G-quadruplexe ( Kd = 490 nM )
Pyridostatin is a small molecule that binds to G-quadruplex DNA structures. [1]

Cell cycle arrest is induced by pyridoglostin (RR82) (10 μM; 48 hours) [1]. A tiny chemical called pyridoglotin selectively binds to the G-quadruplex structure in DNA, forming complexes with it to stabilize it. Dose-dependent neuronal death, neurite retraction, and synapse loss are brought on by piridostatin. In primary neurons that are cultivated, pyridoglostin causes DNA double strand breaks to develop. Significantly, BRCA1 protein (which safeguards and fixes the neuronal genome) is transcriptionally downregulated by pyridoglotin (1–5 μM, over night) [3].

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In FRET-melting assays, pyridostatin and its alkyne analogue (pyridostatin-a) promoted similar melting profiles for a set of known G-quadruplex DNA motifs. [1]
- Circular dichroism spectroscopy of G-quadruplex sequences from the SRC gene showed characteristic molar ellipticity maxima at 265 nm (parallel conformation) and/or 298 nm (antiparallel conformation), indicating folded G-quadruplex structures. [1]
- NMR spectroscopy of G-quadruplex sequences from SRC revealed imino proton signals between 10.5 and 12.5 ppm, characteristic of Hoogsteen hydrogen bonding in stacked G-quartets. After addition of 1.1 mole equivalents of pyridostatin, global line broadening and an upfield shift of imino proton signals were observed, consistent with pyridostatin binding to the top G-quartet via stacking interactions. [1]
- In cellulo chemical labeling of pyridostatin-a (alkyne analogue) via copper-catalyzed click chemistry produced fluorescently labeled pyridostatin (3), which formed nuclear foci and larger patterns consistent with nucleoli staining. [1]

Pyridostatin has anti‐tumoral activity against BRCA2‐deficient xenografts[4]
Compounds that bind and stabilise G4s have been shown to be active against BRCA1/2‐deficient xenograft tumours established in mice (RHPS4 and CX‐5461). However, these have not yet been demonstrated to benefit patients with BRCA mutations. Moreover, BRCA‐mutated tumours are difficult to treat because they rapidly develop resistance to targeted therapies (e.g. PARP inhibitors; PARPi). Therefore, it is imperative to identify new G4 ligands that not only eliminate BRCA‐deficient tumours but also counteract resistant disease. Our previously published results (Zimmer et al, 2016) demonstrated that the G4 ligand pyridostatin is specifically toxic to BRCA2‐deficient cells in vitro. In this study, researchers evaluated the potential of pyridostatin in eliminating BRCA2‐deficient xenograft tumours in vivo. To address this, researchers generated xenografts in CB17‐SCID mice using the isogenic BRCA2 +/+ (BRCA2‐proficient) and BRCA2 −/− (BRCA2‐deficient) human colorectal adenocarcinoma DLD1 cells (Fig 1A and B). Researchers extensively optimised conditions for in vivo use of pyridostatin and established that a dose schedule of 7.5 mg/kg/day administered intravenously for five consecutive days, followed by a 2‐day break and a second 5‐day treatment were well tolerated, as demonstrated by the lack of significant weight loss with no adverse clinical signs (Appendix Table S1). Using these conditions, researchers found that pyridostatin effectively and specifically inhibited growth of xenograft tumours established from BRCA2‐deficient DLD1 cells (Fig 1B). As a control, researchers used the PARPi talazoparib, known for its ability to eradicate BRCA1/2‐deficient tumours in mice (Shen et al, 2013) and recently licensed for use in metastatic breast cancer patients carrying BRCA1/2 germline mutations (Litton et al, 2018). The anti‐tumoral effect of pyridostatin against the BRCA2‐deficient tumours was similar to talazoparib, and neither drug impaired the growth of BRCA2‐proficient tumours.

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Furthermore, researchers investigated the in vivo response to pyridostatin using a second tumour model, established from isogenic BRCA2 +/+ and BRCA2 −/− colorectal carcinoma HCT116 cells (Xu et al, 2014). Pyridostatin showed selective toxicity against BRCA2‐deficient HCT116 cell‐derived tumours (Appendix Fig S1A and B; Appendix Table S2), similarly to its effect in DLD1 cell‐derived xenografts.[4]
Researchers' previous work showed that pyridostatin treatment causes DNA damage accumulation in cells with compromised HR repair, including BRCA2‐deficient cells (Zimmer et al, 2016). Consistently, immunohistochemical (IHC) analyses revealed that BRCA2‐deficient, but not BRCA2‐proficient, tumours exhibited increased level of the DNA damage marker γH2AX upon exposure to either pyridostatin or talazoparib (Appendix Fig S1C–F). These results indicated that pyridostatin can specifically suppress not only the growth of the cells (Zimmer et al, 2016), but also of tumours lacking BRCA2 and that it acts in vivo by inflicting DNA damage.[4]

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Cell Viability Assay [1]
Cell Types: More than 60 different cancer cell lines
Tested Concentrations: 10 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Mainly accumulated in the G2 phase of the cell cycle of more than 60 different cancer cell lines.

