Chk1 (IC50 = 1.2 nM)
Checkpoint Kinase 1 (CHK1) [1]
Checkpoint Kinase 1 (CHK1) [2]

体外活性：GDC-0575（也称为 ARRY-575、RG7741）是一种新型、有效、选择性的 CHK1 抑制剂，可特异性结合并抑制 CHK1，IC50 为 1.2 nM。这使得肿瘤细胞能够绕过 S 期和 G2/M 期的 CHK1 依赖性细胞周期停滞，从而允许细胞在进入有丝分裂之前进行 DNA 修复。 CHK1 是一种 ATP 依赖性丝氨酸-苏氨酸激酶，可磷酸化 cdc25 磷酸酶以响应 DNA 损伤。因此，CHK1 抑制可能会使肿瘤细胞对某些化疗药物的 DNA 损伤作用敏感。激酶测定：GDC-0575（也称为 ARRY-575、RG7741）是一种新型、有效、选择性的 CHK1 抑制剂，可特异性结合并抑制 CHK1，IC50 为 1.2 nM。细胞测定：在一组黑色素瘤细胞系中，GDC-0575 在促进 DNA 损伤、复制应激和细胞死亡方面比 V158411、LY2603618 和 MK-8776 更有效。 GDC-0575 消除 DNA 损伤诱导的 S 和 G2-M 检查点，加剧 DNA 双链断裂并诱导 STS 细胞凋亡。 GDC-0575与吉西他滨一起具有协同或相加作用。 CHK1 抑制剂 GDC-0575 与 AraC 组合可通过诱导细胞凋亡增强对原代急性髓系白血病细胞的体外杀伤。

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与阿糖胞苷（AraC）联合使用时，GDC-0575 增强了AraC对多种急性髓系白血病（AML）细胞系的细胞毒性，与单独使用AraC相比，细胞代谢活性降低且凋亡率升高；当细胞系接种在照射后的MS5基质层上时，这种增强作用仍然存在[1]
- 在原代AML样本中，GDC-0575 与AraC联合使用提高了AraC的杀伤效果，且不增加对L-LTC-ICs的耗竭作用；细胞周期分析显示其对G0期细胞比例有影响[1]
- 部分黑色素瘤细胞系对 GDC-0575 诱导的细胞死亡高度敏感，该药物可在细胞不进入有丝分裂的情况下触发细胞死亡；GDC-0575 处理仅在高敏感黑色素瘤细胞中引起强烈的RPA2过度磷酸化和DNA损伤增加，这与S期细胞周期检查点缺陷相关[2]

GDC-0575 是一种高选择性口服小分子 CHK1 抑制剂，可导致异种移植模型中的肿瘤缩小和生长延迟，单药剂量为 25 mg/kg 时即可发挥作用，但药物剂量较高时疗效会提高。 GDC-0575有效阻断D20和C002异种移植物中的肿瘤生长，并且在施用最终剂量后效果维持至少10天

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在U937-Luc和HL60-Luc移植瘤小鼠模型中，AraC与 GDC-0575 联合治疗通过体内生物发光成像（BLI）检测显示可抑制肿瘤增殖，并降低移植小鼠骨髓中白血病细胞的比例[1]
- 在移植了原代AML患者样本的小鼠中，无论在中植入（ME）还是高植入（HE）阶段开始治疗，AraC与 GDC-0575 联合使用均降低了骨髓中人源细胞的百分比；该联合治疗不影响正常长期造血干细胞/祖细胞，不会产生新的突变，且能消除AraC诱导的突变克隆[1]
- 在AraC+GDC-0575 联合治疗基础上添加粒细胞集落刺激因子（G-CSF），在体内进一步增强了对AML的细胞毒性，减少了残留白血病细胞，提高了增殖期Ki67+CD45+细胞的比例，并降低了L-LTC-IC频率[1]
- 在具有内源性复制应激升高的黑色素瘤移植瘤模型中，GDC-0575 在体内能有效抑制肿瘤生长[2]

GDC-0575，也称为 ARRY-575 或 RG7741，是一种新型、强效、选择性的 CHK1 抑制剂，可与其特异性结合并抑制它，IC50 为 1.2 nM。

