G-quadruplexe ( Kd = 490 nM )

体外活性：吡啶抑素可降低 MRC-5–SV40 细胞和各种癌细胞系的增殖，并通过 DNA 损伤检查点激活诱导细胞周期停滞。 Pyridostatin 还通过与 SRC 中的 G-四联体基序相互作用，降低 MDA-MB-231 细胞中 SRC 依赖性细胞运动。 Pyridostatin 通过抑制 G-四链体来减少 EBNA1 合成。细胞测定：细胞（MRC-5–SV40 细胞和各种癌细胞系）以相等的汇合度铺板，并且在收获细胞之前的指定时间内不进行处理或连续用 2 μM 吡啶他汀处理。来自各个板的细胞被胰蛋白酶消化并在库尔特计数器中计数。图表代表每个时间间隔的细胞总数，误差线代表SEM数据代表三个独立实验。

Pyridostatin对BRCA2缺陷异种移植物具有抗肿瘤活性[4]
结合和稳定G4s的化合物已被证明对小鼠建立的BRCA1/2缺失异种移植物肿瘤有活性(RHPS4和CX‐5461)。然而，这些尚未被证明对BRCA突变患者有益。此外，BRCA突变的肿瘤很难治疗，因为它们对靶向治疗(例如PARP抑制剂;PARPi)。因此，必须找到新的G4配体，不仅可以消除BRCA缺陷肿瘤，还可以抵抗耐药疾病。我们之前发表的结果(Zimmer et al, 2016)表明，G4配体pyridostatin对体外BRCA2缺陷细胞具有特异性毒性。在这项研究中，研究人员评估了pyridostatin在体内消除BRCA2缺陷异种移植物肿瘤中的潜力。为了解决这个问题，我们使用等基因BRCA2 +/+ (BRCA2‐精通)和BRCA2−/−(BRCA2‐缺乏)人结直肠癌DLD1细胞在CB17‐SCID小鼠中产生异种移植物(图1A和B)。研究人员广泛优化了pyridostatin在体内使用的条件，并确定了7.5 mg/kg/天的剂量计划静脉注射连续5天，然后休息2天，第二个5天的治疗耐受性良好。没有明显的体重减轻，没有不良的临床症状(附录表S1)。在这些条件下，研究人员发现pyridostatin有效且特异性地抑制BRCA2‐缺陷DLD1细胞建立的异种移植物肿瘤的生长(图1B)。作为对照，研究人员使用了PARPi talazoparib，该药物以其根除小鼠BRCA1/2缺失肿瘤的能力而闻名(Shen et al .， 2013)，最近被许可用于携带BRCA1/2种系突变的转移性乳腺癌患者(Litton et al .， 2018)。pyridostatin对BRCA2 -缺陷肿瘤的抗肿瘤作用与talazoparib相似，两种药物都不会损害BRCA2 -精通肿瘤的生长。

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此外，研究人员使用第二种肿瘤模型研究了pyridostatin在体内的反应，该模型是由等基因BRCA2 +/+和BRCA2 - / -结直肠癌HCT116细胞建立的(Xu et al .， 2014)。Pyridostatin显示出对BRCA2缺失HCT116细胞源性肿瘤的选择性毒性(附录图S1A和B;附录表S2)，其作用与DLD1细胞来源的异种移植物相似[4]
研究人员之前的研究表明，pyridostatin治疗会导致HR修复受损的细胞(包括BRCA2缺陷细胞)中DNA损伤的积累(Zimmer等，2016)。一致地，免疫组织化学(IHC)分析显示，BRCA2‐缺乏，但不是BRCA2‐完全，肿瘤在暴露于pyridostatin或talazoparib时显示出DNA损伤标记γH2AX水平增加(附录图S1C-F)。这些结果表明，pyridostatin不仅可以特异性抑制细胞的生长(Zimmer等，2016)，还可以特异性抑制缺乏BRCA2的肿瘤，并通过造成DNA损伤在体内起作用[4]。

稳定端粒DNA g -四联体形成的配体具有治疗癌症的潜力，因为g -四联体结构不能被端粒酶延长，而端粒酶在许多癌细胞中过度表达。因此，了解与这些结构结合的小分子的动力学、热力学和力学性质是很重要的，但经典的系综分析无法同时测量这些。在这项研究中，研究人员使用激光镊子方法来研究这种相互作用。通过力跳跃方法，他们观察到pyridostatin促进端粒g -四联体的折叠。pyridostatin结合g -四联体的机械稳定性增加，使得解离常数K(d)的测定为490±80 nM。从类hess过程中获得的结合自由能变化为pyridostatin提供了相同的K(d)，对于较弱的配体RR110, K(d)为42±3µM。研究人员预计，这种单分子平台可以为配体生物大分子的力学、动力学和热力学性质提供详细的见解，这些特性具有生物学相关性。[1]

