ART558

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]。