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Azd8055

Manufactured by AstraZeneca
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AZD8055 is a selective and potent inhibitor of the mammalian target of rapamycin (mTOR) kinase. It targets the catalytic domain of mTOR, thus inhibiting both mTORC1 and mTORC2 complexes. This compound can be used as a research tool to study the role of mTOR signaling in various biological processes.

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Lab products found in correlation

Azd6244, by AstraZeneca (2 mentions) Puromycin, by Thermo Fisher Scientific (2 mentions) Hygromycin, by Thermo Fisher Scientific (2 mentions) Doxycycline, by Merck Group (2 mentions) Ku006, by Bio-Techne (2 mentions) Wy354, by Bio-Techne (2 mentions) Mln0128, by Bio-Techne (2 mentions) Rad001, by Bio-Techne (2 mentions) Pp242, by Bio-Techne (2 mentions) P akt ser473, by Cell Signaling Technology (1 mentions) Antibodies against erk2, by Santa Cruz Biotechnology (1 mentions) Mcl 1, by BD (1 mentions) P27kip1, by BD (1 mentions) Selumetinib, by AstraZeneca (1 mentions) Mk 2206, by Selleck Chemicals (1 mentions) Rapamycin, by Merck Group (1 mentions) Rapamycin, by Bio-Techne (1 mentions) β tubulin, by GeneTex (1 mentions) Azd8055, by Merck Group (1 mentions) Calcium folinate, by Sandoz (1 mentions)

11 protocols using azd8055

1

Preparing AZD8055 and RAPA Stock Solutions

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Stock solutions of AZD8055 (provided by AstraZeneca, Cambridge, UK) and RAPA (Miltenyi Biotec, Auburn, CA, USA) were prepared in DMSO (vehicle) and used at the indicated final concentrations in culture medium.
Gedaly R., Orozco G., Ancheta A.P., Donoho M., Desai S.N., Chapelin F., Khurana A., Lewis L.J., Zhang C, & Marti F. (2023). Metabolic Disruption Induced by mTOR Signaling Pathway Inhibition in Regulatory T-Cell Expansion for Clinical Application. Cells, 12(16), 2066.
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2

Apoptosis Signaling Pathway Protocol

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Selumetinib (also known as AZD6244 and ARRY-142886) and AZD8055 were provided by AstraZeneca (Macclesfield, UK). ABT-737 was synthesized at the University of Texas MD Anderson Cancer Center based on its published chemical structure (15 (link)). The chemical structures of the above-mentioned reagents are shown at Supplementary Figure S1. Antibodies against human phosphorylated (p)-p44/42 MAPK (ERK1/2)(Thr202/Tyr204), p-AKT(Ser473), p-AKT(Thr308), p-S6K(Ser240/244), p-Rb (Ser780), p-4E-BP1 (Thr37/46), p-Bad (Ser136), p-MEK1/2 were purchased from Cell Signaling Technology (Danvers, MA), as were the antibodies against AKT, S6K, 4E-BP1, Bad, Bid, Bcl-xL, survivin, caspase-8, caspase-9, and cleaved-caspase-3. Antibodies against Bax, Mcl-1, XIAP, and p27Kip-1 were purchased from BD Biosciences (San Jose, CA), antibody against Bcl-2 was purchased from Dako (Carpinteria, CA), antibody against Bak was purchased from Upstate (Lake Placid, NY), antibodies against ERK2, Cdk2, and Cdk4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and antibodies against Bim, cyclin D1, Cdc2, and Puma were purchased from CalBiochem (San Diego, CA).
Zhang W., Ruvolo V.R., Gao C., Zhou L., Bornmann W., Tsao T., Schober W.D., Smith P., Guichard S., Konopleva M, & Andreeff M. (2014). Evaluation of Apoptosis Induction by Concomitant Inhibition of MEK, mTOR, and Bcl-2 in Human Acute Myeloid Leukemia Cells. Molecular cancer therapeutics, 13(7), 1848-1859.
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3

Cytotoxicity Assay for MPNST Cells

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Cytotoxicity of the tested compounds was measured as described previously [26 (link)]. In brief, MPNST cells were seeded in 96-well-plates at 5000 cells per well and treated with the ATP-competitive mTOR inhibitor AZD8055 (AstraZeneca, Cambridge, UK), the allosteric AKT inhibitor MK-2206 (Selleckchem, Munich, Germany), the allosteric MEK1 and MEK2 inhibitor AZD6244 (AstraZeneca, Cambridge, UK), the combination of MK-2206 and AZD8055, or the combination of all three compounds for 72 h. Cytotoxicity was measured by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) assay (Roche, Penzberg, Germany) in quadruplicates.
Schulte A., Ewald F., Spyra M., Smit D.J., Jiang W., Salamon J., Jücker M, & Mautner V.F. (2020). Combined Targeting of AKT and mTOR Inhibits Proliferation of Human NF1-Associated Malignant Peripheral Nerve Sheath Tumour Cells In Vitro but not in a Xenograft Mouse Model In Vivo. International Journal of Molecular Sciences, 21(4), 1548.
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4

