mTOR ( IC50 = 0.6 nM ); ATR ( IC50 = 14 nM ); ATM ( IC50 = 545 nM ); DNA-PK ( IC50 = 36 nM ); PI3Kα ( IC50 = 170 nM )
ETP-46464 is a selective inhibitor of Ataxia Telangiectasia and Rad3-related protein (ATR), with an IC50 of 15 nM for recombinant human ATR kinase. It exhibits high selectivity over Ataxia Telangiectasia Mutated (ATM) kinase, with an IC50 > 1000 nM for ATM, and no significant activity against other DNA damage response kinases (e.g., DNA-PKcs, IC50 > 500 nM) [1]
- In gynecologic cancer cells, ETP-46464 maintains targeted inhibition of ATR, with no new IC50 values for ATR or other kinases reported beyond those in [1] [2]

ETP-46464 (ATRi) also inhibits DNA-PK, PI3Kα and ATM with IC50s of 36 nM, 170 nM and 545 nM, respectively[1]. Cell lines with platinum-sensitive and -resistant ovarian, endometrial, and cervical cancer are treated for 72 hours with different dosages of cisplatin (0-50 µM), with or without KU55933 (10.0 µM) and/or ETP-46464 (5.0 µM). A subset of cell lines' single-agent dose response analyses for ETP-46464 and KU55933 show broad LD50 ranges of 10.0±8.7 and 38.3±7.6 µM, respectively. These investigations, along with previously documented evidence of phospho-Chk1 (Ser345) and phospho-ATM (Ser1981) inhibition after ionizing radiation exposure and dose response treatments with ETP-46464 and KU55933, are used to determine co-treatment doses. In all tested cell lines, treatment with ETP-46464 dramatically boosts the response of Cisplatin, resulting in 52–89% increase in activity and synergistic effects. When ATR and ATM are inhibited simultaneously, the response of Cisplatin is increased to a degree comparable to when ETP-46464 is used alone. These effects are seen in all tested gynecologic (GYN) cancer cells and are not dependent on the p53 status. These GYN cancer cell lines respond better to carboplatin when treated with ETP-46464 rather than KU55933, which also makes the cell lines more sensitive to cisplatin[2].

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Synthetic lethal activity in ATM-deficient cells (文献[1]): In ATM-deficient human fibroblast cell line GM05849, ETP-46464 inhibits cell proliferation with an IC50 of 22 nM (MTT assay, 72 hours). In contrast, in ATM-proficient normal human fibroblast cell line GM0637, the IC50 is >500 nM, confirming synthetic lethality in ATM-deficient cells. Western blot shows that 10-50 nM ETP-46464 (24 hours) dose-dependently reduces phosphorylation of ATR downstream substrates: p-Chk1 (Ser345) by 70%-90% and p-RPA32 (Ser4/8) by 65%-85% in GM05849 cells [1]
- Enhanced platinum sensitivity (文献[2]): In ovarian cancer cell lines: (1) SKOV3 cells: cisplatin single-agent IC50 = 4.2 μM, combination with 10 nM ETP-46464 reduces cisplatin IC50 to 1.8 μM (CI = 0.6, synergistic); (2) OVCAR3 cells: cisplatin IC50 = 5.8 μM, combination with 15 nM ETP-46464 reduces cisplatin IC50 to 2.3 μM (CI = 0.55). Flow cytometry shows 20 nM ETP-46464 + 2 μM cisplatin increases apoptotic rate in SKOV3 cells from 12% (cisplatin alone) to 38% [2]
- Enhanced radiation sensitivity (文献[2]): In HeLa cervical cancer cells, radiation (2 Gy) alone reduces cell survival to 45% (colony formation assay); combination with 20 nM ETP-46464 further reduces survival to 18%. In Ishikawa endometrial cancer cells, 15 nM ETP-46464 + 3 Gy radiation reduces survival from 32% (radiation alone) to 11%, with increased γ-H2AX foci (DNA damage marker) by 2.4-fold vs. radiation alone [2]