在共培养研究中，在共培养开始前两天将饲养细胞接种到 I 型胶原蛋白包被的 96 孔或 6 孔板上，并使其达到汇合。更换培养基，并在共培养开始前一天接受 6.8 Gy 的辐射。首先，在共培养的第 0 天，使用适当的 AmL 培养基以 2 × 10⁵ 细胞/mL 的密度对 AmL 细胞进行铺板。在具有 5% CO₂ 和指定氧气浓度的培养箱中，细胞在 37°C 下培养。在短期培养 (STC) 期间，细胞在缺氧 (5% O₂) 下维持一周，同时用 500 nM AraC 和/或 100 nM GDC-0575 处理[1]。

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AML细胞系处理实验：四种AML细胞系分别用AraC单独处理或AraC+GDC-0575 联合处理1天，以未处理细胞为对照，检测细胞代谢活性和凋亡率，评估细胞毒性作用[1]
- 基质层培养细胞实验：将AML细胞系接种在照射后的MS5基质层上，进行上述相同处理后，测定凋亡细胞比例[1]
- 原代AML样本培养及分析实验：对原代AML样本在不同处理条件下进行短期培养（STC）和长期培养（LTC），通过计数珠计数并归一化到未处理组评估存活率；通过在甲基纤维素中培养细胞2周进行L-LTC-IC/LDA实验，检测L-LTC-IC频率[1]
- 细胞周期分析实验：原代AML样本处理1周后，通过Ki67/DAPI表达检测细胞周期分布，重点关注G0期细胞[1]
- 黑色素瘤细胞敏感性实验：用 GDC-0575 处理黑色素瘤细胞系，评估细胞死亡情况；在高敏感细胞系中，检测 GDC-0575 处理后RPA2磷酸化水平、DNA损伤情况及细胞周期进程，分析细胞对药物的应答[2]

Intravenous injections of 1 × 10⁵-10⁶ AmL and 1-3 × 10⁵ hCB CD34+/hBM CD34⁺ cells are administered to NSG mice. Mice are given a proper 7-day treatment regimen after proving AmL engraftment through FACS analysis of tibia bone marrow aspiration at 9–11 weeks. This includes daily subcutaneous injection of 10 mg/kg AraC, oral gavage of 7.5 mg/kg GDC-0575 suspension every other day, and/or intraperitoneal injection of 300 μg/kg G-CSF every day for 5 days. Mice die from cervical dislocation one week after the last dose. After being dissected, the tibias, pelvis, and femurs are flushed with PBS. Ammonium chloride lyses red blood cells. Human-specific PE-conjugated anti-CD33, PE-FITC-conjugated anti-CD19, PE-Cy7-conjugated anti-CD45, and PERCP-conjugated anti-murine CD45 antibodies are used to stain cells. DAPI staining is used to weed out dead cells and debris. Analysis is done using a BD LSR II flow cytometer. FlowJo software is used for flow cytometry analysis. Over 100,000 DAPI-negative occurrences are gathered. If there is only one population of mCD45^-hCD45⁺CD33⁺CD19^- cells and no mCD45-hCD45⁺CD33^-CD19⁺ cells nearby, it is considered that AmL is engrained[1].

