCN1 levels remained suppressed 7 days post-injection, suggesting a sustained effect (Figures S7B and S7C). We next determined whether FC-A could induce axon regeneration after ONC. Mice received either 1 or 2 intravitreal injections of FC-A (Figure 8A). Cholera toxin β (CTβ) was used to trace RGC axons and GAP43 staining was used to visualize and quantify actively growing fibers. Surgeries and analysis were performed blinded to the experimental condition. Treatment with a single injection of FC-A was insufficient to stimulate axon regeneration; however, a second injection on day 7 resulted in significant regeneration at 100, 200, and 500 μm from the lesion compared to control animals (Figures 8B and 8C). Nearly all CTβ+ axons were also GAP43+, indicating that the axons were in a growth state (Figure 8D). We also assessed RGC density in the retina using Brn3a as a marker. As expected, ONC caused a massive loss of RGCs (∼70% loss of RGCs). Interestingly, FC-A-did not improve RGC survival, indicating that FC-A stimulates axon regeneration independent of cell survival (Figures 8E and 8F). These findings show that therapeutic administration of FC-A can stimulate axon regeneration after CNS injury and open the door to a new class of small molecules that could be further optimized to repair CNS damage.

Kinase Activity Determination[4]
Researchers originally provided ProQinase with fusicoccin A as a stock solution in 100% DMSO and aliquots were further diluted with water in 96-well microliter plates directly before use. A radiometric protein kinase assay (33PanQinase Activity Assay) was used for measuring the kinase activity of 288 recombinant protein kinases as detailed previously. Fusicoccin A was tested in two replicates in each kinase assay, and the final concentration tested was 50 µM.

Cell Growth Inhibition Assay[4]
The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay was used to determine the overall growth rate of each cell line as previously described. The number of living cells was determined after 72 hours of culture in the presence of fusicoccin A, 3′-O-deacetyl fusicoccin A, or 19-O-deacetyl fusicoccin A. Fusicoccin A was also tested on mouse astrocytes. Each experimental condition was performed in triplicate.[4]
To investigate whether fusicoccin A-induced growth inhibitory effects in glioma cells were irreversible or not, U373-MG and Hs683 cells were treated with 100 µM fusicoccin A and were washed after 3, 24, and 48 hours after treatment. The number of viable cells was determined 72 hours after having removed fusicoccin A from the culture media. Each experimental condition was performed in six replicates.[4]

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Determination of In Vitro Cell Death[4]
To evaluate viability in U373-MG cells that were treated with fusicoccin A, an assay measuring DNA fragmentation was used. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay was performed according to a procedure previously described using the APO-Direct Kit. The protocol was performed according to the manufacturer's instructions, including the use of positive and negative controls. Briefly, U373-MG GBM cells were treated with 100 µM fusicoccin A for either 24 or 72 hours in culture media or left untreated. Adherent and nonadherent cells were collected, fixed with 1% paraformaldehyde (1 hour) at 4°C, permeabilized, and stored in 70% ethanol at -20°C. TUNEL labeling was performed for 1 hour at 37°C and the stained cells were analyzed on a CellLab Quanta SC flow cytometer.[4]

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Analyses of Actin Cytoskeletal Organization[4]
U373-MG cells were cultured for 30 hours in the absence (controls) or presence of 100 µM fusicoccin A on glass coverslips as previously described. Fluorescent phalloidin conjugated with the Alexa Fluor 488 fluorochrome was used to label fibrillar actin, and Alexa Fluor 594-conjugated DNAse I was used to stain globular actin. The coverslips were mounted on microscope slides with 10 µl of Moviol agent. Three coverslips per experimental condition were analyzed and three pictures were taken for each coverslip (with the same exposure time) using an AxioCamHRm fluorescent microscope. [4]

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Boyden Chamber Assay[4]
The invasive features of U373-MG cells treated with 50 and 100 µM fusicoccin A for 24 hours in vitro were assessed using the Boyden transwell invasion system as detailed elsewhere. In parallel, the same number of U373-MG cells were seeded in 24-well plates coated with Matrigel, and we treated the cells with 0 (control), 50, or 100 µM fusicoccin A for 24 hours. Tonormalize the number of invasive cells, the number of viable cells in each condition was determined. Each experiment was performed in triplicate.[4]