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MRC5-SV40 fibroblasts and various cancer cell lines treated with pyridostatin (2 μM for 24–72 h) showed decreased proliferation and accumulation in G2 phase of the cell cycle. [1]
- Western blot analysis of pyridostatin-treated cells (2 μM, 24–72 h) revealed phosphorylation of histone H2AX (Ser-139, γH2AX), KAP1 (Ser-824), Chk1 (Ser-345), RPA (Ser-4/8), and DNA-PKcs (Ser-2056), indicating DNA damage response activation. PARP-1 cleavage was observed after 72 h treatment, indicating apoptosis in some cells. [1]
- Immunofluorescence analysis showed γH2AX foci formation in pyridostatin-treated cells (20 μM, 4 h) in G1, S, and G2 phases. Pre-treatment with DRB (transcription inhibitor) prevented γH2AX foci in G1 and G2 cells; additional pre-treatment with aphidicolin (replication inhibitor) further reduced foci. [1]
- Neutral comet assays confirmed DNA double-strand breaks in pyridostatin-treated cells (2 μM, 24 h); tail moments were enhanced by co-treatment with DNA-PKcs inhibitor NU7441. [1]
- Pyridostatin (2 μM) reduced SRC mRNA levels by >95% after 8 h treatment in MRC5-SV40 cells, and reduced SRC protein levels by ~60% after 24 h. [1]
- In MDA-MB-231 breast cancer cells, pyridostatin (2 μM) significantly reduced wound healing in scratch assays after 48 h, while doxorubicin (100 nM) did not affect migration despite similar growth inhibition. [1]
- Chromatin immunoprecipitation (ChIP) of γH2AX followed by high-throughput sequencing (ChIP-Seq) identified pyridostatin-induced DNA damage sites enriched in gene bodies containing clusters of sequences with G-quadruplex-forming potential. [1]
- In U2OS cells stably expressing GFP-hPif1α, fluorescence microscopy showed colocalization of GFP-hPif1α with fluorescently labeled pyridostatin in cells fixed prior to drug addition, indicating overlapping targeting of genomic structures. [1]

CB17‐SCID mice
7.5 mg/kg
i.v.

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In vivo xenograft experiments[4]
CB17‐SCID mice (CB17/Icr‐Prkdcscid/IcrIcoCrl, male or female), FVB female mice were purchased from Charles River Laboratories. The mice were maintained in high‐efficiency, particulate air HEPA‐filtered racks and were fed autoclaved laboratory rodent diet.[4]
To generate xenografts derived from DLD1 and HCT116 BRCA2‐proficient or ‐deficient cells, CB17‐SCID male mice 6 weeks old were injected intramuscularly, into the hind leg muscles, with 5 × 106 cells per mouse. When a tumour volume of approximately 250 mm3 was evident, mice were randomised to start the treatments.[4]
To generate the PARPi‐resistant mouse tumour model, FVB female mice 6 weeks old were injected intramuscularly into the hind leg muscles with 4 × 106 KP3.33 (Brca1 +/+) cells or KB1PM5 (Brca1 −/− Tp53bp1 −/−) mouse mammary tumour cells. Each experimental group included five mice. When a tumour volume of approximately 250 mm3 was evident, mice were randomised and the treatment started.[4]
To generate xenografts derived from MDA‐MB‐436 cells, CB17‐SCID female mice 6 weeks old were injected intramuscularly with 4 × 106 cells per mouse. When a tumour volume of approximately 220 mm3 was evident (6 days after cell injection), treatment was initiated. Each experimental group included five mice.[4]

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Talazoparib (BMN 673, Selleckchem) was dissolved in 10% of dimethylacetamide, 6% of solutol HS, 84% of PBS and administered orally at doses of 0.33 mg/kg/day for five consecutive days, followed by 2‐day break and five more days of treatment (Wang et al, 2016). pyridostatin was dissolved in saline solution and administered intravenously at doses of 7.5 mg/kg/day for five consecutive days, followed by 2‐day break and five more days of treatment. NU‐7441 (Selleckchem) was dissolved in 5% of DMSO, 40% PEG300, 5% of Tween‐80 and administered intraperitoneally at doses of 10 mg/kg/day for five consecutive days, followed by 2‐day break and five more days of treatment (Zhao et al, 2006). Paclitaxel was dissolved in saline solution and administered intravenously at doses of 20 mg/kg/day at day 1 and day 8 of treatment (Bizzaro et al, 2018). When combined with other compounds, paclitaxel was administered intravenously at day 5 and 12 of treatment, pyridostatin and NU‐7441 were administered intravenously and intraperitoneally, respectively, for four consecutive days, followed by a 3‐day break and four more days of treatment. NU‐7441 was administered 2 h before pyridostatin. At indicated time points, tumour volumes were measured in two dimensions using a caliper and tumour weight was estimated from tumour volume (1 mg = 1 mm3). The student’s t‐test (unpaired, two‐tailed) was used for single pair‐wise comparisons. Differences were considered statistically significant when P < 0.05. Survival curves of mice were processed using the Kaplan–Meier method, and statistical significance was assessed by log‐rank test. Data were plotted using GraphPad Prism Software 8.3.[4]

