Background
LY2606368, an ATP-competitive CHK1 inhibitor, is currently in the clinical stage with a Ki of 0.9 nM and an IC50 of <1 nM. LY2606368 inhibits CHK2 (IC50=8 nM) and RSK1 (IC50=9 nM)[2].
Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe[1].LY2606368 triggered S-phase DNA damage, such as pH2AX (S139) and TUNEL positive staining cells significantly increased in S-phase cells. LY2606368 also requires CDC25A and CDK2 to trigger DNA damage. In addition, LY2606368 causes replication catastrophe[1]. In primary patient-derived osteosarcoma cells, LY2606368 alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double-stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations[3]. To investigate the combined effect of the Chk1 inhibitor LY2606368and antitumor drugs (GEM and S 1) on pancreatic cancer cell line SUIT 2. Acombination of LY2606368 and GS showed effective induction of apoptosis[4]. HGSOC cell lines were also sensitive to LY2606368, associated with induction of DNA damage and replication stress. LY2606368 also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability[5]. BRCAwt HGSOC develops resistance to LY2606368 monotherapy via a prolonged G2 delay induced by lower CDK1/CyclinB1 activity, thus preventing cells from mitotic catastrophe and cell death[6]. PLK1 or CHEK1 inhibitors (BI-2536 or LY2606368) were found to exert a superior anticancer effect against cell lines at low nanomolar concentrations and induce cell-cycle arrest[7].
Up to 72.3% inhibition of tumor growth was observed in the three doses of LY2606368 tested, and mice lost no more than 3% of their body weight, indicating that LY2606368 was well tolerated in any treatment group. In addition, tumor regrowth was slow in the highest dose group during the 28-day recovery period, indicating a durable tumor response to LY2606368[1].
参考文献:
[1]: King C, Diaz HB,et,al. Barda D, Marshall MS. LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms. Mol Cancer Ther. 2015 Sep;14(9):2004-13. doi: 10.1158/1535-7163.MCT-14-1037. Epub 2015 Jul 3. PMID: 26141948.
[2]: Yin Y, Shen Q, et,al. Chk1 inhibition potentiates the therapeutic efficacy of PARP inhibitor BMN673 in gastric cancer. Am J Cancer Res. 2017 Mar 1;7(3):473-483. PMID: 28401005; PMCID: PMC5385637.
[3]: Heidler CL, Roth EK, et,al. Prexasertib (LY2606368) reduces clonogenic survival by inducing apoptosis in primary patient-derived osteosarcoma cells and synergizes with cisplatin and talazoparib. Int J Cancer. 2020 Aug 15;147(4):1059-1070. doi: 10.1002/ijc.32814. Epub 2019 Dec 19. PMID: 31782150; PMCID: PMC7384073.
[4]: Morimoto Y, Takada K, et,al. Prexasertib increases the sensitivity of pancreatic cancer cells to gemcitabine and S?1. Oncol Rep. 2020 Feb;43(2):689-699. doi: 10.3892/or.2019.7421. Epub 2019 Nov 28. PMID: 31789403.
[5]: Parmar K, Kochupurakkal BS, et,al. The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition. Clin Cancer Res. 2019 Oct 15;25(20):6127-6140. doi: 10.1158/1078-0432.CCR-19-0448. Epub 2019 Aug 13. PMID: 31409614; PMCID: PMC6801076.
[6]: Nair J, Huang TT, et,al. Resistance to the CHK1 inhibitor prexasertib involves functionally distinct CHK1 activities in BRCA wild-type ovarian cancer. Oncogene. 2020 Aug;39(33):5520-5535. doi: 10.1038/s41388-020-1383-4. Epub 2020 Jul 9. PMID: 32647134; PMCID: PMC7426265.
[7]: Yoshida K, Yokoi A, et,al. Aberrant Activation of Cell-Cycle-Related Kinases and the Potential Therapeutic Impact of PLK1 or CHEK1 Inhibition in Uterine Leiomyosarcoma. Clin Cancer Res. 2022 May 13;28(10):2147-2159. doi: 10.1158/1078-0432.CCR-22-0100. PMID: 35302600.
