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Purity: ≥98%
AZD0156 is a novel, orally bioactive, potent and selective inhibitor of ATM (ataxia telangiectasia mutated) kinase with an IC50 of 0.58 nM and with potential chemo-/radio-sensitizing and antineoplastic activities. In addition to blocking DNA damage checkpoint activation and disrupting DNA damage repair, AZD0156 inhibits the kinase activity of ATM and ATM-mediated signaling. This results in tumor cell apoptosis and ultimately leads to cell death in ATM-overexpressing tumor cells. Based on its sub-nanomolar potency in cell-based assays for ATM inhibition, AZD0156 was found to be the first oral ATM inhibitor of its kind. Furthermore, AZD0156 exhibited selectivities over 1000 times greater than those of other PIKK family enzymes.
Targets |
ATM
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ln Vitro |
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].
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. 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]. |
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ln Vivo |
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].
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). 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). 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. |
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Enzyme Assay |
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.
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Cell Assay |
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.
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Animal Protocol |
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References |
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Additional Infomation |
ATM Kinase Inhibitor AZD0156 is an orally bioavailable ataxia telangiectasia mutated (ATM) kinase inhibitor, with potential chemo-/radio-sensitizing and antineoplastic activities. Upon oral administration, AZD0156 targets and binds to ATM, thereby inhibiting the kinase activity of ATM and ATM-mediated signaling. This prevents DNA damage checkpoint activation, disrupts DNA damage repair, induces tumor cell apoptosis, and leads to cell death of ATM-overexpressing tumor cells. In addition, AZD0156 sensitizes tumor cells to chemo- and radiotherapy. ATM, a serine/threonine protein kinase, is upregulated in a variety of cancer cell types; it is activated in response to DNA damage and plays a key role in DNA-strand repair.
Ataxia telangiectasia mutant (ATM) is a serine/threonine protein kinase from the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases (also comprising ATR, DNA-PKcs, mTOR etc.) and plays a crucial role in the cellular DNA damage response signalling activated by DNA double strand breaks (DSB). Activated ATM promotes DNA repair and S/G1-cell cycle checkpoints to prevent premature mitosis, maintain genomic integrity and promote appropriate cell survival or death pathways. DSBs arise intrinsically through the collapse of stalled replication forks, which are induced by a wide range of chemotherapies, or extrinsically through exposure to ionizing radiation. Therefore, ATM inhibition represents an exciting clinical opportunity as a target to hyper-sensitize tumors to chemo/radiotherapy. Herein, we describe our efforts to identify multiple, novel series of ATM inhibitors. We will describe our optimization efforts with particular attention given to improving the potency of the compounds in cellular systems and the selectivity of the compounds over other closely related proteins (e.g. ATR, mTOR etc.). We will also describe our efforts to optimize both the physicochemical properties of the molecules as well as the pharmacokinetic profile to enable low dose, oral administration in the clinic. These efforts culminated in the discovery of the clinical candidate AZD0156, a first in class orally available ATM inhibitor. AZD0156 shows sub-nanomolar potency in cell based assays of ATM inhibition with selectivities of greater than 1000 fold over other members of the PIKK family of enzymes. AZD0156 is a permeable, highly soluble compound with excellent preclinical pharmacokinetic properties including oral bioavailability. AZD0156 shows robust efficacy in mouse xenograft models after oral administration when combined with DSB inducing agents. AZD0156 is currently undergoing early clinical assessment.[1] Clinical efficacy has been seen with olaparib in patients with tumors containing BRCA mutations, and our studies using TNBC patient-derived xenograft models demonstrate that AZD0156 enhances the antitumor activity of olaparib irrespective of intrinsic olaparib sensitivity and DDR deficiencies. Here we present data demonstrating that intermittent dosing of AZD0156 enhances olaparib activity in vivo, which should prove valuable in facilitating the development of well-tolerated combination regimes in the clinic. Our data provides proof-of-concept for the assessment of AZD0156 and olaparib combinations in the clinic. Furthermore, AZD0156 provides a valuable tool for preclinical target validation and research into the roles of ATM in the DDR and noncanonical pathways.[3] |
Molecular Formula |
C26H31N5O3
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Molecular Weight |
461.56
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Exact Mass |
461.242
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Elemental Analysis |
C, 67.66; H, 6.77; N, 15.17; O, 10.40
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CAS # |
1821428-35-6
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Related CAS # |
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PubChem CID |
118502708
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Appearance |
White to off-white solid powder
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Density |
1.2±0.1 g/cm3
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Boiling Point |
628.3±55.0 °C at 760 mmHg
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Flash Point |
333.8±31.5 °C
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Vapour Pressure |
0.0±1.8 mmHg at 25°C
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Index of Refraction |
1.626
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LogP |
2.4
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Hydrogen Bond Donor Count |
0
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Hydrogen Bond Acceptor Count |
6
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Rotatable Bond Count |
7
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Heavy Atom Count |
34
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Complexity |
687
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Defined Atom Stereocenter Count |
0
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SMILES |
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
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InChi Key |
AOTRIQLYUAFVSC-UHFFFAOYSA-N
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InChi Code |
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
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Chemical Name |
8-[6-[3-(dimethylamino)propoxy]pyridin-3-yl]-3-methyl-1-(oxan-4-yl)imidazo[4,5-c]quinolin-2-one
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Synonyms |
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
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Solubility (In Vivo) |
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. 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. View More
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. |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.1666 mL | 10.8328 mL | 21.6657 mL | |
5 mM | 0.4333 mL | 2.1666 mL | 4.3331 mL | |
10 mM | 0.2167 mL | 1.0833 mL | 2.1666 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
NCT02588105 | Completed | Drug: AZD0156 Drug: Olaparib |
Advanced Solid Tumours | AstraZeneca | November 10, 2015 | Phase 1 |