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U937-Luc and HL60-Luc xenograft model: Mice were injected with Luc-marked tumor cells and treated with AraC and GDC-0575 according to a specific regimen. In vivo bioluminescence imaging (BLI) was performed 15 days (for U937-Luc) and 30 days (for HL60-Luc) after cell injection to quantify tumor proliferation kinetics; the proportion of tumor cells in the bone marrow was detected 3 days (D15 for U937-Luc) and 30 days (D30 for HL60-Luc) post-treatment [1]
- Primary AML patient sample xenograft model: Mice were transplanted with primary AML patient samples and treated with AraC, GDC-0575 alone or in combination, starting at either medium engraftment (ME) or high engraftment (HE). Mice were euthanized 1 week post-treatment, and the percentage of human cells in the bone marrow was detected; body weight changes of AML6-injected mice were monitored, and mice were euthanized when body weight decreased by 20%; hCD45/CD33+ cells from AML6-injected mice 1 week post-treatment were collected for secondary transplantation [1]
- Normal hematopoietic stem cell transplantation model: Mice were transplanted with human cord blood CD34+ cells and treated with the combination of AraC and GDC-0575. The percentage of human cells, HSPCs (Lin−CD34+CD38−) and HPCs (Lin−CD34+CD38+) in the bone marrow was detected 1 week and 8 weeks post-treatment; hCD45+ cells from mice 1 week post-treatment were collected for secondary transplantation [1]
- Triple combination therapy model: Mice transplanted with primary AML samples were treated with AraC+GDC-0575+G-CSF starting at high engraftment, and the percentage of human cells in the bone marrow was detected 1 week post-treatment; for AML7-transplanted mice, residual leukemic cells were detected by hematoxylin & eosin (H&E) staining and immunofluorescence, and the ratio of Ki67+CD45+ cells was analyzed; hCD45+ cells from AML7-injected mice were collected for L-LTC-IC assay, and hCD45+ cells from AML8-injected mice were collected for secondary transplantation [1]
- Human bone marrow transplantation model: Mice were transplanted with human bone marrow cells and treated with G-CSF, and the percentage of human cells in the bone marrow was detected 1 week and 8 weeks post-treatment [1]
- Melanoma xenograft model: Mice were transplanted with melanoma cells to establish xenografts, then treated with GDC-0575 to observe the inhibitory effect on tumor growth [2]

The combination of AraC and GDC-0575 did not affect normal long-term hematopoietic stem/progenitors and did not induce de novo mutations [1]
- Body weight changes of AML6-injected mice were monitored during treatment, and mice were euthanized when a 20% reduction in body weight was reached[1]

[1]. The combination of CHK1 inhibitor with G-CSF overrides cytarabine resistance in human acute myeloid leukemia. Nat Commun. 2017 Nov 22;8(1):1679.

[2]. Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo. Clin Cancer Res. 2018 Jun 15;24(12):2901-2912.

GDC-0575 can override cytarabine resistance in human acute myeloid leukemia when combined with G-CSF, and its mechanism may be related to inhibiting DNA repair; persistent residual leukemic cells are quiescent, and G-CSF can force these cells into the cell cycle, thereby enhancing the efficacy of the triple combination therapy (AraC+GDC-0575+G-CSF), which provides a more efficient treatment option for AML in the clinic [1]
- Endogenous replication stress marks melanomas sensitive to GDC-0575; GDC-0575 induces cell death in sensitive melanomas by increasing replication stress and DNA damage; the number and intensity of pRPA2 Ser4/8 foci in untreated tumors can serve as a useful marker of replication stress in vivo to predict the sensitivity of melanomas to GDC-0575 [2]

C17H23BRCL2N4O

450.200721025467

449.038

1657014-42-0

GDC-0575;1196541-47-5;GDC0575 hydrochloride;1196504-54-7

131749450

Light yellow to yellow solid

0

87

5

4

3

25

460

1

C1C[C@H](CN(C1)C2=C3C(=CNC3=NC=C2Br)NC(=O)C4CC4)N.Cl.Cl

OYMHZTORKRPOBI-YQFADDPSSA-N

InChI=1S/C16H20BrN5O.2ClH/c17-11-6-19-15-13(14(11)22-5-1-2-10(18)8-22)12(7-20-15)21-16(23)9-3-4-9;;/h6-7,9-10H,1-5,8,18H2,(H,19,20)(H,21,23);2*1H/t10-;;/m1../s1

N-[4-[(3R)-3-aminopiperidin-1-yl]-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl]cyclopropanecarboxamide;dihydrochloride

ARRY-575 2HCl; ARRY-575; ARRY575; RG7741; RG-7741 2HCl; RG 7741; GDC-0575; GDC 0575 2HCl; GDC0575; AK 687476 2HCl; AK-687476; AK687476 dihydrochloric acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.17 mg/mL (4.81 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 21.7 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.17 mg/mL (4.81 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 21.7 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.17 mg/mL (4.81 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 21.7 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 10 mg/mL (22.16 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。