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Generation of PDTX models[4]
Fresh tumour samples from patients with gBRCA breast cancer were prospectively collected for implantation into mice under an institutional IRB‐approved protocol and the associated informed consent, or by the National Research Ethics Service, Cambridgeshire 2 REC (REC reference number: 08/H0308/178) (Bruna et al, 2016).The VHI0179 patient‐derived tumour xenografts (PDTXs) were generated from a patient breast tumour with a BRCA1 germline truncation and resistant to Olaparib due to REV7 mutation. Written informed consent was obtained from all patients and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Frozen tumour fragments (15–20 mm3) were coated in Matrigel and implanted using a small incision in a subcutaneous pocket made in one side of the lower back into one CB17‐SCID female mice 6 weeks old. When the tumour reached approximately 400 mm3, tumour was explanted from the sacrificed mouse, cut into fragments of about 15–20 mm3 and implanted again subcutaneously in fourteen CB17‐ SCID female mice. When the tumour reached approximately 200 mm3, mice were randomised in vehicle and treated group to start the treatments. Each experimental group included seven mice.

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Pyridostatin induced DNA damage and cell cycle arrest in human cancer cells. [1]
- Long-term treatment (72 h or longer) caused apoptosis in some cells, as indicated by PARP-1 cleavage. [1]
- DNA-PKcs-deficient MO59J cells were more sensitive to pyridostatin than DNA-PKcs-proficient MO59K cells. [1]

[1]. Small-molecule-induced DNA damage identifies alternative DNA structures in human genes. Nat Chem Biol. 2012;8(3):301-310. Published 2012 Feb 5.

[2]. A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands. Nat Chem. 2011;3(10):782-787. Published 2011 Aug 28.

[3]. The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons. Aging (Albany NY). 2017;9(9):1957-1970.

Pyridostatin is a highly selective G-quadruplex binding small molecule designed to target polymorphic G-quadruplex structures regardless of sequence variability. [1]
- The compound induces replication- and transcription-dependent DNA damage, leading to cell cycle arrest and transcriptional downregulation of genes containing G-quadruplex-forming sequences. [1]
- ChIP-Seq analysis revealed that pyridostatin targets gene bodies with clusters of potential G-quadruplex sequences, including the proto-oncogene SRC. [1]
- Pyridostatin reduced SRC protein levels and SRC-dependent cell motility in breast cancer cells, validating SRC as a target. [1]
- The study provides evidence for the existence of pre-folded G-quadruplex structures in unperturbed human cells, based on colocalization of labeled pyridostatin with the G-quadruplex helicase hPif1 in fixed cells. [1]
- The copper-catalyzed alkyne-azide cycloaddition (click chemistry) was used to fluorescently label pyridostatin in cells, enabling visualization of its localization. [1]

C35H34N10O7

706.70726

596.249

1085412-37-8

Pyridostatin hydrochloride;1781882-65-2; 1472611-44-1 (TFA salt)

25227847

Typically exists as solid at room temperature

1.4±0.1 g/cm3

753.8±60.0 °C at 760 mmHg

409.7±32.9 °C

0.0±2.5 mmHg at 25°C

1.726

0.59

5

11

13

44

850

0

C1=CC=C2C(=C1)C(=CC(=N2)NC(=O)C3=CC(=CC(=N3)C(=O)NC4=NC5=CC=CC=C5C(=C4)OCCN)OCCN)OCCN

VGHSATQVJCTKEF-UHFFFAOYSA-N

InChI=1S/C31H32N8O5/c32-9-12-42-19-15-24(30(40)38-28-17-26(43-13-10-33)20-5-1-3-7-22(20)36-28)35-25(16-19)31(41)39-29-18-27(44-14-11-34)21-6-2-4-8-23(21)37-29/h1-8,15-18H,9-14,32-34H2,(H,36,38,40)(H,37,39,41)

4-(2-aminoethoxy)-2-N,6-N-bis[4-(2-aminoethoxy)quinolin-2-yl]pyridine-2,6-dicarboxamide

Pyridostatin; 1085412-37-8; Pyridostain; 4-(2-Aminoethoxy)-N2,N6-bis(4-(2-aminoethoxy)quinolin-2-yl)pyridine-2,6-dicarboxamide; RR-82; RR 82; 4-(2-aminoethoxy)-2-N,6-N-bis[4-(2-aminoethoxy)quinolin-2-yl]pyridine-2,6-dicarboxamide; RR82 hydrochloride;

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.)