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]
The primary target of GC7 Sulfate is deoxyhypusine synthase (DHPS/DHS), as well as related enzymes in spermidine synthesis and metabolic pathways. DHPS catalyzes the first step of the conversion of a specific lysine residue (Lys50 in human eIF5A) to the intermediate deoxyhypusine modification, which is the rate-limiting step for eIF5A activation. eIF5A is the only known substrate protein of DHPS in eukaryotic cells, and its hypusination promotes the translation of a subset of mRNAs involved in cytokine responses, cell proliferation, differentiation and stress responses. GC7 competitively inhibits the binding of spermidine to DHPS by specifically interacting with the active site of DHPS, with a Ki value of 9.7 nM. Additionally, GC7 indirectly regulates cell cycle progression through the p21/Cdk4/Rb signaling pathway.

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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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]

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GC7 Sulfate exhibits significant antiproliferative activity against various cell lines in vitro. After 72 hours of treatment in MYCN2 and BE(2)-C neuroblastoma cells, GC7 significantly reduces viable cell numbers in a dose-dependent manner within the concentration range of 0.1 to 100 μM. In MYCN2 cells, 5 μM GC7 reduces cell viability by approximately 40%-60%; whereas the drug-resistant, MYCN-amplified BE(2)-C cell line requires 25 μM to reduce cell viability by approximately 50%. In hepatocellular carcinoma (HCC) cells, between 0 and 20 μM, GC7 induces little cytotoxicity, while higher concentrations (50 to 100 μM) significantly inhibit the viability of all five tested HCC cell lines. Western blot analysis shows that treatment with ≥5 μM GC7 for 72 hours reduces total retinoblastoma protein (Rb), phosphorylated Rb and cyclin-dependent kinase 4 (Cdk4), while increasing the expression of the cell cycle inhibitor p21. The mechanism acts primarily through the p21/Cdk4/Rb signaling axis to induce cell cycle arrest. Additionally, in CHO cells, 1 μM GC7 inhibits hypusine production.

GC7 Sulfate demonstrates therapeutic potential in various animal models. In obese diabetic C57BLKS/J-db/db mice, a 2-week treatment with GC7 improved glucose tolerance, increased insulin release, and enhanced β cell mass. In a humanized T1D mouse model, inhibition of eIF5A hypusination by GC7 altered the pancreatic microenvironment by reducing Th1/Th17 response, increasing Treg numbers, decreasing serum IL-17 and IL-21 cytokine levels, reducing anti-GAD65 antibodies, and ablating ER stress to improve β-cell function. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. In a Melan-a Tm5 murine melanoma model, administration at a dose of 0.9 mg/kg reduced tumor growth. In a stroke model, a single GC7 pre- or post-treatment significantly reduced infarct volume and improved mouse performance in rotarod and Morris water-maze tests. However, the therapeutic application of GC7 is limited by low selectivity and restricted bioavailability.

The in vitro enzymatic inhibition assay for GC7 Sulfate is typically performed as follows: Recombinant DHPS enzyme is incubated in a buffer system containing 50 mM HEPES (pH 7.5), 1 mM DTT. Increasing concentrations of GC7 (0.1 nM–100 μM) are added along with spermidine and eIF5A substrate peptides. The reaction is carried out at 37°C for 30-60 minutes. NAD⁺ is added as a cofactor to initiate the reaction. The production of the reaction product deoxyhypusine is quantified by HPLC or radiolabeling detection (³H-spermidine or ¹⁴C-spermidine incorporation assay) to calculate inhibitory activity. The IC₅₀ values of GC7 against mammalian DHPS range from 17-50 nM, with a Ki value of 9.7 nM.

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.

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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]

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In vitro cell-based assays typically use neuroblastoma cell lines (such as MYCN2, BE(2)-C, SH-SY5Y) or other relevant tumor cell lines. Cells are cultured in DMEM or RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37°C. GC7 Sulfate powder is first dissolved in sterile water to prepare a stock solution (typically 10-50 mM), then diluted with culture medium to working concentrations (0.1–100 μM). Cells are seeded in 96-well plates (approximately 5 × 10³ cells per well) and cultured overnight before being treated with various concentrations of GC7 for 72 hours. Cell viability is measured using MTS or CCK-8 reagents: MTS reagent is added to each well, incubated at 37°C for 1-4 hours, and absorbance is read at 490 nm or 450 nm. Flow cytometry is used to analyze cell cycle progression and apoptosis. Western blot is performed to detect signaling pathway proteins (such as p21, Rb, p-Rb, Cdk4). Additionally, ³H-spermidine incorporation can be measured to evaluate eIF5A hypusination inhibition.

