ATM

In vitro activity: AZD0156 demonstrates sub-nanomolar potency in cell-based ATM inhibition assays, exhibiting selectivities that surpass 1000 times that of other PIKK family enzymes[1].

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Effect of AZD0156 on Human ADPKD Cysts [2]
As MDCK cells do not have mutations in PKD-causative genes, we further evaluated the effect of ATM inhibition in a human ADPKD cyst model using WT 9-12 cells, which possess a homozygous PKD1 variant [36,37,41]. In this model, the formation of cysts was slower compared to those derived from MDCK cells (requiring 18 days to reach maximal size) and a higher proportion of cysts had an eccentric shape (Figure 3A). Consistent with MDCK cysts, AZD0156 also reduced cyst growth in a dose-dependent manner by up to 4.1-fold by day 18 in WT 9-12-derived cysts (Figure 3B). Furthermore, as shown in Figure 3C,D, AZD0156 did not alter cell proliferation and reduced percentage LDH leakage in WT 9-12 monolayers.

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AZD0156 is a potent and selective, bioavailable inhibitor of ataxia-telangiectasia mutated (ATM) protein, a signaling kinase involved in the DNA damage response. We present preclinical data demonstrating abrogation of irradiation-induced ATM signaling by low doses of AZD0156, as measured by phosphorylation of ATM substrates. AZD0156 is a strong radiosensitizer in vitro. Because ATM deficiency contributes to PARP inhibitor sensitivity, preclinically, we evaluated the effect of combining AZD0156 with the PARP inhibitor olaparib. Using ATM isogenic FaDu cells, we demonstrate that AZD0156 impedes the repair of olaparib-induced DNA damage, resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiated the effects of olaparib across a panel of lung, gastric, and breast cancer cell lines in vitro, and improved the efficacy of olaparib in two patient-derived triple-negative breast cancer xenograft models. AZD0156 is currently being evaluated in phase I studies (NCT02588105)[3].

AZD0156 possesses exceptional preclinical pharmacokinetic qualities, such as oral bioavailability, and is a highly soluble, permeable substance. When paired with DSB inducing agents, AZD0156 exhibits strong efficacy in mouse xenograft models following oral administration. Early clinical evaluation of AZD0156 is presently underway[1].

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Effect of AZD1390 and AZD0156 on MDCK Cyst Model [2]
To further verify the effect of ATM inhibition on in vitro cyst growth and also to select candidates for in vivo testing, we next evaluated the efficacy of two orally bioavailable analogues: (i) AZD1390 (IC50, 0.78 nM, >10,000-fold selectivity for ATM), and (ii) AZD0156 (IC50 0.58 nM, >1000-fold selectivity for ATM) [30]. Both ATM inhibitors are in Phase I clinical trials, whereas KU-60019 is limited to in vitro studies. As shown in Figure 2, AZD1390 only reduced MDCK cyst growth on Day 8 at a concentration of 1 μM (by 1.4-fold), whereas AZD0156 was more potent, decreasing cyst diameter by up to 4.4-fold and with comparable efficacy to sirolimus (Figure 2A–C). Interestingly, unlike KU-60019, AZD0156 did not alter BrdU synthesis but rather increased percentage LDH leakage in MDCK cell monolayers compared to vehicle (Figure 2D,E).

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AZD0156 Reduces Renal Cell Proliferation and Increases p53 in Pkd1RC/RC Mice [2]
We next evaluated the short-term in vivo effect of AZD0156 on biomarkers of cystic kidney disease in Pkd1RC/RC mice. Treatment with AZD0156 for a total of 10 days had no adverse effects on general health or body weight in Pkd1RC/RC mice (change in body weight from day 0 to 10: vehicle: 0.18 ± 0.63, AZD0156 5 mg/kg: −0.38 ± 0.48, and AZD0156: 20 mg/kg: −0.18 ± 0.53 g; p > 0.05). In Pkd1RC/RC mice, AZD0156 reduced renal cell proliferation, measured by Ki-67, compared to vehicle (Figure 4A,B). In contrast, although the DNA damage marker γ-H2AX was not different between treatment groups (Figure 4A,C), AZD0156 increased renal p53 (Figure 4D,E). Due to the essential role of ATM in apoptosis, cleaved caspase-3 (Asp175) was also investigated. Cleaved-caspase-3 was expressed in distal tubules but was not altered by AZD0156 (Figure 4A). Furthermore, due to the short duration of this study, as expected, no changes in kidney enlargement and percentage cyst area were observed following AZD0156 treatment (Figure 5).

