cIAP1 (IC50 = 15 nM); cIAP2 (IC50 = 21 nM); XIAP (IC50 = 15 nM)
In H1975 NSCLC cells, AZD5582 (20 nM; 48 hours) inhibits cell viability by working with IFN or viral double-stranded RNA (dsRNA)[2].
Inducing the cleavage of caspase-3 and caspase-7, AZD5582 (20 nM; 17 or 25 hours) downregulates cIAP-1, activates RIPK1 (the upstream regulator of caspase-8), and activates the extrinsic (caspase-8) and intrinsic (caspase-9) apoptosis pathways[2].
Due to the co-treatment of HCC827 NSCLC cells with AZD5582 and IFN, which results in the induction of cell death and active caspase-3/8 activities, AZD5582 (20 nM; 48 hours) causes apoptosis[2].

In H1975 NSCLC cells, AZD5582 (20 nM; 48 hours) inhibits cell viability by working with IFN or viral double-stranded RNA (dsRNA)[2].
Inducing the cleavage of caspase-3 and caspase-7, AZD5582 (20 nM; 17 or 25 hours) downregulates cIAP-1, activates RIPK1 (the upstream regulator of caspase-8), and activates the extrinsic (caspase-8) and intrinsic (caspase-9) apoptosis pathways[2].
Due to the co-treatment of HCC827 NSCLC cells with AZD5582 and IFN, which results in the induction of cell death and active caspase-3/8 activities, AZD5582 (20 nM; 48 hours) causes apoptosis[2].

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AZD5582 alone at 20 nM slightly inhibited the viability of the H1975 non-small cell lung cancer (NSCLC) cell line. [2]
AZD5582 (20 nM) cooperated strongly with interferon-gamma (IFNγ) at concentrations as low as 1 ng/ml to profoundly induce cell death in H1975 cells. In contrast, combinations of AZD5582 with TNFα, IFNα, or IFNλ had minimal effects. [2]
AZD5582 (20 nM) also cooperated with the double-stranded RNA analog polyinosinic-polycytidylic acid (poly(I:C)) to induce cell death in H1975 cells, but the effect was weaker than with IFNγ. AZD5582 showed minor synergistic effects with cisplatin or TRAIL. [2]
The synergistic cell death induced by AZD5582 plus IFNγ was also observed in other NSCLC cell lines, including HCC827 and H1437, but was weak in A549 cells and absent in Calu-3 and H441 cells. Normal human alveolar epithelial cells (HAECs) were not harmed by the combination. [2]
AZD5582 (20 nM) combined with IFNγ (1 or 5 ng/ml) completely eradicated colony formation of H1975 cells in a long-term (4-week) clonogenic assay, whereas AZD5582 alone or combined with poly(I:C) did not. [2]
Western blot analysis showed that AZD5582 (20 nM) alone downregulated cIAP1 protein levels and induced phosphorylation of RIPK1 and cleavage (activation) of caspase-8, caspase-9, caspase-3, caspase-7, and PARP in H1975 cells. These apoptotic markers were markedly enhanced when AZD5582 was combined with IFNγ (10 ng/ml). [2]
In HCC827 cells, AZD5582 (20 nM) plus IFNγ (5 ng/ml) synergistically induced time-dependent cleavage of caspase-8 and PARP, and significantly increased caspase-3 and caspase-8 enzymatic activities. Annexin V staining confirmed apoptosis induction. [2]
The cell death induced by AZD5582/IFNγ combination was almost completely blocked by a JAK kinase inhibitor, and significantly prevented by the general caspase inhibitor Z-VAD-FMK, the caspase-8 specific inhibitor Z-IETD-FMK, and the RIPK1 inhibitor necrostatin-1. It was not affected by a TNFα neutralizing antibody, an NF-κB inhibitor, an EGFR inhibitor, or inhibitors of necroptosis (RIPK3 inhibitor GSK872, MLKL inhibitor necrosulfonamide), indicating a TNFα-independent apoptotic mechanism mediated by JAK kinase, caspase-8, and RIPK1. [2]
Sensitivity to the AZD5582/IFNγ combination correlated with cellular competence in IFNγ signaling (expression of IFNγ receptor-1 and STAT1) and low expression levels of the IAP proteins survivin and livin. [2]

After two weeks of treatment with AZD5582 (intravenous injection; 0.1-3.0 mg/kg; once a week; two weeks), the tumors had mostly disappeared. When the mice are given a medium dose of the drug (0.5 mg/kg), cIAP1 degrades after administration, but it takes some time to reach an apoptosis-inducing effect[1].

Cell death is assessed by trypan blue exclusion using at least 200~500 cells, depending on whether the cells were treated with AZD5582, siRNA, or DNA. Pancreatic cancer cells are seeded at 1~3 × 104 cells per well in a 96-well microtiter plate for the MTS assay. The next day, cells are given various doses of the IAP antagonist AZD5582 for 72 hours. The MTS assay is carried out in accordance with the MTS assay protocol.

