ART558
目录号 : GC62553
ART558是一种纳米摩尔强效、选择性、低分子量(MW)的DNA聚合酶Polθ的抑制剂，IC50为7.9 nM。
Cas No.:2603528-97-6
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
Mouse embryonic stem cells (mESC) |
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Preparation Method |
Wild-type and Pol-θ-deficient mESC were pretreated with ART558 at different concentrations and transfected with Cas9-wild type (WT) and sgHPRT_ex3.1. |
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Reaction Conditions |
0-50μM;2days |
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Applications |
ART558 exhibited maximal reduction in Cas9-induced mutation frequency at a concentration of 10 μM in wild-type cells, without exerting additional effects on the already reduced mutation frequency observed in Polϴ-deficient cells. |
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References: |
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ART558 is a potent and selective low-molecular-weight allosteric inhibitor of DNA polymerase Polθ, with a molecular weight (MW) in the nanomolar range and an IC50 of 7.9 nM. It functions by stabilizing the closed conformation of Polθ while bound to DNA, effectively blocking its polymerase activity. ART558 specifically targets Theta-Mediated End Joining, a major DNA repair process mediated by Polθ, while sparing Non-Homologous End Joining mechanisms [1-3].
ART558 (0-50 μM; 2 days) demonstrated maximal reduction of Cas9-induced mutation frequency at 10 μM in wild-type cells. Additionally, ART558(10 μM) attenuates mutagenic repair at Cas9-induced breaks in mouse embryonic stem cells [3]. ART558 (1-3 μM) induces Polθ-dependent radiosensitization in cancer cell lines. The cell-cycle-related effects of Polθ inhibition by ART558 in combination with radiation are also observed [4]. In the absence of Polθ, RAD52 accumulations suppress single-stranded DNA (ssDNA) gap-filling during the G2/M phase and promote MRE11 nuclease accumulation. Conversely, the survival of BRCA1-deficient cells treated with the Polθ inhibitor ART558 (10 μM) is not rescued by RAD52 suppression. Instead, ssDNA gap-filling is hindered by the direct inhibition of the polymerase activity itself [5]. Furthermore, ART558 (0–25 μM; 2 days) sensitized both pancreatic cell lines (Capan-1 and HTERT HPNE cells) to sub-lethal doses of cisplatin [6].
References:
[1]. Zatreanu, D., Robinson, et al. Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance. Nat Commun 12, 3636 (2021). https://doi.org/10.1038/s41467-021-23463-8
[2]. Stockley ML, Ferdinand A, et al. Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta. J Med Chem. 2022 Oct 27;65(20):13879-13891. doi: 10.1021/acs.jmedchem.2c01142. Epub 2022 Oct 6. Erratum in: J Med Chem. 2023 Feb 23. doi: 10.1021/acs.jmedchem.3c00204. PMID: 36200480.
[3]. Schimmel J, Muñoz-Subirana N, et al. Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways. Cell Rep. 2023 Feb 28;42(2):112019. doi: 10.1016/j.celrep.2023.112019. Epub 2023 Jan 25. PMID: 36701230
[4]. Rodriguez-Berriguete G, et al. Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models. Clin Cancer Res. 2023 Apr 14;29(8):1631-1642. doi: 10.1158/1078-0432.CCR-22-2977. PMID: 36689546; PMCID: PMC10102842.
[5]. Ronson GE, Starowicz K, et al. Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting. Nat Commun. 2023 Nov 29;14(1):7834. doi: 10.1038/s41467-023-43677-2. PMID: 38030626; PMCID: PMC10687250.
[6]. Bugbee T, Gathoni M, et al. Inhibition of p300 increases cytotoxicity of cisplatin in pancreatic cancer cells. Gene. 2023 Dec 20;888:147762. doi: 10.1016/j.gene.2023.147762. Epub 2023 Sep 2. PMID: 37666373; PMCID: PMC10563798.
ART558是一种纳米摩尔强效、选择性、低分子量(MW)的DNA聚合酶Polθ的抑制剂，IC50为7.9 nM。ART558通过稳定其与DNA结合时的封闭构象来阻断Polϴ的聚合酶活性。ART558主要抑制由polθ介导的DNA修复中的末端连接,但不靶向非同源末端连接[1-3]。
ART558(0-50μM;2days)在野生型细胞中, 10 μM时最大限度地降低了Cas9诱导的突变频率。在小鼠胚胎干细胞中，ART558(10 μM )减少Cas9诱导的断裂的突变修复[3]。ART558(1-3μM)诱导癌细胞polθ依赖性放射致敏。ART558联合放疗抑制Polθ的细胞周期相关效应[4]。在缺乏Polθ的情况下，RAD52的积累抑制了G2/M期单链DNA (ssDNA)的间隙填充，促进了MRE11核酸酶的积累。用Polθ抑制剂ART558 (10 μM)处理的BRCA1缺陷细胞的存活不能通过RAD52抑制而恢复。相反，直接抑制聚合酶活性本身会阻碍ssDNA的缺口填充[5]。此外，ART558 (0-25 μM;2days)使两种胰腺细胞系(Capan-1和HTERT HPNE细胞)对亚致死剂量的顺铂致敏[6]。
| Cas No. | 2603528-97-6 | SDF | |
| 分子式 | C21H21F3N4O2 | 分子量 | 418.41 |
| 溶解度 | 85 mg/mL in DMSO(Need ultrasonic) | 储存条件 | 4°C, away from moisture and light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.39 mL | 11.95 mL | 23.9 mL |
| 5 mM | 0.478 mL | 2.39 mL | 4.78 mL |
| 10 mM | 0.239 mL | 1.195 mL | 2.39 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance
Nat Commun 2021 Jun 17;12(1):3636.PMID:34140467DOI:10.1038/s41467-021-23463-8.
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.
Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta
J Med Chem 2022 Oct 27;65(20):13879-13891.PMID:36200480DOI:10.1021/acs.jmedchem.2c01142.
Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.
Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
Clin Cancer Res 2023 Apr 14;29(8):1631-1642.PMID:36689546DOI:10.1158/1078-0432.CCR-22-2977.
Purpose: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. Experimental design: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. Results: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. Conclusions: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.
Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways
Cell Rep 2023 Jan 25;42(2):112019.PMID:36701230DOI:10.1016/j.celrep.2023.112019.
Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.