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.

---

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
7.5 mg/kg
i.v.

---

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]
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]

---

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.

[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 that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. Here, we have used a laser tweezers method to investigate such interactions. With a force jump approach, we observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant K(d) of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical K(d) for pyridostatin and a K(d) of 42 ± 3 µM for a weaker ligand RR110. We anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance.[1]

---

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.[2]

---

Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.[3]

---

The cells with compromised BRCA1 or BRCA2 (BRCA1/2) function accumulate stalled replication forks, which leads to replication-associated DNA damage and genomic instability, a signature of BRCA1/2-mutated tumours. Targeted therapies against BRCA1/2-mutated tumours exploit this vulnerability by introducing additional DNA lesions. Because homologous recombination (HR) repair is abrogated in the absence of BRCA1 or BRCA2, these lesions are specifically lethal to tumour cells, but not to the healthy tissue. Ligands that bind and stabilise G-quadruplexes (G4s) have recently emerged as a class of compounds that selectively eliminate the cells and tumours lacking BRCA1 or BRCA2. Pyridostatin is a small molecule that binds G4s and is specifically toxic to BRCA1/2-deficient cells in vitro. However, its in vivo potential has not yet been evaluated. Here, we demonstrate that pyridostatin exhibits a high specific activity against BRCA1/2-deficient tumours, including patient-derived xenograft tumours that have acquired PARP inhibitor (PARPi) resistance. Mechanistically, we demonstrate that pyridostatin disrupts replication leading to DNA double-stranded breaks (DSBs) that can be repaired in the absence of BRCA1/2 by canonical non-homologous end joining (C-NHEJ). Consistent with this, chemical inhibitors of DNA-PKcs, a core component of C-NHEJ kinase activity, act synergistically with pyridostatin in eliminating BRCA1/2-deficient cells and tumours. Furthermore, we demonstrate that pyridostatin triggers cGAS/STING-dependent innate immune responses when BRCA1 or BRCA2 is abrogated. Paclitaxel, a drug routinely used in cancer chemotherapy, potentiates the in vivo toxicity of pyridostatin. Overall, our results demonstrate that pyridostatin is a compound suitable for further therapeutic development, alone or in combination with paclitaxel and DNA-PKcs inhibitors, for the benefit of cancer patients carrying 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.

---

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