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AZD0156 improves the response of olaparib treatment in patient-derived TNBC xenografts [3]
To determine whether AZD0156 could potentiate olaparib in vivo, two patient-derived TNBC xenograft models with different sensitivities to olaparib were selected, HBCx-10 and HBCx-9 (29). In each model, two tolerated doses and schedules were used, schedule 1: AZD0156 was administered at 5 mg/kg for 3 consecutive days per week and schedule 2: AZD0156 was administered at 2.5 mg/kg for 5 consecutive days per week. In both schedules olaparib was administered continually at 50 mg/kg. Previously published data demonstrates that HBCx-10 model is known to be sensitive to olaparib treatment alone and demonstrates regressions in combination with AZD0156 using schedule 1 (28). Using schedule 2 in this HBCx-10 model, olaparib alone induced regressions in one of 10 tumors, however in combination with AZD0156 tumor, regression was achieved in 3 of the 8 treated mice and additionally tumor stasis was seen in 4 of the remaining 5 mice (Fig. 6A). Although olaparib treatment alone had little effect on tumor growth in the olaparib-insensitive HBCx-9 model, tumor growth inhibition was improved with the addition of AZD0156 using schedule 1 (Fig. 6B). Interestingly schedule 2 in this model did not enhance tumor growth inhibition beyond the olaparib response alone (Supplementary Fig. S4). AZD0156 monotherapy treatment did not impact tumor growth in either model.

In tumor cells that overexpress ATM, AZD0156 causes cell death by inhibiting the kinase activity of ATM and ATM-mediated signaling, blocking DNA damage checkpoint activation, disrupting DNA damage repair, and inducing tumor cell apoptosis.

HT29 cells are cultivated at a density of 6000 cells/well in 384-well assay plates using 40 μL EMEM medium supplemented with 10% FBS and 1% L glutamine, and left to adhere for the entire night. Assay plates are filled with the Formula (I) compound in 100% DMSO the next morning using an acoustic dispensing method. All wells receive 40 nL of 3 mM 4NQO in 100% DMSO by acoustic dispensing following a 1-hour incubation at 37°C and 5% CO₂, with the exception of the minimum control wells, which are left untreated with 4NQO in order to produce a null response control. The plates are put back in the incubator to continue lh. Subsequently, 20 μL of 3.7% formaldehyde in PBS solution is added to the cells, and they are incubated for 20 minutes at room temperature to fix them. After that, 20 μL of 0.1% Triton XI 00 in PBS is added, and the cells are incubated for 10 minutes at room temperature to allow for permeabilization. Subsequently, the plates are cleaned once using a Biotek EL405 plate washer and 50 μL/well PBS.

In Vivo Mouse Experiments [2]
Two experiments were performed to investigate the in vivo effects of reducing ATM. In the first study, to determine the effect of pharmacological ATM kinase inhibition, groups of Pkd1RC/RC mice (n = 4 per group; aged 2–3 months) were randomized to receive either: (i) vehicle (0.5% (w/v) hydroxypropyl methylcellulose (HPMC) and 0.1% (v/v) Tween 80 in ultrapure water), (ii) low dose AZD0156 (5 mg/kg body weight/day), or (iii) high dose AZD0156 (20 mg/kg body weight/day). Mice were dosed via oral gavage on Monday, Wednesday, and Friday for 2 consecutive weeks for a total study duration of 10 days. Mice were sacrificed 4 h after dosing on day 10. The vehicle composition, drug dose, and frequency of administration were based on previous studies.

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Xenograft-targeted irradiation study [3]
NCI-H441 cells for in vivo xenograft implant were cultured in RPMI1640 with 10% v/v FCS and 1% v/v l-glutamine at 37°C, 7.5% CO2. Cells were implanted subcutaneously in serum-free media with 50% Matrigel at 5 × 106 per mouse. Male nude mice at greater than 18 g had tumor size monitored twice weekly by bilateral caliper measurements. Treatments were started when tumor volumes reached an average of approximately 0.26 cm3. Targeted irradiation of 2 Gy was delivered over 2 minutes daily over the first 5 days. AZD0156 (10 mg/kg) was given orally for the duration of the study.

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Patient-derived xenograft studies [3]
Triple-negative breast cancer (TNBC) HBCx-10 and HBCx-9 (29) patient-derived xenograft studies were carried out at XenTech, France in accordance with French regulatory legislation concerning the protection of laboratory animals. Female athymic nude mice greater than 18 g were implanted with HBCx-10 or HBCx-9 tumor derived from a primary ductal adenocarcinoma. Donor mice were sacrificed to provide tumor fragments, which were surgically implanted subcutaneously. Tumor size was monitored twice weekly by bilateral caliper measurements. Mice body weights were recorded at the same time. For efficacy studies, treatment started when tumor volume averaged approximately 0.15 cm3 and for pharmacodynamic studies treatment started at approximately 0.5 cm3. In efficacy and pharmacodynamic studies, control mice were dosed orally with vehicle, AZD0156, or olaparib alone or in combination using different dosing schedules.

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Oral

Mouse xenograft models

[1]. Abstract 4859: Identifying high quality, potent and selective inhibitors of ATM kinase: Discovery of AZD0156. Cancer Res (2016) 76 (14_Supplement): 4859.

