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GC7 Sulfate, the sulfate salt of GC-7 (N1-guanyl-1,7-diaminoheptane), is a novel and potent deoxyhypusine synthase (DHPS) inhibitor that inhibits Neuroblastoma (NB) cell proliferation in a dose-dependent manner, through induction of the cell cycle inhibitor p21 and reduction of total and phosphorylated Rb proteins.
| Targets |
DHPS; p21
GC7 Sulfate is a deoxyhypusine synthase (DHPS) inhibitor that exhibits the ability to suppress MYCN-amplified and chemotherapy-resistant NB cell proliferation. It also modulates multiple cell cycle-regulating proteins and initiates arrest through the p21/Cdk4/Rb signaling axis, even when MYCN is present.[1] |
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| ln Vitro |
GC7 Sulfate is a deoxyhypusine synthase (DHPS) inhibitor that exhibits the ability to suppress MYCN-amplified and chemotherapy-resistant NB cell proliferation. It also modulates multiple cell cycle-regulating proteins and initiates arrest through the p21/Cdk4/Rb signaling axis, even when MYCN is present.[1]
GC7 inhibited the proliferation of human neuroblastoma cell lines MYCN2 (with or without doxycycline-induced MYCN expression) and BE(2)-C in a dose-dependent manner, as measured by MTS viability assay after 72 hours of treatment. In MYCN2 cells without MYCN expression, 5 µM GC7 inhibited viability by ~40%, and in MYCN2 cells with MYCN expression, 5 µM GC7 inhibited viability by ~60%. The drug-resistant, MYCN-amplified BE(2)-C cell line required 25 µM GC7 to reduce viability by ~50%. [1] Western blot analysis showed that treatment with ≥5 µM GC7 for 72 hours reduced protein levels of total retinoblastoma (Rb) and cyclin-dependent kinase 4 (Cdk4), and induced hypophosphorylation of Rb (detected using phospho-specific antibodies for Ser807/811 and Ser795) in both MYCN2 and BE(2)-C cells. Concurrently, GC7 treatment increased the expression of the cell cycle inhibitor p21 in MYCN2 cells, but p21 induction was very weak in BE(2)-C cells. [1] Time-course experiments (24, 48, 72 hours) with GC7 (5, 10, 25 µM) on MYCN2 cells confirmed that changes in Rb, p-Rb, Cdk4, and p21 protein levels became prominent after 48 and 72 hours. [1] Immunofluorescence microscopy corroborated the Western blot findings, showing decreased levels of total Rb, phosphorylated Rb, and Cdk4, and increased levels of p21 protein in MYCN2 cells treated with 10 and 100 µM GC7 for 72 hours. The reduction in cell number (nuclei count) was also evident. [1] Analysis of proteins in the apoptosis pathway (PARP, caspase 3, caspase 9) showed no activation upon GC7 treatment, suggesting its effect is not primarily via inducing apoptosis. [1] |
| Cell Assay |
The MTS assay kit is used to assess cell viability in compliance with the manufacturer's instructions. To summarize, cells are treated with GC7 Sulfate in 96-well plates for 72 hours at different concentrations (0.1 to 100 μM). Following this, they are immediately incubated with the MTS dye reagent for 1 hour at 37°C in 5% CO2 atmosphere. Using a reader, the absorbance is measured at 490 nm.
For cell proliferation assays, neuroblastoma cells (MYCN2, BE(2)-C) were seeded in 96-well plates. Sixteen hours after seeding, cells were treated with GC7 at various concentrations (0.1 to 100 µM) for 72 hours. Cell viability was analyzed using an MTS assay kit according to the manufacturer's protocol. After incubation with the MTS reagent for 1 hour at 37°C in 5% CO₂, absorbance was measured at 490 nm. [1] For Western blot analysis, cells were treated with GC7 for specified durations. Cell lysates were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined using a Bradford-based assay. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific primary antibodies (e.g., against Rb, p-Rb, Cdk4, p21, GAPDH). Horseradish peroxidase or fluorescent dye-conjugated secondary antibodies were used for detection, and signals were captured using appropriate imaging systems. [1] For immunofluorescence microscopy, cells were seeded on 96-well plates, treated, then fixed with paraformaldehyde, permeabilized, and blocked. Cells were incubated overnight at 4°C with primary antibodies (diluted 1:100), washed, and incubated with fluorescent dye-conjugated secondary antibodies (diluted 1:2000). Nuclei were stained with Hoechst 33342. Images were acquired using a high-content imaging system with a 20X objective. [1] During incubation with GC7, the culture medium was supplemented with 0.5 mM aminoguanidine to prevent the formation of toxic aldehydes and peroxides by the catalytic action of serum diamine oxidase on GC7. [1] |
| References |
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| Additional Infomation |
GC7 (N¹-guanosine-1,7-diaminoheptane) is an inhibitor of deoxyxanthine synthase (DHPS). DHPS, in conjunction with spermidine, is crucial for the hypoxanthineization and activation of eukaryotic translation initiation factor 5A (eIF5A). [1] In neuroblastoma, high expression of DHPS mRNA in tumor samples (n=88) was significantly associated with poor clinical parameters, including short overall survival, age at diagnosis greater than 18 months, and MYCN gene amplification. In multiple independent neuroblastoma datasets, DHPS expression was also positively correlated with MYCN and ODC1 mRNA expression. [1] The antiproliferative effect of GC7 in neuroblastoma cells was associated with the induction of cell cycle inhibitor p21, the reduction of total Rb protein and phosphorylated Rb protein, and the reduction of Cdk4, suggesting that it triggers cell cycle arrest through the p21/Cdk4/Rb signaling pathway. [1] This study suggests that DHPS may be a potential therapeutic target for neuroblastoma, and GC7 may be a candidate drug, especially for high-risk neuroblastoma with MYCN amplification. [1]
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| Molecular Formula |
C8H22N4O4S
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|---|---|
| Molecular Weight |
270.349680423737
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| Exact Mass |
270.14
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| Elemental Analysis |
C, 35.54; H, 8.20; N, 20.72; O, 23.67; S, 11.86
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| CAS # |
150417-90-6
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| PubChem CID |
131842088
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
17
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| Complexity |
200
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(O)(O)(=O)=O.C(CNC(N)=N)CCCCCN
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| InChi Key |
MDDOWYFCKAAANU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C8H20N4.H2O4S/c9-6-4-2-1-3-5-7-12-8(10)11;1-5(2,3)4/h1-7,9H2,(H4,10,11,12);(H2,1,2,3,4)
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| Chemical Name |
2-(7-aminoheptyl)guanidine;sulfuric acid
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| Synonyms |
N1-Guanyl-1,7-diaminoheptane; GC7 (sulfate)
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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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
Water : 3 mg/mL
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 5 mg/mL (18.49 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6989 mL | 18.4945 mL | 36.9891 mL | |
| 5 mM | 0.7398 mL | 3.6989 mL | 7.3978 mL | |
| 10 mM | 0.3699 mL | 1.8495 mL | 3.6989 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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