Telomeric G-quadruplex (G4) structures. [1]
Telomerase (indirectly, by stabilizing G4). In vitro telomerase inhibition IC50 = 0.31 μM (measured by TRAP-G4 assay). Selectivity ratio for telomerase vs. Taq polymerase inhibition > 50-100. [1]
G-quadruplex stabilization: ΔTm > 21°C (measured by G4 FRET assay). Selectivity index for G4 over B-DNA > 50-100. [1]

360A suppresses telomerase activity and stabilizes G-quadruplexes with an IC50 of 300 nM for telomerase in the TRAP-G4 test. The viability of glioma cell lines, including T98G, CB193, U118-MG, SAOS-2, and primary astrocytes, is decreased by 360A, with IC50 values of 4.8 ± 1.1 μM, 3.9 ± 0.4 μM, 8.4 ± 0.5 μM, > 15 μM, and 17.4 ± 1.2 μM, respectively[1]. 360A results in sister-telomere fusion that is dependent on DNA PKcs and generates Rad51-dependent telomere abnormalities that preferentially affect lagging strand telomeres, such as telomere loss or telomeric doublets [2].

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Inhibited cell proliferation and induced massive apoptosis in a dose-dependent manner in three telomerase-positive glioma cell lines (T98G, CB193, U118-MG) and the ALT cell line SAOS-2 after several days of treatment. No effect was observed after 72h of treatment. [1]
- In T98G cells, treatment with 1 μM 360A induced a marked decrease in the G0/G1 phase and a dramatic increase in S phase (from 21% in control to >55% after 9 days), followed by a massive increase in sub-G1 phase (apoptosis) after 12 days. [1]
- In CB193 cells, treatment with 5 μM 360A induced S-phase arrest followed by massive apoptosis, shown by increased sub-G1 fraction, >20% TUNEL-positive cells (20.6% on day 11, 39% on day 14), and cells containing cleaved caspase-3 (21.9% on day 11, 35.2% on day 14). [1]
- In SAOS-2 cells, 360A induced apoptosis associated with a moderate increase of cells in S and G2/M phases. [1]
- Time-lapse video microscopy of T98G cells treated with 1 μM 360A for 7 days showed a dramatic increase of metaphase duration (some cells arrested for >500 min vs. ~200 min for normal division) and major cytokinesis alterations. [1]
- Treatment with 360A did not induce a senescence program (no SA-β-galactosidase-positive cells) in T98G or CB193 cells. [1]
- Continuous treatment of T98G cells with 1 μM 360A led to a slight but significant decrease of the telomeric G-overhang signal (27% decrease after 10 days). [1]
- 360A induced telomere instability in T98G and CB193 cells, evidenced by frequent detection of dicentric chromosomes, ring chromosomes, and anaphase bridges. [1]

G-quadruplex stabilization assay (G4-FRET): The ability of 360A to stabilize a telomeric G-quadruplex was assessed using a FRET (Fluorescence Resonance Energy Transfer) melting assay. A fluorescently labeled telomeric oligonucleotide (F21T) was used. The change in melting temperature (ΔTm) of the G-quadruplex upon addition of the ligand was measured. A ΔTm > 21°C was observed for 360A, indicating strong interaction with the G4 structure. [1]
- Telomerase inhibition assay (TRAP-G4): Telomerase inhibition was measured using a modified TRAP (Telomeric Repeat Amplification Protocol) assay called TRAP-G4. This assay includes an additional step where the telomerase product is incubated with the ligand to force G-quadruplex formation during the PCR step, making the assay sensitive to G4 ligands. The IC50 value for telomerase inhibition by 360A was 0.31 μM. The selectivity of the compound was assessed by comparing its IC50 for telomerase inhibition to its IC50 for inhibition of an internal Taq polymerase PCR control. The selectivity ratio was >50-100. [1]