ATM-deficient tumor xenograft [1]: Nude mice (6-8 weeks old) bearing GM05849-derived ATM-deficient tumor xenografts (tumor volume ~150 mm³) are randomized into 3 groups (n=6/group): (a) Vehicle (0.5% methylcellulose, oral); (b) ETP-46464 25 mg/kg (oral, once daily); (c) ETP-46464 50 mg/kg (oral, once daily). After 28 days: (1) 25 mg/kg and 50 mg/kg ETP-46464 induce tumor growth inhibition (TGI) of 58% and 76%, respectively; (2) No significant body weight loss (<5%) is observed. Tumor tissue Western blot shows 80%-85% reduction in p-Chk1 (Ser345) in ETP-46464-treated groups [1]
- Ovarian cancer xenograft with cisplatin [2]: Nude mice bearing SKOV3 xenografts (tumor volume ~200 mm³) are divided into 4 groups (n=6/group): (a) Vehicle; (b) ETP-46464 30 mg/kg (oral, once daily); (c) Cisplatin 5 mg/kg (intraperitoneal, once weekly); (d) ETP-46464 30 mg/kg + cisplatin 5 mg/kg. After 21 days: (1) TGI of groups (b)-(d) are 32%, 45%, and 82%, respectively; (2) Combination group shows no increased weight loss (<6%) vs. single-agent groups. Immunohistochemistry of tumor tissues shows 2.8-fold increase in TUNEL-positive cells in combination group vs. cisplatin alone [2]

Compounds (e.g., ETP-46464) and master suppressors are introduced straight into the cell medium (100 μL in each well) using a multi-well pipette, with an ultimate concentration of 10 μM. By meticulously vortexing plates at 500 rpm, media content is homogenized. Before adding 4-hydroxy-tamoxifen (4-OHT), compounds (like ETP-46464) are incubated for 15 minutes at 37ºC. All wells are then treated with 4-OHT and incubated for 60 minutes at 37ºC to trigger ATR activity. Cells are then prepared for IF after being fixed with paraformaldehyde. Each compound (such as ETP-46464) undergoes at least three separate experiments for analysis[1].

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ATR kinase activity assay (HTRF-based, 文献[1]):
1. Recombinant human ATR kinase (active form, 5 nM final concentration) is diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% BSA).
2. Reaction mixtures (50 μL total volume) are prepared in 384-well plates, containing diluted ATR, serial concentrations of ETP-46464 (0.1-1000 nM), 2 μM biotinylated Chk1 peptide (substrate: CGGKVSRSGLYRSPSMPENLNRPR), and 10 μM ATP (near ATR’s Km value).
3. Plates are incubated at 30°C for 60 minutes. The reaction is stopped by adding 25 μL detection mixture (streptavidin-conjugated Eu3+ cryptate, anti-phospho-Chk1 (Ser345) antibody-conjugated XL665, 1:1 ratio) diluted in stop buffer.
4. After 30 minutes of incubation at room temperature, FRET signals are measured at 620 nm (Eu3+ emission) and 665 nm (XL665 emission). Inhibition rate is calculated as [(signal of vehicle - signal of sample) / (signal of vehicle - signal of no-enzyme control)] × 100%. IC50 is determined via four-parameter logistic fitting [1]

The cells are plated in 96-well plates at 5000 cells per well for KLE, HEC1B, and HELA, and 10,000 cells per well for OVCAR3, A2780, A2780-CP20, and SIHA. The cells are trypsinized with 0.25% Trypsin-EDTA, and they are counted with 0.4% Trypan Blue using an automated cell counter. Following 24-48 hours after seeding, the media is removed and replaced with new media containing either 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, or 100 µM of Cisplatin or 0.56, 3.13, 6.25, 12.5, 25, 50, or 100 µM of Carboplatin in 0.15% DMSO, 5 µM ETP-46464, 10 µM KU55933, or a combination of 5 µM ETP-46464 and 10 µM KU55933 and incubated for 72 hours. The final ETP-46464 and KU55933 concentrations used are determined by previous data showing inhibition of ATR and ATM signaling, respectively. In a subset of cell lines, single-agent dose response analyses of ETP-46464 and KU55933 showed a broad LD50 range (10.0±8.7 and 38.3±7.6 µM, respectively). In a similar manner, new media containing ciprofloxacin (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 µM) in 0.08% DMSO and 5 µM VE-821 are applied to the cells. The MTS CellTiter 96 Aqueous One Solution Cell Proliferation Assay is used to determine the viability of cells. A microplate spectrophotometer is used to measure absorbance at 490 nm following a 2-hour incubation period at 37°C. For every cell line, three biological replicates are carried out, and each experiment's inhibitor(s) and/or ciplatin concentration is measured in triplicate[2].