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Cell Viability/Proliferation Assay: NSCLC cells were seeded into 48-well plates, grown to subconfluence, and then treated with AZD5582 (various concentrations, commonly 20 nM) alone or in combination with cytokines (e.g., IFNγ, TNFα) or other agents (e.g., poly(I:C), TRAIL, cisplatin) for indicated time periods (e.g., 48-72 hours). Cell viability was assessed using an MTS assay reagent. Absorbance was measured, and cell survival rates were calculated relative to DMSO-treated control cells. Viable cell counts were also performed using trypan blue exclusion and an automated cell counter. [2]
Clonogenic (Colony Formation) Assay: Cells were seeded into 12-well plates at a low density (500 cells/well). After overnight attachment, cells were incubated with AZD5582 (20 nM) and/or IFNγ (1 or 5 ng/ml) for 4 weeks, with media changes every 5 days. Colonies were then fixed, stained with crystal violet, and counted visually. The stain was also eluted and quantified by measuring absorbance at 540 nm. [2]
Western Blot Analysis: Cells were treated with AZD5582 and/or IFNγ, washed with PBS, and lysed. Protein concentrations were determined. Equal amounts of protein were resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies against target proteins (e.g., cleaved caspases, PARP, cIAP1, XIAP, p-RIPK1, STAT1, survivin, livin). After incubation with HRP-conjugated secondary antibodies, signals were detected using enhanced chemiluminescence substrate. [2]
Apoptosis Assay (Annexin V Flow Cytometry): Cells were treated with AZD5582 (20 nM) and IFNγ (5 ng/ml) for 48 hours. Both adherent and detached cells were collected. Cells were then stained with FITC-conjugated Annexin V and analyzed by flow cytometry to identify apoptotic cells. [2]
Caspase Activity Assay: Cells were treated with AZD5582 and IFNγ, then lysed. Cell lysates (containing 100 µg protein) were incubated with colorimetric caspase-3 substrate (DEVD-pNA) or caspase-8 substrate (IETD-pNA) in reaction buffer. The release of the chromophore p-nitroaniline (pNA) was measured by absorbance at 405 nm, and activity was expressed as OD405 per amount of protein. [2]
Mechanistic Studies using Inhibitors: To investigate the death pathway, cells were pre-treated or co-treated with various pharmacological inhibitors (e.g., Z-VAD-FMK, Z-IETD-FMK, necrostatin-1, JAK kinase inhibitor, TNFα neutralizing antibody, GSK872, necrosulfonamide) alongside AZD5582 and IFNγ. Cell viability was subsequently assessed by MTS assay to determine the effect of pathway inhibition on the combination-induced cell death. [2]

[1]. Discovery of a novel class of dimeric Smac mimetics as potent IAP antagonists resulting in a clinical candidate for the treatment of cancer (AZD5582). J Med Chem. 2013 Dec 27;56(24):9897-919.

[2]. IF-γ and Smac mimetics synergize to induce apoptosis of lung cancer cells in a TNFα-independent manner,Cancer Cell Int. 2018; 18: 84.

AZD5582 has been described as a novel dimeric Smac mimic. [2]
This study found that AZD5582, as a monotherapy, exhibited limited antiproliferative activity in various non-small cell lung cancer (NSCLC) cell lines, consistent with the characteristics of previously reported IAP inhibitors. However, its therapeutic potential was significantly enhanced when used in combination with IFNγ. [2]
This study suggests that the AZD5582/IFNγ combination therapy represents a novel apoptosis-targeting therapy for a subset of NSCLC patients. Biomarkers predicting efficacy may include functional IFNγ signaling pathway components (expression/activation of IFNγR1 and STAT1) and low expression of specific IAP proteins (survivin and livin). [2]

C58H78N8O8

1015.2887

1014.594

C, 68.61; H, 7.74; N, 11.04; O, 12.61

1258392-53-8

AZD5582 dihydrochloride;1883545-51-4

49847690

white solid powder

1.3±0.1 g/cm3

1207.3±65.0 °C at 760 mmHg

683.9±34.3 °C

0.0±0.3 mmHg at 25°C

1.622

9.22

6

10

19

74

1950

10

O=C([C@H](C1CCCCC1)NC([C@H](C)NC)=O)N1CCC[C@H]1C(N[C@H]1C2=CC=CC=C2C[C@H]1OCC#CC#CCO[C@@H]1CC2=CC=CC=C2[C@@H]1NC([C@@H]1CCCN1C([C@H](C1CCCCC1)NC([C@H](C)NC)=O)=O)=O)=O

WLMCRYCCYXHPQF-ZVMUOSSASA-N

InChI=1S/C58H78N8O8/c1-37(59-3)53(67)61-49(39-21-9-7-10-22-39)57(71)65-31-19-29-45(65)55(69)63-51-43-27-15-13-25-41(43)35-47(51)73-33-17-5-6-18-34-74-48-36-42-26-14-16-28-44(42)52(48)64-56(70)46-30-20-32-66(46)58(72)50(40-23-11-8-12-24-40)62-54(68)38(2)60-4/h13-16,25-28,37-40,45-52,59-60H,7-12,19-24,29-36H2,1-4H3,(H,61,67)(H,62,68)(H,63,69)

(2S)-1-[(2S)-2-cyclohexyl-2-[[(2S)-2-(methylamino)propanoyl]amino]acetyl]-N-[(1S,2R)-2-[6-[[(1S,2R)-1-[[(2S)-1-[(2S)-2-cyclohexyl-2-[[(2S)-2-(methylamino)propanoyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]-2,3-dihydro-1H-inden-2-yl]oxy]hexa-2,4-diynoxy]-2,3-dihydro-1H-inden-1-yl]pyrrolidine-2-carboxamide

AZD-5582; AZD 5582; AZD5582

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~98.5 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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