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In Vitro Adhesion Assay[4]
The adhesion assay was performed as described previously [31] with modifications. Briefly, glass coverslips in 24-well plates were precoated with Matrigel (diluted 1:3 in RPMI culture media with 10% FCS) for 1 hour at 37°C. The wells were washed and nonspecific binding was then blocked with 0.1% BSA in phosphate-buffered saline for 30 minutes. U373-MG cells (20,000 cells per well) were allowed to adhere for 24 hours at 37°C in the presence of either 0 (control), 50, or 100 µM fusicoccin A, after which nonadherent cells were gently washed away with warm phosphate-buffered saline. Adherent cells were methanol-fixed, hematoxylin-stained, and counted in 10 fields per well at a G x 10 magnification using an Olympus microscope. Each experimental condition was performed in triplicate.

Dorsal hemisection spinal cord injuries[3]
Adult female C57BL/6 mice (8-10 weeks of age) were deeply anesthetized with ketamine:xylazine:acepromazine (50:5:1 mg/kg) and a laminectomy was done to expose the T9 thoracic spinal cord. Dorsal hemisection of the spinal cord was performed with spring micro-scissors to cut through the central canal. 200 μg fusicoccin-A (FC-A) solubilized in a 1:3 stock of ethanol:PBS was diluted in a thrombin solution into a final volume of 25 μL, quickly mixed with 25 μL of a fibrinogen solution and immediately applied directly onto the injury site, forming a viscous gel. Thrombin and fibrinogen solutions were purchased as a kit (Evicel Fibrin Sealant). Mice received 0.2 μL bilateral injections of 10% biotinylated dextran amine (BDA) into the sensorimotor cortex with a glass pipette. On day 21, mice were transcardially perfused with 4% PFA and the spinal cord was cryoprotected in 30% sucrose. 25 μm longitudinal sections were collected on slides, incubated in 0.6% hydrogen peroxide for 3hr, and overnight with streptavidin complex (Vector Laboratories). Sections were reacted with diaminobenzidine to visualize the streptavidin–biotin–dextranamine complex and counterstained with methyl green. Scar borders were determined from the methyl green stain and quantification of axonal die-back distance was assessed blinded to the experimental condition by measuring and averaging the distances between the 5 closest end-bulbs from the edge of the lesion. Animals that had incomplete lesions were excluded from the study. Sample sizes were based on previous spinal cord injury studies (Liu et al., 2010). All surgeries were randomized and performed blinded to the experimental condition in two independent cohorts.[3]

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Optic nerve crush[3]
Age and sex matched C57BL/6 mice (male and female, 8-14 weeks of age) were anesthetized with isoflurane and an incision was made above the left orbit. Under a surgical microscope, the extra-ocular muscles were resected to expose the underlying optic nerve. The optic nerve was crushed 0.5-1 mm behind the optic nerve head with fine forceps (Dumont #5) for 10 s. Care was taken to avoid damaging the ophthalmic artery. Vascular integrity of the retina was assessed by a fundus examination. For intravitreal injections, a small puncture was made in the sclera with a 30-gauge needle and 2 μL of PBS orfusicoccin-A (FC-A) (1 μg/μL solution in PBS) was injected into the wound using a Hamilton syringe. The needle was held in place for ∼2 min to avoid reflux and the puncture was sealed with surgical glue. Mice that had significant reflux were excluded from the study. Injections of PBS or fusicoccin-A (FC-A) were performed immediately after injury and again 7 days after injury. Cholera toxin injections (1 μL) were performed on day 12-13. Mice were transcardially perfused with 4% PFA on day 14. Optic nerves and eyes were harvested on day 14 and post-fixed in 4% PFA for 2hr. Retinal flat mounts were prepared and stained with anti-Brn3a (0.3 μg/mL, RRID: AB_2167511). Optic nerves were cryoprotected in 30% sucrose at 4°C overnight and embedded in OCT. Longitudinal sections (14 μm thickness) were collected on slides, permeabilized and blocked with 0.3% Triton-X in 5% BSA for 1hr at room temperature, stained with anti-GAP43 (1:1000, RRID: AB_10005026) overnight at 4°C, washed 3 times with PBS and stained with Alexa 488-conjugated secondary antibody (1:1000). Sample sizes were based on previous optic nerve injury studies (Chandran et al., 2016). All surgeries and injections were randomized and performed blinded to the experimental condition.

