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| Targets |
The action target of Cucurbitacin D is heat shock protein 90 (HSP90), specifically inhibiting the ATPase activity of HSP90. The IC50 value for recombinant human HSP90α ATPase activity is 0.12 ± 0.02 μM [1]
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| ln Vitro |
1. Inhibition of HSP90 ATPase activity: Cucurbitacin D was tested for its effect on the ATPase activity of recombinant human HSP90α using the ADP-Glo ATPase assay. The compound exhibited concentration-dependent inhibition, with an IC50 of 0.12 ± 0.02 μM. At a concentration of 1 μM, it inhibited HSP90 ATPase activity by over 90%, which was comparable to the positive control geldanamycin (IC50: 0.05 μM) [1]
2. Disruption of HSP90 chaperone function: Human lung adenocarcinoma A549 cells and breast cancer MCF-7 cells were treated with Cucurbitacin D (0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM) for 24 hours. Western blot analysis showed that Cucurbitacin D dose-dependently reduced the protein levels of HSP90 client proteins, including Akt, ErbB2, and Raf-1 (e.g., 0.1 μM reduced Akt protein by ~65% in A549 cells). Additionally, native PAGE analysis revealed that Cucurbitacin D inhibited the formation of HSP90 oligomers (a key form for chaperone activity) in A549 cells, with 0.5 μM completely blocking oligomer formation [1] 3. Antiproliferative activity against cancer cells: Cucurbitacin D was evaluated for antiproliferative activity in multiple human cancer cell lines (A549, MCF-7, HepG2, HCT116) using the MTT assay. The compound inhibited cell proliferation in a concentration-dependent manner, with IC50 values of 0.08 ± 0.01 μM (A549), 0.15 ± 0.03 μM (MCF-7), 0.11 ± 0.02 μM (HepG2), and 0.09 ± 0.01 μM (HCT116) after 72 hours of treatment [1] |
| Enzyme Assay |
HSP90 ATPase activity assay: The reaction system (25 μL total volume) contained 50 mM Tris-HCl buffer (pH 7.5), 5 mM MgCl2, 2 mM DTT, 0.2 μg recombinant human HSP90α, 100 μM ATP, and different concentrations of Cucurbitacin D (0.01 μM to 10 μM). The mixture was incubated at 37°C for 1 hour to allow ATP hydrolysis. Then, 25 μL of ADP-Glo reagent was added to terminate the reaction and deplete remaining ATP, followed by incubation at room temperature for 40 minutes. Next, 50 μL of kinase detection reagent was added to convert ADP to ATP and generate luminescence, which was measured using a microplate reader. The luminescence intensity was inversely proportional to the amount of ADP produced (i.e., HSP90 ATPase activity). The inhibition rate was calculated by comparing with the vehicle control, and the IC50 value was determined by nonlinear regression analysis of the concentration-inhibition curve [1]
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| Cell Assay |
1. Cancer cell proliferation assay (MTT assay): Human cancer cells (A549, MCF-7, HepG2, HCT116) were seeded in 96-well plates at a density of 3×10^3 cells per well and cultured in RPMI 1640 medium containing 10% fetal bovine serum overnight. The medium was replaced with fresh medium containing different concentrations of Cucurbitacin D (0.001 μM to 1 μM), and the cells were incubated for 72 hours at 37°C in a 5% CO2 incubator. After incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for another 4 hours. The supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the IC50 value was calculated based on the cell viability curve [1]
2. Western blot analysis of HSP90 client proteins: A549 or MCF-7 cells were seeded in 6-well plates at a density of 5×10^5 cells per well and cultured overnight. The cells were treated with Cucurbitacin D (0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM) for 24 hours, then washed with cold PBS and lysed with RIPA buffer containing protease inhibitors. The protein concentration was determined using a BCA kit. Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies against Akt, ErbB2, Raf-1, and β-actin (loading control) overnight at 4°C. After incubation with secondary antibodies, the bands were visualized using an enhanced chemiluminescence (ECL) system, and the band intensity was quantified using image analysis software [1] 3. Native PAGE analysis of HSP90 oligomers: A549 cells were treated with Cucurbitacin D (0.1 μM, 0.5 μM) for 24 hours, lysed with native lysis buffer (without SDS or reducing agents), and centrifuged to collect the supernatant. Equal amounts of protein were loaded onto a 4-12% native PAGE gel and electrophoresed at 4°C. The gel was transferred to a PVDF membrane and probed with an anti-HSP90 antibody to detect HSP90 monomers and oligomers. The results showed that Cucurbitacin D reduced the intensity of HSP90 oligomer bands in a concentration-dependent manner [1] |
