G-quadruplexe (Kd = 490 nM)

Pyridostatin (RR82) hydrochloride (10 μM; 48 hours) induces cell cycle arrest[1].
Pyridostatin hydrochloride is a small molecule that selectively binds to G-quadruplex DNA, forming a complex that stabilizes the G-quadruplex structure. Neurite retraction, synaptic loss, and dose-dependent neuronal death are brought on by pyridostatin hydrochloride. Pyridostatin hydrochloride induces the formation of DNA double-strand breaks in primary neurons in culture. The BRCA1 protein, which protects and fixes the neuronal genome, is remarkably downregulated at the transcriptional level by pyridogallostatin hydrochloride (1–5 μM, overnight)[3].

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]

Cell Line: Over 60 different cancer cell lines
Concentration: 10 μM
Incubation Time: 48 hours
Result: Predominantly accumulated in the G2 phase of the cell cycle over 60 different cancer cell lines.

CB17‐SCID mice
\n7.5 mg/kg
\ni.v.

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\n\nIn vivo xenograft experiments[4]
\nCB17‐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]
\n\nTo 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]
\n\nTo 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]
\n\nTo 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]
\n\nTalazoparib (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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\n\nGeneration of PDTX models[4]
\nFresh 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).\n\nThe 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.

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

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

[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.

[4]. Anti-tumoural activity of the G-quadruplex ligand pyridostatin against BRCA1/2-deficient tumours. EMBO Mol Med . 2022 Mar 7;14(3):e14501.

Ligands capable of stabilizing telomeric DNA G-quadruplex formation hold potential as cancer therapeutics because telomerase (an enzyme overexpressed in many cancer cells) cannot elongate G-quadruplex structures. Therefore, understanding the kinetics, thermodynamics, and mechanical properties of small molecules binding to these structures is crucial, but conventional monolithic analytical methods cannot simultaneously measure these properties. In this paper, we investigated such interactions using laser tweezers. Using a kinetic spur, we observed that pyridostatine promotes the folding of telomeric G-quadruplexes. The enhanced mechanical stability of the pyridostatine-bound G-quadruplex allowed us to determine its dissociation constant K(d) to be 490 ± 80 nM. The binding free energy change obtained via a Hess-like process confirmed that K(d) for pyridostatine was consistent with the above results, while the K(d) for the weaker ligand RR110 was 42 ± 3 µM. We anticipate that this single-molecule platform will provide detailed information on the biologically significant mechanical, kinetic, and thermodynamic properties of ligand-bound biomacromolecules. [1]

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Guinein-rich DNA sequences can form non-Watson-Crick base structures in vitro, which are ubiquitous in the human genome. However, whether such structures are normally present in mammalian cells has been a hot topic of research for decades. This paper shows that the G-quadruplex-interacting drug pyridostatine promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. Chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX revealed the distribution of pyridostatine-induced damage sites across the genome and showed that pyridostatine targets genes containing clusters of G-quadruplex sequences. Thus, pyridostatine regulates the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatine reduced the abundance of SRC protein and SRC-dependent cell motility in human breast cancer cells, confirming that SRC is the target of the drug. We used an unbiased approach to determine the genomic site of action of the drug, establishing a framework for discovering functional DNA-drug interactions. [2] Viruses capable of establishing latent infection have evolved unique mechanisms to evade host immune recognition. These viruses' maintenance proteins regulate their synthesis levels to maintain persistent infection but below the host's immune detection threshold. The mechanisms controlling this fine-tuning of viral latency remain unclear. In this study, we discovered that the mRNA encoding gamma herpesvirus maintenance proteins contains clusters of specialized structural elements—G-quadruplexes—within their open reading frames (ORFs), which are responsible for cis-regulating viral mRNA translation. By studying the nuclear antigen 1 (EBNA1) mRNA encoded by Epstein-Barr virus, we found that disrupting G-quadruplexes with antisense oligonucleotides increased EBNA1 mRNA translation. Conversely, pretreatment with the G-quadruplex stabilizer pyridostazine decreased EBNA1 synthesis, highlighting the importance of G-quadruplexes in viral transcripts as unique regulatory signals for translation and immune escape. Furthermore, these findings suggest alternative therapeutic strategies targeting viral open reading frame (ORF) RNA structures. [3]

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Impaired BRCA1 or BRCA2 (BRCA1/2) function leads to the accumulation of arrested replication forks, resulting in replication-related DNA damage and genomic instability, characteristic of BRCA1/2 mutant tumors. Targeted therapies for BRCA1/2 mutant tumors exploit this vulnerability by introducing additional DNA damage. Since BRCA1 or BRCA2 deficiency leads to impaired homologous recombination (HR) repair, this damage is specifically lethal to tumor cells but harmless to healthy tissues. In recent years, ligands that bind to and stabilize G-quadruplexes (G4) have emerged as a class of compounds capable of selectively eliminating BRCA1 or BRCA2-deficient cells and tumors. Pyridostatine is a small molecule that binds to G4 and exhibits specific toxicity to BRCA1/2-deficient cells in vitro. However, its in vivo activity has not been evaluated. In this paper, we demonstrate that pyridostatine exhibits highly specific activity against BRCA1/2-deficient tumors, including patient-derived xenografts that have acquired resistance to PARP inhibitors (PARPi). Mechanistically, we demonstrated that pyridostatine interferes with DNA replication, leading to DNA double-strand breaks (DSBs), which can be repaired via the classical non-homologous end joining (C-NHEJ) pathway in the presence of BRCA1/2 deficiency. Consistent with this, chemical inhibitors of DNA-PKcs (a core component of C-NHEJ kinase activity) synergistically with pyridostatine to eliminate BRCA1/2-deficient cells and tumors. Furthermore, we demonstrated that pyridostatine triggers a cGAS/STING-dependent innate immune response when BRCA1 or BRCA2 is knocked out. Paclitaxel, a commonly used chemotherapy drug for cancer, enhances the in vivo toxicity of pyridostatine. In summary, our results suggest that pyridostatine is a compound suitable for further therapeutic development, either alone or in combination with paclitaxel and DNA-PKcs inhibitors, to benefit cancer patients with BRCA1/2 mutations. [4]

C31H37CL5N8O5

778.9411

778.13

1781882-65-2

Pyridostatin;1085412-37-8;Pyridostatin TFA;1472611-44-1

78243739

White to off-white solid

10

11

13

49

850

0

SRIZPFGTXSQRFM-UHFFFAOYSA-N

InChI=1S/C31H32N8O5.5ClH/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);5*1H

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

RR82 hydrochloride; Pyridostatin hydrochloride; 1781882-65-2; Pyridostatin (hydrochloride); RR82 hydrochloride; 4-(2-aminoethoxy)-2-N,6-N-bis[4-(2-aminoethoxy)quinolin-2-yl]pyridine-2,6-dicarboxamide;pentahydrochloride; Pyridostatin pentahydrochloride; RR-82 hydrochloride; 4-(2-aminoethoxy)-N2,N6-bis[4-(2-aminoethoxy)quinolin-2-yl]pyridine-2,6-dicarboxamide pentahydrochloride

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 (e.g. under nitrogen), 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)

H2O: ~50 mg/mL (~64.2 mM)

Solubility in Formulation 1: 100 mg/mL (128.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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