In in vivo experiments, GC7 Sulfate is typically administered via intraperitoneal injection. Using C57BLKS/J-db/db obese diabetic mice as an example: GC7 is dissolved in sterile saline or PBS and administered at a dose of 1-5 mg/kg/day via intraperitoneal injection for 2 consecutive weeks. Control groups receive an equal volume of vehicle. Body weight and blood glucose levels are monitored weekly during treatment. On day 14, an oral glucose tolerance test (OGTT) or intraperitoneal glucose tolerance test (IPGTT) is performed: after overnight fasting, mice receive a glucose solution (1-2 g/kg body weight), and blood samples are collected at 0, 15, 30, 60, 90, and 120 minutes to measure blood glucose and insulin levels. After euthanasia, pancreatic tissues are isolated for H&E staining and immunohistochemistry analysis to assess changes in β cell mass. For pharmacokinetic evaluation, mice receive a single intraperitoneal injection of GC7 (2 mg/kg), and plasma samples are collected at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 hours; drug concentrations are determined by LC-MS/MS. In the Melan-a Tm5 murine melanoma model, GC7 (0.9 mg/kg) is administered daily via tail vein or intraperitoneal injection for 14-21 days, with tumor volume and body weight measured every 3 days. Additionally, in a renal ischemia-reperfusion injury model, rats receive an intraperitoneal injection of GC7 (8 mg/kg) 30 minutes before surgery, followed by an identical dose after surgery; renal tissues are collected for Western blot analysis of hypusinated eIF5A levels.

Due to the polar guanidino group in the molecule, GC7 has low lipophilicity and poor oral bioavailability; intraperitoneal injection is the preferred route of administration in animal experiments (offering superior bioavailability to oral administration). In vitro solubility assays have shown that GC7 Sulfate has a solubility of ≥12.78 mg/mL in water with ultrasonic assistance, is insoluble in DMSO, and insoluble in ethanol. In in vitro cell experiments, GC7 concentration typically ranges from 0.1-100 μM; in animal models, the commonly used intraperitoneal injection dose ranges from 0.9-5 mg/kg/day. Comprehensive pharmacokinetic parameters (including half-life T₁/₂, clearance Cl, steady-state volume of distribution Vdss, maximum plasma concentration Cmax, time to peak concentration Tmax, and oral bioavailability F%) have not yet been systematically reported. Please refer to the latest literature for specific values.

Toxicological information for GC7 Sulfate mainly comes from in vitro cytotoxicity assessments and material safety data sheets. In in vitro experiments, between 0-20 μM, GC7 induces little cytotoxicity in HCC cells, while higher concentrations (50-100 μM) significantly inhibit cell viability, indicating concentration-dependent cytotoxicity. Safety data sheets indicate that under fire conditions, the compound may decompose and emit toxic fumes; direct inhalation, eye contact, and skin contact should be avoided. Appropriate exhaust ventilation should be used during handling, and dust and aerosol formation should be avoided. GC7 Sulfate toxicity classification: irritating to skin, risk of serious damage to eyes, and prolonged exposure may cause serious damage to health. This compound is not intended for human diagnostic or therapeutic use and is limited to basic research use only. It is recommended to operate in a fume hood, wearing protective gloves and goggles, and to store at -20°C in a dry, dark environment. Systematic long-term in vivo toxicology data are currently not available.

[1]. Deoxyhypusine synthase (DHPS) inhibitor GC7 induces p21/Rb-mediated inhibition of tumor cell growth and DHPS expression correlates with poor prognosis in neuroblastoma patients. Cell Oncol (Dordr). 2014 Dec;37(6):387-98.

[2]. N1-guanyl-1,7-diaminoheptane (GC7) enhances the therapeutic efficacy of doxorubicin by inhibiting activation of eukaryotic translation initiation factor 5A2 (eIF5A2) and preventing the epithelial-mesenchymal transition in hepatocellular carcinoma cells. Exp Cell Res. 2013 Oct 15;319(17):2708-17.

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]

C8H22N4O4S

270.349680423737

270.14

C, 35.54; H, 8.20; N, 20.72; O, 23.67; S, 11.86

150417-90-6

150333-69-0

131842088

White to off-white solid powder

5

6

7

17

200

0

S(O)(O)(=O)=O.C(CNC(N)=N)CCCCCN

MDDOWYFCKAAANU-UHFFFAOYSA-N

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)

2-(7-aminoheptyl)guanidine;sulfuric acid

N1-Guanyl-1,7-diaminoheptane; GC7 (sulfate)

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Water : 3 mg/mL

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).

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