| References | |
| Additional Infomation |
Cucurbitacin D is a cucurbitacin whose lanostane skeleton is multiplely substituted with hydroxyl, methyl and oxo substituents, and is unsaturated at the 5 and 23 positions. It is a cucurbitacin, a secondary α-hydroxy ketone and a tertiary α-hydroxy ketone. It is derived from the hydride of lanostane. Cucurbitacin D has been reported to be found in Trichosanthes tricuspidata, Elaeocarpus chinensis and other organisms with relevant data. 1. Cucurbitacin D is a triterpenoid compound that is naturally found in Cucurbitaceae plants (e.g., Cucurbita pepo, Momordica charantia) that have traditionally been used in folk medicine to treat inflammation and tumors[1]. 2. Mechanism of action: Cucurbitacin D disrupts the HSP90 molecular chaperone mechanism through two main pathways: first, it directly inhibits the ATPase activity of HSP90 (a core step in molecular chaperone function); second, it prevents the formation of HSP90 oligomers (oligomers are essential for the efficient folding of substrate proteins). This dual effect leads to the misfolding and degradation of HSP90 client proteins (most of which are oncogenic proteins, such as Akt and ErbB2), thereby inhibiting cancer cell proliferation [1]. 3. Significance of research: As HSP90 is a well-established anticancer target, cucurbitacin D shows potential as a lead compound for developing novel HSP90 inhibitors. Its unique mechanism (simultaneous inhibition of ATPase activity and oligomerization) distinguishes it from existing HSP90 inhibitors (such as gerdemycin, which targets only ATPase activity), providing a new direction for the development of anticancer drugs [1].
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| Molecular Formula |
C30H44O7
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|---|---|
| Molecular Weight |
516.6662
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| Exact Mass |
516.308
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| CAS # |
3877-86-9
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| PubChem CID |
5281318
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
684.0±55.0 °C at 760 mmHg
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| Melting Point |
151-152ºC
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| Flash Point |
381.4±28.0 °C
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| Vapour Pressure |
0.0±4.8 mmHg at 25°C
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| Index of Refraction |
1.582
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| LogP |
1.48
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
37
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| Complexity |
1100
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| Defined Atom Stereocenter Count |
9
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| SMILES |
C[C@@]12C[C@H]([C@@H]([C@]1(CC(=O)[C@@]3([C@H]2CC=C4[C@H]3C[C@@H](C(=O)C4(C)C)O)C)C)[C@](C)(C(=O)/C=C/C(C)(C)O)O)O
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| InChi Key |
SRPHMISUTWFFKJ-QJNWWGCFSA-N
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| InChi Code |
InChI=1S/C30H44O7/c1-25(2,36)12-11-21(33)30(8,37)23-19(32)14-27(5)20-10-9-16-17(13-18(31)24(35)26(16,3)4)29(20,7)22(34)15-28(23,27)6/h9,11-12,17-20,23,31-32,36-37H,10,13-15H2,1-8H3/b12-11+/t17-,18+,19-,20+,23+,27+,28-,29+,30+/m1/s1
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| Chemical Name |
(2S,8S,9R,10R,13R,14S,16R,17R)-17-[(E,2R)-2,6-dihydroxy-6-methyl-3-oxohept-4-en-2-yl]-2,16-dihydroxy-4,4,9,13,14-pentamethyl-2,7,8,10,12,15,16,17-octahydro-1H-cyclopenta[a]phenanthrene-3,11-dione
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~110 mg/mL (~212.90 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.75 mg/mL (5.32 mM) in 10% DMSO + 90% (20% SBE-β-CD in 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 27.5 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 2: ≥ 2.75 mg/mL (5.32 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 27.5 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 | 1.9355 mL | 9.6774 mL | 19.3547 mL | |
| 5 mM | 0.3871 mL | 1.9355 mL | 3.8709 mL | |
| 10 mM | 0.1935 mL | 0.9677 mL | 1.9355 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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