Inhibiting mTOR Pathways in HTR8 Cells

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Rapamycin (Tocris-Bioscience; 53123-88-9) was used to inhibit the mTORC1 pathway, and the ATP competitor AZD8055 (AstraZeneca; 2525) was used for inhibition of the mTORC1 and mTORC2 pathways. Cells were treated with 100 nM Rapamycin or 10 μM AZD8055, diluted in dimethyl sulfoxide (Sigma-Aldrich: D-2650), for 24 h before infection. These concentrations proved to be nontoxic to HTR8 cells (see Fig. S4 in the supplemental material). Afterward, cells were infected at an MOI of 3 with each viral strain; supernatants were collected (24 hpi) for virus yield determination by focus-forming assay. In addition, viral protein expression was detected using the anti-NS3 mouse MAb 1ED8 and β-tubulin (GeneTex; GTX-101279) as load control. An anti-mouse antibody conjugated to HRP (Jackson Immuno-Research; 15-035-003) was used as a secondary antibody.
Viettri M., Caraballo G., Sanchez M.E., Espejel-Nuñez A., Betanzos A., Ortiz-Navarrete V., Estrada-Gutierrez G., Nava P, & Ludert J.E. (2023). Comparative Infections of Zika, Dengue, and Yellow Fever Viruses in Human Cytotrophoblast-Derived Cells Suggest a Gating Role for the Cytotrophoblast in Zika Virus Placental Invasion. Microbiology Spectrum, 11(3), e00630-23.
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5

Breast Cancer Cell Line Maintenance Protocol

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All cell lines were obtained from the American Type Culture Collection (ATCC). MCF-7 and MDA-MB-468 (ATCC catalog number: HTB-22 and HTB-132 respectively) breast cancer cell lines were maintained in a 1:1 mixture of DME: F12 medium supplemented with 4 mM glutamine, 100 units/mL each of penicillin and streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in 5% CO2. The MDA-MB-468 inducible expression cells were maintained in the same medium with addition of 50 µg/ml hygromycin and 0.2 µg/mL Puromycin. The HEK-293 cells (ATCC catalog number: CRL-1573) were maintained in DMEM medium with glutamine, antibiotics and 10% FBS. The cell lines resulted negative for mycoplasma contamination. AZD8055 was obtained from AstraZeneca Pharmaceuticals, rapamycin was purchased from EMD Bioscience. RAD001, KU006, WY354, PP242, MLN0128 were purchased from Tocris. Doxycycline was purchased from Sigma Aldrich. Puromycin and hygromycin stock solution were purchased from Invitrogen. Drugs were dissolved in DMSO to yield 10 mM stock and stored at −20°C.
Rodrik-Outmezguine V.S., Okaniwa M., Yao Z., Novotny C.J., McWhirter C., Banaji A., Won H., Wong W., Berger M., de Stanchina E., Barratt D.G., Cosulich S., Klinowska T., Rosen N, & Shokat K.M. (2016). Overcoming mTOR Resistance Mutations with a New Generation mTOR Inhibitor. Nature, 534(7606), 272-276.
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6

Breast Cancer Cell Line Maintenance Protocol

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All cell lines were obtained from the American Type Culture Collection (ATCC). MCF-7 and MDA-MB-468 (ATCC catalog number: HTB-22 and HTB-132 respectively) breast cancer cell lines were maintained in a 1:1 mixture of DME: F12 medium supplemented with 4 mM glutamine, 100 units/mL each of penicillin and streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in 5% CO2. The MDA-MB-468 inducible expression cells were maintained in the same medium with addition of 50 µg/ml hygromycin and 0.2 µg/mL Puromycin. The HEK-293 cells (ATCC catalog number: CRL-1573) were maintained in DMEM medium with glutamine, antibiotics and 10% FBS. The cell lines resulted negative for mycoplasma contamination. AZD8055 was obtained from AstraZeneca Pharmaceuticals, rapamycin was purchased from EMD Bioscience. RAD001, KU006, WY354, PP242, MLN0128 were purchased from Tocris. Doxycycline was purchased from Sigma Aldrich. Puromycin and hygromycin stock solution were purchased from Invitrogen. Drugs were dissolved in DMSO to yield 10 mM stock and stored at −20°C.
Rodrik-Outmezguine V.S., Okaniwa M., Yao Z., Novotny C.J., McWhirter C., Banaji A., Won H., Wong W., Berger M., de Stanchina E., Barratt D.G., Cosulich S., Klinowska T., Rosen N, & Shokat K.M. (2016). Overcoming mTOR Resistance Mutations with a New Generation mTOR Inhibitor. Nature, 534(7606), 272-276.
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7