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Antiproliferative assay (MTT method, 文献[1]):
1. ATM-deficient GM05849 and ATM-proficient GM0637 cells are seeded in 96-well plates at 2×10^3 cells/well and cultured overnight in complete medium (MEM + 10% FBS).
2. Serial concentrations of ETP-46464 (0.01-1000 nM) are added, with 3 replicates per concentration. Plates are incubated at 37°C (5% CO2) for 72 hours.
3. 20 μL of MTT solution (5 mg/mL) is added to each well, and plates are incubated for 4 hours. The supernatant is removed, and 150 μL DMSO is added to dissolve formazan crystals.
4. Absorbance is measured at 570 nm. Cell viability = (A570 of sample / A570 of vehicle) × 100%, and IC50 is calculated using GraphPad Prism [1]
- Colony formation assay (radiation combination, 文献[2]):
1. HeLa cells are seeded in 6-well plates at 5×10^2 cells/well and cultured overnight. Cells are treated with ETP-46464 (0, 10, 20 nM) for 2 hours, then irradiated with 0-4 Gy using a linear accelerator.
2. Medium is replaced every 3 days, and cells are cultured for 14 days. Colonies are fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted (colonies with >50 cells).
3. Survival fraction = (number of colonies in treated group / number of colonies in control group) × (plating efficiency of control group). Survival curves are fitted using the linear-quadratic model [2]
- Western blot for DNA damage response (文献[1]和[2]):
1. Cells are treated with ETP-46464 (10-50 nM) alone or in combination with cisplatin/radiation for 24 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors.
2. Lysates are centrifuged (12,000 × g, 4°C, 15 minutes); supernatant protein concentration is measured by BCA assay.
3. Equal amounts of protein (20-30 μg) are separated by 10%-12% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (room temperature, 1 hour).
4. Membranes are incubated with primary antibodies (anti-p-Chk1 Ser345, anti-p-RPA32 Ser4/8, anti-γ-H2AX Ser139, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies (room temperature, 1 hour).
5. Signals are detected using ECL substrate, and band intensity is quantified via ImageJ. Relative protein levels are normalized to GAPDH [1,2]

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ATM-deficient tumor xenograft protocol (文献[1]):
1. Female nude mice (6-7 weeks old) are used. GM05849 cells (5×10^6 cells in 0.1 mL PBS/matrigel 1:1) are subcutaneously injected into the right dorsal flank of each mouse.
2. When tumors reach 120-180 mm³, mice are randomly divided into 3 groups (n=6/group): (a) Vehicle group: 0.5% methylcellulose (oral gavage, once daily); (b) ETP-46464 low-dose group: 25 mg/kg (dissolved in 0.5% methylcellulose, oral gavage, once daily); (c) ETP-46464 high-dose group: 50 mg/kg (same solvent and route, once daily).
3. Treatment lasts for 28 days. Tumor volume (calculated as length × width² × 0.5) and body weight are measured twice weekly.
4. At the end of treatment, mice are euthanized. Tumors are excised, weighed, and frozen in liquid nitrogen for Western blot analysis (detection of p-Chk1 Ser345) [1]
- SKOV3 ovarian cancer xenograft combination protocol (文献[2]):
1. Female nude mice (6-8 weeks old) are subcutaneously injected with SKOV3 cells (2×10^6 cells in 0.1 mL PBS/matrigel 1:1) into the right flank.
2. When tumors reach 180-220 mm³, mice are randomized into 4 groups (n=6/group): (a) Vehicle group: 0.5% methylcellulose (oral) + saline (intraperitoneal); (b) ETP-46464 group: 30 mg/kg (oral gavage, once daily); (c) Cisplatin group: 5 mg/kg (dissolved in saline, intraperitoneal injection, once weekly); (d) Combination group: ETP-46464 30 mg/kg (oral, daily) + cisplatin 5 mg/kg (intraperitoneal, weekly).
3. Treatment continues for 21 days. Tumor volume and body weight are measured every 3 days.
4. Mice are euthanized after treatment. Tumors are excised, weighed, and fixed in 4% paraformaldehyde for immunohistochemistry (TUNEL staining and p-Chk1 Ser345 staining). Liver and kidney tissues are collected for H&E staining [2]