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建立潜伏感染的病毒已经进化出独特的机制来避免宿主免疫识别。这些病毒的维持蛋白将其合成调节到足以维持持续感染的水平，但低于宿主免疫检测的阈值水平。控制这种病毒潜伏期精细调节的机制尚不清楚。在这项研究中，研究人员发现编码γ疱疹病毒维持蛋白的mRNA在其开放阅读框中含有异常结构元件g -四联体簇，这些结构元件负责顺式调节病毒mRNA翻译。通过研究Epstein-Barr病毒编码的核抗原1 (EBNA1) mRNA，研究人员发现，使用反义寡核苷酸破坏g -四联体的稳定性可以增加EBNA1 mRNA的翻译。相比之下，用稳定g -四联体的小分子pyridostatin< strong>预处理会降低EBNA1的合成，这突出了病毒编码转录物中g -四联体作为翻译控制和免疫逃避的独特调节信号的重要性。此外，这些发现提示了针对病毒orf内RNA结构的替代治疗策略。[3]

克隆生存试验[4]
BRCA2 +/+和BRCA2 - / - DLD1细胞在6孔板中以每孔200至4,000个细胞的密度进行技术复制。在细胞粘附后开始药物治疗。10-14天后，用0.5%结晶紫在50%甲醇和20%乙醇的dH2O中染色。相对于同一细胞系未处理的细胞，表达细胞存活率。 将人MDA - MB - 436细胞以每皿1 × 105个细胞的密度接种于60 - mm培养皿中。第二天，用5µM NU‐7441、0.3µM pyridostatin或0.1-3 nM紫杉醇处理细胞24 h。对于NU‐7441 + pyridostatin或NU‐7441 +紫杉醇联合用药，同时加药24 h。对于pyridostatin +紫杉醇联合用药，细胞用pyridostatin处理24小时，随后用紫杉醇处理24小时。对于NU - 7441 + pyridostatin +紫杉醇组合，细胞用NU - 7441 + pyridostatin处理24小时，随后用紫杉醇处理24小时。为了评估细胞集落形成能力，在处理结束时，用胰蛋白酶将细胞提出来，按每皿1000个细胞的密度进行三次技术复制。13天后，用2%亚甲蓝和60%乙醇对菌落进行染色并计数(> 50个细胞等于一个菌落)。

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在体外可以采用非沃森-克里克结构的富含鸟嘌呤的DNA序列在人类基因组中普遍存在。然而，这种结构是否在哺乳动物细胞中正常存在，几十年来一直是活跃研究的主题。在这里，我们发现g -四重体相互作用的药物pyridostatin通过诱导复制和转录依赖性DNA损伤来促进人类癌细胞的生长停滞。DNA损伤标记γH2AX的染色质免疫沉淀测序分析提供了pyridostatin诱导的损伤位点的全基因组分布，并揭示了pyridostatin靶向含有g -四重体形成倾向的序列簇的基因体。因此，pyridostatin调节这些基因的表达，包括原癌基因SRC。我们观察到pyridostatin降低了人乳腺癌细胞中SRC蛋白的丰度和SRC依赖的细胞运动，验证了SRC作为该药物的靶点。我们不偏不倚的方法来定义药物的基因组作用位点，为发现功能dna -药物相互作用建立了框架。[2]

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将细胞铺板至同等汇合后，在收获前不进行处理或用 2 μM pyridostatin连续处理指定的时间。使用库尔特计数器完成胰蛋白酶消化和单个板细胞的计数。图表上的误差线显示每个时间间隔的 sem 和细胞总数。三个独立的实验由数据表示。

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]
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.

[1]. Nat Chem . 2011 Aug 28;3(10):782-7.

[2]. Nat Chem Biol . 2012 Feb 5;8(3):301-10.

[3]. Nat Chem Biol . 2014 May;10(5):358-64.

[4]. EMBO Mol Med . 2022 Mar 7;14(3):e14501.

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]

C37H35F9N8O11

938.72

710.242

C, 47.34; H, 3.76; F, 18.21; N, 11.94; O, 18.75

1472611-44-1

1781882-65-2; 1629261-49-9 (HCl); 1085412-37-8; 1472611-44-1 (TFA)

117064504

White solid powder

240Ų

FC(C(=O)O)(F)F.FC(C(=O)O)(F)F.FC(C(=O)O)(F)F.O(CCN)C1=CC(NC(C2C=C(C=C(C(NC3C=C(C4=CC=CC=C4N=3)OCCN)=O)N=2)OCCN)=O)=NC2=CC=CC=C12

CYYZQGUDHAKBIQ-UHFFFAOYSA-N

InChI=1S/C31H32N8O5.3C2HF3O2/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;3*3-2(4,5)1(6)7/h1-8,15-18H,9-14,32-34H2,(H,36,38,40)(H,37,39,41);3*(H,6,7)

-(2-aminoethoxy)-N2,N6-bis(4-(2-aminoethoxy)quinolin-2-yl)pyridine-2,6-dicarboxamide tris(2,2,2-trifluoroacetate)

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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