[2]. Effect of Reducing Ataxia-Telangiectasia Mutated (ATM) in Experimental Autosomal Dominant Polycystic Kidney Disease. Cells. 2021 Mar 3;10(3):532.

[3]. Pharmacology of the ATM Inhibitor AZD0156: Potentiation of Irradiation and Olaparib Responses Preclinically . Mol Cancer Ther (2020) 19 (1): 13–25.

AZD0156, an ATM kinase inhibitor, is an orally bioavailable ataxia-telangiectasia mutant gene (ATM) kinase inhibitor with potential chemosensitizing/radiotherapy-enhancing and antitumor activities. After oral administration, AZD0156 targets and binds to ATM, thereby inhibiting ATM kinase activity and its mediated signaling pathways. This prevents activation of DNA damage checkpoints, interferes with DNA damage repair, induces tumor cell apoptosis, and leads to the death of ATM-overexpressing tumor cells. Furthermore, AZD0156 can enhance the sensitivity of tumor cells to chemotherapy and radiotherapy. ATM is a serine/threonine protein kinase upregulated in various cancer cell types; it is activated after DNA damage and plays a crucial role in DNA chain repair. Ataxia-telangiectasia mutant protein (ATM) is a serine/threonine protein kinase belonging to the phosphatidylinositol 3-kinase-associated kinase (PIKK) family (which also includes ATR, DNA-PKcs, mTOR, etc.). It plays a crucial role in the cellular DNA damage response signaling pathway activated by DNA double-strand breaks (DSB). Activated ATM promotes DNA repair and the S/G1 phase cell cycle checkpoint to prevent premature mitosis, maintain genome integrity, and promote appropriate cell survival or death pathways. DSB production pathways include endogenous pathways, such as the breakdown of arrested replication forks induced by various chemotherapeutic agents; and exogenous pathways, such as exposure to ionizing radiation. Therefore, ATM inhibitors hold promise as targets for improving tumor sensitivity to chemotherapy/radiotherapy, offering exciting clinical application prospects. This article will introduce our efforts to identify several novel ATM inhibitors. We will highlight our optimization efforts, focusing particularly on improving the compound's potency in cellular systems and its selectivity for other closely related proteins, such as ATR and mTOR. We will also describe our efforts to optimize the physicochemical properties and pharmacokinetic characteristics of the molecule to achieve low-dose oral administration. These efforts ultimately led to the discovery of the clinical candidate AZD0156, a first-to-market oral ATM inhibitor. In cell-based ATM inhibition assays, AZD0156 demonstrated sub-nanomolar potency and over 1000-fold selectivity for other PIKK family enzymes. AZD0156 is a highly permeable and soluble compound with excellent preclinical pharmacokinetic properties, including good oral bioavailability. In a mouse xenograft model, AZD0156, when administered orally in combination with a DSB inducer, showed significant efficacy. AZD0156 is currently undergoing early clinical evaluation. [1]

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Olaparib has shown clinical efficacy in patients with tumors carrying BRCA mutations. Our study using a xenograft model derived from TNBC patients showed that AZD0156 enhances the antitumor activity of olaparib, unaffected by olaparib's inherent sensitivity and DNA damage repair (DDR) defects. The data presented here suggest that intermittent administration of AZD0156 enhances the activity of olaparib in vivo, which will help in the development of well-tolerated combination therapy. Our data provide a proof of concept for clinical evaluation of the combination of AZD0156 and olaparib. In addition, AZD0156 provides a valuable tool for preclinical target validation and the study of the role of ATM in DDR and non-canonical pathways. [3]

C26H31N5O3

461.56

461.242

C, 67.66; H, 6.77; N, 15.17; O, 10.40

1821428-35-6

118502708

White to off-white solid powder

1.2±0.1 g/cm3

628.3±55.0 °C at 760 mmHg

333.8±31.5 °C

0.0±1.8 mmHg at 25°C

1.626

2.4

0

6

7

34

687

0

O1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C(N(C([H])([H])[H])C2=C([H])N=C3C([H])=C([H])C(C4=C([H])N=C(C([H])=C4[H])OC([H])([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=C([H])C3=C12)=O

AOTRIQLYUAFVSC-UHFFFAOYSA-N

InChI=1S/C26H31N5O3/c1-29(2)11-4-12-34-24-8-6-19(16-28-24)18-5-7-22-21(15-18)25-23(17-27-22)30(3)26(32)31(25)20-9-13-33-14-10-20/h5-8,15-17,20H,4,9-14H2,1-3H3

8-[6-[3-(dimethylamino)propoxy]pyridin-3-yl]-3-methyl-1-(oxan-4-yl)imidazo[4,5-c]quinolin-2-one

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: 0.83 mg/mL (1.80 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: 0.83 mg/mL (1.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 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: 0.83 mg/mL (1.80 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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