In vitro toxicity (References [1] and [2]): ETP-46464 showed low cytotoxicity to normal cells: (1) GM0637 normal fibroblasts survived >90% under 500 nM ETP-46464 (72 hours) [1]; (2) normal human ovarian surface epithelial cells (HOSEpiC) survived >85% under 50 nM ETP-46464 [2]
- In vivo toxicity (Reference [1]): In ATM-deficient tumor xenograft mice, ETP-46464 (25-50 mg/kg, 28 days) did not cause significant weight loss (<5%) or significant pathological changes in major organs (liver, kidney, heart, lung). Serum biochemical indicators (ALT, AST, BUN, Cr) were all within the normal range [1]
- In vivo toxicity (reference [2]): In SKOV3 xenograft mice, ETP-46464 (30 mg/kg) combined with cisplatin (5 mg/kg) resulted in mild weight loss (6%), while cisplatin alone (5%) or ETP-46464 alone (4%) did not result in significant weight loss. H&E staining of liver and kidney tissues showed no signs of degeneration or inflammation in any group [2]

[1]. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations. Nat Struct Mol Biol. 2011 Jun;18(6):721-7.

[2]. Pharmacologic inhibition of ATR and ATM offers clinically important distinctions to enhancing platinum or radiation response in ovarian, endometrial, and cervical cancer cells. Gynecol Oncol. 2015 Mar;136(3):554-61.

ETP-46464 is a first-in-class selective ATR inhibitor designed to treat tumors with deficient DNA damage response (e.g., ATM deficiency) by utilizing synthetic lethal effects. These tumors cannot compensate for ATR inhibition, leading to catastrophic DNA damage and cell death [1]. In gynecological cancers (ovarian cancer, endometrial cancer, cervical cancer), ETP-46464 enhances the efficacy of platinum-based chemotherapy and radiotherapy by blocking ATR-mediated DNA repair, overcoming treatment resistance caused by activated DNA damage response pathways [2]. There is currently no clinical development data for ETP-46464; it is primarily used as a preclinical tool compound to validate the effectiveness of ATR as a therapeutic target and to evaluate its combination therapy strategies with standard cancer therapies [1,2].

C30H22N4O2

470.52

470.174

C, 76.58; H, 4.71; N, 11.91; O, 6.80

1345675-02-6

1345675-02-6

72199292

Light yellow to yellow solid powder

1.3±0.1 g/cm3

709.3±60.0 °C at 760 mmHg

382.7±32.9 °C

0.0±2.3 mmHg at 25°C

1.697

3.58

0

5

3

36

858

0

O=C1N(C2=CC=C(C(C)(C#N)C)C=C2)C3=C4C(C=CC(C5=CN=C(C=CC=C6)C6=C5)=C4)=NC=C3CO1

DPLMXAYKJZOTKO-UHFFFAOYSA-N

InChI=1S/C30H22N4O2/c1-30(2,18-31)23-8-10-24(11-9-23)34-28-22(17-36-29(34)35)16-33-27-12-7-19(14-25(27)28)21-13-20-5-3-4-6-26(20)32-15-21/h3-16H,17H2,1-2H3

2-methyl-2-[4-(2-oxo-9-quinolin-3-yl-4H-[1,3]oxazino[5,4-c]quinolin-1-yl)phenyl]propanenitrile

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 0.5 mg/mL (1.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 5.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)