Cell viability assay (WST-1): Cells were seeded in 96-well plates and treated with various concentrations of 360A (0.1 - 20 μM) or DMSO control for 7 days at 37°C. The medium was changed on day 3. After 7 days, the WST-1 reagent was added, and cell viability/proliferation was measured. The IC50 values (concentration causing 50% inhibition) were calculated. For T98G cells, IC50 = 4.8±1.1 μM; for CB193, IC50 = 3.9±0.4 μM; for U118-MG, IC50 = 8.4±0.5 μM; for primary astrocytes, IC50 = 17.4±1.2 μM; for SAOS-2 cells, IC50 > 15 μM. [1]
- Long-term population doubling assay: Cells (5x10^5) were seeded in 75 cm² flasks and treated with 360A or DMSO. Every 3-4 days, cells were trypsinized, counted using trypan blue to assess viability, and re-seeded at 5x10^5 cells/flask. Cumulative population doublings were calculated over time. This assay showed that 360A decreased the rate of cell proliferation and induced growth arrest and cell death in T98G, U118-MG, CB193, and SAOS-2 cells after 1-3 weeks. [1]
- Cell cycle analysis by flow cytometry: Cells (0.5-1x10^6) were fixed in 70% ethanol, then stained with propidium iodide and RNase. The cell suspension was analyzed by flow cytometry to determine the percentage of cells in sub-G1, G0/G1, S, and G2/M phases. Treatment with 360A induced S-phase arrest and subsequent apoptosis in glioma cell lines. [1]
- TUNEL assay for apoptosis: Apoptotic cells were detected using the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method. Cells were stained and analyzed by flow cytometry or fluorescence microscopy. The percentage of TUNEL-positive cells was calculated from at least 200 cells per sample. 360A induced a significant increase in TUNEL-positive cells in glioma cell lines (e.g., >20% in CB193). [1]
- Cleaved caspase-3 detection: Cells were fixed, permeabilized, and incubated with a rabbit antibody specific for the cleaved (active) form of caspase-3. After washing, cells were incubated with a fluorescently labeled secondary antibody and counterstained with DAPI. The percentage of cells with activated caspase-3 was determined by fluorescence microscopy. 360A treatment increased cleaved caspase-3 positive cells in CB193 cultures (e.g., 35.2% on day 14). [1]
- Telomere length analysis (Southern blot): High-molecular weight genomic DNA was digested with HinfI restriction enzyme. Digested DNA was separated by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with a 32P-labeled telomeric probe (CCCTAA)4. The mean telomere restriction fragment (TRF) length was analyzed. No significant telomere shortening was detected in cells treated with 360A. [1]
- Telomeric G-overhang assay (non-denaturing hybridization): Undigested genomic DNA (2.5 μg) was hybridized with a 33P-labeled 21C oligonucleotide probe in a sodium/magnesium buffer. Samples were run on an agarose gel containing ethidium bromide. The gel was dried, and the ethidium bromide fluorescence and radioactivity were scanned. The relative G-overhang signal was normalized to the ethidium bromide signal. Treatment with 360A led to a 27% decrease in the G-overhang signal after 10 days. [1]
- Metaphase and anaphase chromosome analysis: Metaphase spreads of glioma cells were prepared and counterstained with Giemsa. Anaphase cells were grown in chamber slides, fixed with formaldehyde, and stained with Giemsa. Chromosome end-to-end associations (dicentric and ring chromosomes) and anaphase bridges were scored. 360A treatment induced these telomeric instability markers (e.g., in T98G, 55% of metaphases had dicentric chromosomes after 10 days of treatment with 1 μM 360A). [1]
- Time-lapse video microscopy: Cells were placed in an incubator chamber at 37°C on an inverted microscope. Phase-contrast images were taken every 10 minutes for 18 hours. T98G cells treated with 1 μM 360A for 7 days showed a dramatic increase in metaphase duration (>500 min) and cytokinesis defects. [1]

360A (up to 20 μM) showed only a reduced effect on primary human astrocyte cultures (IC50 = 17.4±1.2 μM after 7 days) compared to cancer cell lines, suggesting lower sensitivity of these telomerase-negative normal cells. [1]
- Treatment with 360A did not induce a senescence program (no SA-β-galactosidase-positive cells) in T98G or CB193 glioma cell lines. [1]

[1]. Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands. Oncogene. 2005 Apr 21;24(18):2917-28.

[2]. Rad51 and DNA-PKcs are involved in the generation of specific telomere aberrations induced by the quadruplex ligand 360A that impair mitotic cell progression and lead to cell death. Cell Mol Life Sci. 2012 Feb;69(4):629-40.

360A is a 2,6-pyridine-dicarboxamide derivative selected for its highly selective interaction with telomeric G-quadruplex structures over double-stranded DNA and for its potent telomerase inhibition. [1]
- The cellular effects of 360A (growth arrest and apoptosis) are believed to be related to the stabilization of G-quadruplex structures at telomeres, leading to telomere instability (chromosome end fusions, anaphase bridges) and cell cycle alterations (S-phase arrest, increased metaphase duration, cytokinesis defects), rather than global telomere shortening. [1]
- 360A was effective against both telomerase-positive glioma cells and an ALT (Alternative Lengthening of Telomeres) cell line (SAOS-2). [1]
- The compound induced apoptosis in glioma cell lines that are mutated for p53 (e.g., T98G). [1]
- These pyridine-based G-quadruplex ligands, including 360A, are proposed as promising agents for the treatment of various tumors, including malignant gliomas. [1]

C27H23N5O2

449.503825426102

449.185

794458-56-3

360A iodide;737763-37-0

11430774

Typically exists as solid at room temperature

4

2

3

4

34

674

0

C[N+]1=CC(=CC2=CC=CC=C21)NC(=O)C3=NC(=CC=C3)C(=O)NC4=CC5=CC=CC=C5[N+](=C4)C

KPOOEJJPTYXNTN-UHFFFAOYSA-P

InChI=1S/C27H21N5O2/c1-31-16-20(14-18-8-3-5-12-24(18)31)28-26(33)22-10-7-11-23(30-22)27(34)29-21-15-19-9-4-6-13-25(19)32(2)17-21/h3-17H,1-2H3/p+2

3,3'-((pyridine-2,6-dicarbonyl)bis(azanediyl))bis(1-methylquinolin-1-ium) iodide (2,6-N,N′-Methyl-quinolinio-3-yl)-pyridine dicarboxamide

360-A 360A 360 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.)