ETP-46464 is an effective mTOR and ATR inhibitor with IC₅₀s of 0.6 and 14 nM, respectively.

ETP-46464 (ATRi) also inhibits DNA-PK, PI3Kα and ATM with IC₅₀s of 36 nM, 170 nM and 545 nM, respectively^[1]. Platinum-sensitive and -resistant ovarian, endometrial and cervical cancer cell lines are treated with varying levels of Cisplatin (0-50 µM) with or without the ETP-46464 (5.0 µM) and/or the KU55933 (10.0 µM) for 72 h. Single-agent dose response analyses of ETP-46464 and KU55933 in a subset of cell lines reveal a wide LD₅₀ range of 10.0±8.7 and 38.3±7.6 µM respectively. Co-treatment doses are chosen based on these studies and previously published evidence of phospho-Chk1 (Ser345) and phospho-ATM (Ser1981) inhibition following ionizing radiation exposure and dose response treatments with ETP-46464 and KU55933. Treatment with ETP-46464 significantly increases the response of Cisplatin in all cell lines tested, resulting in 52-89% enhancement in activity and are synergistic. The combined inhibition of ATR and ATM enhances the response of Cisplatin to a level equivalent to that observed using ETP-46464 alone. These effects are independent of p53 status, and are observed in all gynecologic (GYN) cancer cells tested. Treatment with ETP-46464, but not KU55933, not only sensitizes these GYN cancer cell lines to Cisplatin, but also enhances the response of Carboplatin^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

470.52

C₃₀H₂₂N₄O₂

1345675-02-6

Room temperature in continental US; may vary elsewhere.

DMSO : 5 mg/mL (10.63 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 0.5 mg/mL (1.06 mM); Clear solution

  此方案可获得 ≥ 0.5 mg/mL (1.06 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 5.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• [1]. Toledo LI, et al. 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]. Teng PN, et al. 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.

Compounds (e.g., ETP-46464) and control inhibitors are delivered directly to cell media (100 μL per well) with a multi-well pipette at a final concentration of 10 μM. Media content is homogenized by carefully vortexing plates at 500 rpm. Prior to 4-hydroxy-tamoxifen (4-OHT) addition, Compounds (e.g., ETP-46464 ) are incubated at 37ºC for 15 minutes. Next, to induce ATR activity, 4-OHT is added to all wells and incubated for 60 minutes at 37ºC. Finally, cells are fixed with paraformaldehyde and processed for IF. Every compound (e.g., ETP-46464) is analyzed at least in three independent experiments^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Cells are trypsinized with 0.25% Trypsin-EDTA and counted with 0.4% Trypan Blue using an automated cell counter and 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. After cells attach and reach approximately 60% confluency (24-48 h post seeding), media is removed and replaced with fresh media containing Cisplatin (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 µM) or Carboplatin (0, 1.56, 3.13, 6.25, 12.5, 25, 50 or 100 µM) 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 h. Final concentrations of ETP-46464 and KU55933 utilized are based on prior evidence indicating inhibition of ATR and ATM signaling, respectively. Single-agent dose response analyses of ETP-46464 and KU55933 in a subset of cell lines revealed a wide LD₅₀ range (10.0±8.7 and 38.3±7.6 µM, respectively). Similarly, cells are treated with fresh media containing Cisplatin (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 µM) in 0.08% DMSO and 5 µM VE-821. Cell viability is assessed using the MTS CellTiter 96 Aqueous One Solution Cell Proliferation Assay. After a 2 h incubation at 37°C, absorbance is measured at 490 nm using a microplate spectrophotometer. Three biological replicates are performed for each cell line where each inhibitor(s)/Cisplatin concentration is assayed in triplicate for each experiment^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Toledo LI, et al. 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]. Teng PN, et al. 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-46321 is a potent and orally bioavailable PI3Kα and PI3Kδ inhibitor with K_iapps of 2.3 and 14.2 nM, respectively.

ETP-46321 is selected to be screened against other PI3K isoforms. ETP-46321 is more potent against isoform α (K_iapp=2.3 nM). ETP-4632, has been profiled and shown to be a potent PI3K α and δ inhibitor, highly selective versus mTOR and 288 representative kinases. ETP-46321 is also tested against three of the p110α mutant enzymes detected in human cancers (E542K, E545K and H1047R), being equipotent against these mutants when compared to the wild type protein (K_iapp=2.33, 1.77 and 1.89 nM for PI3Kα-H1047R, PI3Kα-E545K and PI3Kα-E542K, respectively). ETP-46321 inhibits the phosphorilation of AKT in U2OS cell line with an IC₅₀ of 8.3 nM^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

ETP-46321, is selected for in vivo studies based on its appealing pharmacokinetic profile in BALB-C mice, low in vivo Clearance (0.6 L/h/Kg) and good oral bioavailability (90%). ETP-46321 demonstrates a good pharmacokinetic profile in mice and is selected for preliminary in vivo evaluation in a lung tumor mouse model driven by a K-RasG12V oncogenic mutation, showing significant tumor growth inhibition, and reduction of the tumor metabolic activity as measured by positron emission tomography (PET) techniques^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

473.55

C₂₀H₂₇N₉O₃S

1252594-99-2

Room temperature in continental US; may vary elsewhere.

DMSO : ≥ 33 mg/mL (69.69 mM)

* “≥” means soluble, but saturation unknown.

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

• [1]. Martínez González S, et al. Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor. Bioorg Med Chem Lett. 2012 May 15;22(10):3460-6.

Mice^[1]
BALB/C mice are treated daily with ETP-46321 (50 mg/kg, p.o.) for three weeks. Tumor volumes of four mice in each treatment group are measured and compared to the starting volume at the beginning of the treatment.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Martínez González S, et al. Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor. Bioorg Med Chem Lett. 2012 May 15;22(10):3460-6.