Evaluating mTOR Inhibitors' Impact on Breast Cancer Cell Growth

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Cells were seeded overnight at 40,000 cells/well (24-well plate) in their respective growth media. Cells were grown for seven days with 1 nM to 1,000 nM of the mTOR inhibitors AZD8055 or RAD001 (gifts from AstraZeneca) or appropriate vehicle control (dimethyl sulphoxide (DMSO)). Cell growth was evaluated by trypsin dispersion of cell monolayers and cell number was measured using a Coulter Counter. All TamR, MCF-7, MCF7-X, T47D-tamR and T47D-fasR experiments were performed at least in triplicate. In combination studies, fulvestrant was routinely used at 100 nM, a concentration shown previously to be growth inhibitory in TamR and MCF7-X models
[2 (link),3 (link)]. The growth impact of AZD8055 (0 to 100 nM) alongside oestrogen deprivation (using X cell medium as described above) or 10-7 M 4-OH tamoxifen was also evaluated in MCF-7 cells.
Jordan N.J., Dutkowski C.M., Barrow D., Mottram H.J., Hutcheson I.R., Nicholson R.I., Guichard S.M, & Gee J.M. (2014). Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro. Breast Cancer Research : BCR, 16(1), R12.
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8

Evaluation of mTOR Inhibitors in Cancer

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Irinotecan (Hospira, 20 mg/mL), 5-fluorouracil (5FU) (Accord, 50 mg/mL), oxaliplatin (Hospira, 5 mg/mL), and calcium folinate (leucovorin, LV) (Sandoz, 10 mg/mL) were provided by the Pharmacy Department of the Hôpitaux Universitaires de Strasbourg. Rapamycin was purchased from LC laboratories (R-5000), LY294002 from Calbiochem (440202), and AZD8055 from Selleckchem (S1555). The AZD2014 compound and the AZD8055 congener [24 (link)] were provided from AstraZeneca (Material Transfer Agreement, July 2015).
Reita D., Bour C., Benbrika R., Groh A., Pencreach E., Guérin E, & Guenot D. (2019). Synergistic Anti-Tumor Effect of mTOR Inhibitors with Irinotecan on Colon Cancer Cells. Cancers, 11(10), 1581.
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9

Solubilization of mTORC1/2 Inhibitors

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The dual mTORC1/2 inhibitor, AZD8055 (AstraZeneca, London, UK), was prepared for in vitro assays by dissolution in DMSO to 10 mM (4.65 mg/mL), per manufacturer instructions. The selective mTORC1 inhibitor, sirolimus (Selleckchem, Houston, TX), was prepared for in vitro assays by dissolution in 100% ethanol to 10.9 mM (10 mg/mL). For in vivo assays, AZD8055 was dissolved by sonication in 30% Captisol (CyDex Pharmaceuticals, Lenexa, KS) to a working concentration of 2 mg/ml and pH of 5.0 per manufacturer instructions. For in vivo assays, sirolimus was dissolved in 5% Tween-80 (Sigma Aldrich) and 5% PEG-400 (Hampton Research, Aliso Viejo, CA) to a working concentration of 0.4 mg/ml. Doses of ~ 200 μl drugs were administered to each animal.
Kauffman E.C., Lang M., Rais-Bahrami S., Gupta G.N., Wei D., Yang Y., Sourbier C, & Srinivasan R. (2019). Preclinical efficacy of dual mTORC1/2 inhibitor AZD8055 in renal cell carcinoma harboring a TFE3 gene fusion. BMC Cancer, 19, 917.
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10

Molecular Signaling Pathway Analysis

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Primary antibodies were as follows: Myo1F HPA055242 (Sigma-Aldrich), GAPDH/sc-322 (Santa Cruz Biotechnology®), TNF-α #11948, pSTAT1-Tyr701 #9167, pS6 #15967, pAkt-Ser473 #4060, pAkt-Ser308 #4056, Akt1 #2967 from Cell Signaling Technology®, CD86-PE-CY5 #15-0862-82 (eBioscience), and CD80-PE #553769 (BD pharmingen). Alexa conjugated antibodies and affiniPure goat and rabbit anti-horseradish peroxidase (HRP) were obtained from Thermo Fisher Scientific and Jackson Immunoresearch, respectively. 4′,6-Diamidino-2-phenylindole (DAPI) sc-3598 from Santa Cruz Biotechnology. Recombinant mouse IFN-γ from PeproTech was dissolved in 0.002% mouse serum albumin (MSA; Sigma-Aldrich) and used at 2.5 μg/kg of mice weight. Lipopolysaccharide from Escherichia coli 0111:B4 (Sigma-Aldrich) was dissolved in 0.002% mouse serum albumin and used at 100 μg/mice. AZD8055 [(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol] from Astra Zeneca was dissolved in DMSO and use at 20 nM. Dextran sulfate sodium (DSS) YD318041799 from Carbosynth was dissolved at 2.5% in tap water.
Piedra-Quintero Z.L., Serrano C., Villegas-Sepúlveda N., Maravillas-Montero J.L., Romero-Ramírez S., Shibayama M., Medina-Contreras O., Nava P, & Santos-Argumedo L. (2019). Myosin 1F Regulates M1-Polarization by Stimulating Intercellular Adhesion in Macrophages. Frontiers in Immunology, 9, 3118.
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