LY2606368 是一种 ATP 竞争性 CHK1 抑制剂，目前处于临床阶段，Ki 为 0.9 nM，IC50 <1 nM。 LY2606368 抑制 CHK2 (IC50=8 nM) 和 RSK1 (IC50=9 nM)[2]。
用 LY2606368 处理细胞导致 TUNEL 和 pH2AX- 快速出现S 期细胞群中的阳性双链 DNA 断裂。半胱天冬酶抑制剂 Z-VAD-FMK 对细胞凋亡的抑制对染色体断裂没有影响，表明 LY2606368 导致复制灾难[1]。LY2606368 引发 S 期 DNA 损伤，如 pH2AX (S139) S期细胞中TUNEL阳性染色细胞明显增多。 LY2606368 还需要 CDC25A 和 CDK2 来触发 DNA 损伤。此外，LY2606368 会导致复制灾难[1]。在原发性患者来源的骨肉瘤细胞中，LY2606368 单独导致低纳摩尔浓度下的克隆形成存活率大大降低，并通过影响细胞周期进程、诱导细胞凋亡和诱导双链 DNA 断裂而起作用，浓度远低于临床可耐受和安全的血浆浓度[3]。研究Chk1抑制剂LY2606368和抗肿瘤药物（GEM和S 1）联合作用对胰腺癌细胞系SUIT 2的影响。LY2606368和GS联用显示出有效诱导细胞凋亡[4]。 HGSOC 细胞系也对 LY2606368 敏感，与 DNA 损伤和复制应激的诱导有关。 LY2606368 还使这些细胞系对 PARP 抑制敏感，并损害 HR 修复和复制叉稳定性[5]。 BRCAwt HGSOC 通过较低的 CDK1/CyclinB1 活性诱导的延长的 G2 延迟对 LY2606368 单一疗法产生耐药性，从而防止细胞发生有丝分裂灾难和细胞死亡[6]。 PLK1 或 CHEK1 抑制剂（BI-2536 或 LY2606368）被发现在低纳摩尔浓度下对细胞系发挥优异的抗癌作用，并诱导细胞周期停滞[7]。
在所测试的三个剂量的 LY2606368 中观察到高达 72.3% 的肿瘤生长抑制，小鼠体重减轻不超过 3%，表明 LY2606368 在任何治疗组中都具有良好的耐受性。此外，在 28 天的恢复期内，最高剂量组的肿瘤再生缓慢，表明对 LY2606368[1] 的持久肿瘤反应。
Protocol
| Kinase experiment [1]: |
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Preparation Method
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Purified CHK1 was incubated with LY2606368 and substrate was added to detect whether LY2606368 was inhibited.
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Reaction Conditions
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10-6-10nM LY2606368
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Applications
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LY2606368 is an ATP-competitive protein kinase inhibitor with a Ki of 0.9 nmol/L against purified CHK1.
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| Cell experiment [2]: |
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Cell lines
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HeLa cells
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Preparation Method
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Cells were treated with LY2606368 for 7 hours and stained for evidence of DNA DSB using both TUNEL and an antibody for H2AX phosphorylated on serine 139.
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Reaction Conditions
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33 nmol/L LY2606368 for 7 hours
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Applications
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LY2606368 triggered S-phase DNA damage, such as pH2AX (S139) and TUNEL positive staining cells significantly increased in S-phase cells. LY2606368 also requires CDC25A and CDK2 to trigger DNA damage. In addition, LY2606368 causes replication catastrophe.
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| Animal experiment [3]: |
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Animal models
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Female CD-1nu-/nu mice carrying CALU-6 tumors (26-28 g)
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Preparation Method
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Vehicle consisting of 20% Captisol (CyDex Inc) pH4 or LY2606368 was administered by subcutaneous injection in a volume of 200 ml(10 mmol/L). 4,8, 12, 24, and 48 hours after drug administration, blood for plasma drug exposure was extracted
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Dosage form
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1, 3.3, or 10 mg/kg LY2606368 twice daily for 2 days
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Applications
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Up to 72.3% inhibition of tumor growth was observed in the three doses of LY2606368 tested, and mice lost no more than 3% of their body weight, indicating that LY2606368 was well tolerated in any treatment group. In addition, tumor regrowth was slow in the highest dose group during the 28-day recovery period, indicating a durable tumor response to LY2606368.
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参考文献:
[1]. King C, Diaz HB, et,al. LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms. Mol Cancer Ther. 2015 Sep;14(9):2004-13. doi: 10.1158/1535-7163.MCT-14-1037. Epub 2015 Jul 3. PMID: 26141948.
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