[1]. The phytotoxin fusicoccin, a selective stabilizer of 14-3-3 interactions?. IUBMB Life. 2013;65(6):513-517.

[2]. Small-molecule stabilization of the p53-14-3-3 protein-protein interaction. FEBS Lett. 2017;591(16):2449-2457.

[3]. Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration. Neuron. 2017;93(5):1082-1093.e5.

[4]. Fusicoccin a, a phytotoxic carbotricyclic diterpene glucoside of fungal origin, reduces proliferation and invasion of glioblastoma cells by targeting multiple tyrosine kinases. Transl Oncol. 2013;6(2):112-123.

Fusicoccin is an acetate ester. It has a role as a toxin.
Fusicoccin has been reported in Diaporthe amygdali with data available.

C36H56O12

680.82264

680.377

C, 63.51; H, 8.29; O, 28.20

20108-30-9

447573

White to off-white solid powder

1.2±0.1 g/cm3

760.2±60.0 °C at 760 mmHg

227.0±26.4 °C

0.0±5.8 mmHg at 25°C

1.555

Fusicoccum amygdali

3.28

4

12

14

48

1240

13

C[C@@H]1[C@@H]\2CC[C@@H](/C2=C/[C@]3([C@H](CC(=C3[C@H]([C@@H]1O)O[C@@H]4[C@@H]([C@H]([C@@H]([C@H](O4)COC(C)(C)C=C)O)OC(=O)C)O)[C@H](C)COC(=O)C)O)C)COC

KXTYBXCEQOANSX-WYKQKOHHSA-N

InChI=1S/C36H56O12/c1-10-35(6,7)45-17-26-30(41)33(46-21(5)38)31(42)34(47-26)48-32-28-24(18(2)15-44-20(4)37)13-27(39)36(28,8)14-25-22(16-43-9)11-12-23(25)19(3)29(32)40/h10,14,18-19,22-23,26-27,29-34,39-42H,1,11-13,15-17H2,2-9H3/b25-14-/t18-,19-,22-,23+,26-,27+,29-,30-,31-,32-,33+,34-,36+/m1/s1

[(2S)-2-[(1E,3R,4S,8R,9R,10R,11S,14S)-8-[(2S,3R,4S,5R,6R)-4-acetyloxy-3,5-dihydroxy-6-(2-methylbut-3-en-2-yloxymethyl)oxan-2-yl]oxy-4,9-dihydroxy-14-(methoxymethyl)-3,10-dimethyl-6-tricyclo[9.3.0.03,7]tetradeca-1,6-dienyl]propyl] acetate

Fusicoccin; 20108-30-9; Fusicoccin-A; Fusicoccin A; CHEMBL4244843; (2S)-2-[(1S,4R,5R,6R,6aS,9S,9aE,10aR)-4-{[3-O-acetyl-6-O-(1,1-dimethylprop-2-en-1-yl)-alpha-D-glucopyranosyl]oxy}-1,5-dihydroxy-9-(methoxymethyl)-6,10a-dimethyl-1,2,4,5,6,6a,7,8,9,10a-decahydrodicyclopenta[a,d][8]annulen-3-yl]propyl acetate; [(2S)-2-[(1E,3R,4S,8R,9R,10R,11S,14S)-8-[(2S,3R,4S,5R,6R)-4-acetyloxy-3,5-dihydroxy-6-(2-methylbut-3-en-2-yloxymethyl)oxan-2-yl]oxy-4,9-dihydroxy-14-(methoxymethyl)-3,10-dimethyl-6-tricyclo[9.3.0.03,7]tetradeca-1,6-dienyl]propyl] acetate; fuscicoccin A;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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