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

GDC-0575 (7.5 mg/kg, p.o.) in conjunction with AraC virtually totally eliminates the leukemic burden in mice receiving transplants of U937-Luc cells and exhibits more effective activity than AraC alone. Moreover, in various primary AML models in vivo, GDC-0575 increases the cytotoxicity of AraC[1]. In D20 and C002 xenografts, GDC-0575 (25, 50 mg/kg, p.o.) dose-dependently inhibits the growth of the tumor[2].

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In U937-Luc and HL60-Luc xenograft mouse models, the combination of AraC and GDC-0575 inhibited tumor proliferation as detected by in vivo bioluminescence imaging (BLI) and reduced the proportion of leukemic cells in the bone marrow of transplanted mice [1]
- In mice transplanted with primary AML patient samples, whether treatment was initiated at medium engraftment (ME) or high engraftment (HE), the combination of AraC and GDC-0575 reduced the percentage of human cells in the bone marrow; this combination treatment did not affect normal long-term hematopoietic stem/progenitors, did not generate de novo mutations, and eliminated mutant clones induced by AraC [1]
- The addition of granulocyte colony-stimulating factor (G-CSF) to the AraC+GDC-0575 combination further enhanced the cytotoxicity against AML in vivo, reducing residual leukemic cells, increasing the ratio of cycling Ki67+CD45+ cells, and decreasing the L-LTC-IC frequency [1]
- GDC-0575 effectively inhibited tumor growth in vivo in melanoma xenograft models with elevated endogenous replication stress [2]

GDC-0575, also known as ARRY-575 or RG7741, is a novel, strong, and selective inhibitor of CHK1 that binds to it specifically and inhibits it with an IC50 of 1.2 nM.

In co-culture studies, feeder cells are plated onto type-I collagen-coated 96-well or 6-well plates two days prior to the start of the co-culture and allowed to reach confluence. The culture media is exchanged and they receive a 6.8 Gy radiation one day prior to the start of co-culture. First, AmL cells are plated using the appropriate AmL medium at a density of 2 × 10⁵ cells/mL on day 0 of the co-culture. In incubators with 5% CO₂ and indicated oxygen concentrations, cells are cultured at 37°C. Cells are maintained in hypoxia (5% O₂) for one week while being treated with 500 nM AraC and/or 100 nM GDC-0575 during short-term culture (STC)[1].

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AML cell line treatment assay: Four AML cell lines were treated with AraC alone or AraC+GDC-0575 for 1 day, with untreated cells as the control. Cell metabolic activity and apoptosis rate were detected to evaluate the cytotoxic effect [1]
- Stromal layer culture cell assay: AML cell lines were seeded on an irradiated MS5 stromal layer and treated as above, then the proportion of apoptotic cells was determined [1]
- Primary AML sample culture and analysis assay: Primary AML samples were subjected to short-term culture (STC) and long-term culture (LTC) under different treatment conditions. The survival rate was assessed by counting beads and normalized to the untreated group; L-LTC-IC/LDA assays were performed by culturing cells in methylcellulose for 2 weeks to detect L-LTC-IC frequency [1]
- Cell cycle analysis assay: Primary AML samples were treated for 1 week, and then cell cycle distribution was detected by Ki67/DAPI expression, with a focus on cells in the G0 phase [1]
- Melanoma cell sensitivity assay: Melanoma cell lines were treated with GDC-0575 to evaluate cell death; in hypersensitive cell lines, RPA2 phosphorylation level, DNA damage, and cell cycle progression were detected to analyze the cellular response to the drug [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 does not affect normal long-term hematopoietic stem cells/progenitor cells, nor does it induce new mutations [1]
- During treatment, the weight changes of mice injected with AML6 were monitored, and the mice were euthanized when their weight decreased by 20% [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.

The combined use of GDC-0575 and G-CSF can overcome cytarabine resistance in human acute myeloid leukemia. Its mechanism may be related to the inhibition of DNA repair. Persistent residual leukemia cells are in a quiescent state, while G-CSF can promote these cells to enter the cell cycle, thereby enhancing the efficacy of triple therapy (AraC+GDC-0575+G-CSF) and providing a more effective option for clinical AML treatment [1]. - Endogenous replication stress is a marker of melanoma sensitivity to GDC-0575. GDC-0575 induces the death of sensitive melanoma cells by increasing replication stress and DNA damage. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors can be used as effective markers of replication stress in vivo to predict the sensitivity of melanoma 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

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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.17 mg/mL (4.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.17 mg/mL (4.81 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.17 mg/mL (4.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (22.16 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)