Xevinapant (AT406)

Catalog No.S2754 Batch:S275404

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Technical Data

Formula

C32H43N5O4

Molecular Weight 561.71 CAS No. 1071992-99-8
Solubility (25°C)* In vitro DMSO 100 mg/mL (178.02 mM)
Ethanol 100 mg/mL (178.02 mM)
Water Insoluble
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description Xevinapant (AT406, ARRY-334543, Debio1143, SM-406) is a potent Smac mimetic and an antagonist of IAP (inhibitor of apoptosis protein via E3 ubiquitin ligase), binding to XIAP-BIR3, cIAP1-BIR3 and cIAP2-BIR3 with Ki of 66.4 nM, 1.9 nM, and 5.1 nM, 50- to 100-fold higher affinities than the Smac AVPI peptide. Phase 1.
Targets
cIAP1-BIR3 [1]
(cell-free assay)
cIAP2-BIR3 [1]
(cell-free assay)
XIAP-BIR3 [1]
(cell-free assay)
1.9 nM(Ki) 5.1 nM(Ki) 66.4 nM(Ki)
In vitro AT-406 is a Smac mimetic and appears to mimic closely the AVPI peptide in both hydrogen bonding and hydrophobic interactions with XIAP, with additional hydrophobic contacts with W323 of XIAP. AT-406 is more sensitive to these IAPs than Smac AVPI peptide with 50-100 fold binding affinities. AT-406 (at 1 μM) completely restores the activity of caspase-9, which is suppressed by 500 nM XIAP BIR3 in a cell-free system. In MDA-MB-231 cell, AT-406 induces rapid cellular cIAP1 degradation and also pulls down the cellular XIAP protein. AT-406 effectively inhibits lots of human cancer cell lines and shows IC50 of 144 and 142 nM in MDA-MB-231 cell and SK-OV-3 ovarian cell, with low toxicity against normal-like human breast epithelial MCF-12F cells and primary human normal prostate epithelial cells. AT-406 induces apoptosis in MDA-MB-231 cell by inducing activation of caspase-3 and cleavage of PARP. [1]
In vivo AT-406 has good pharmacokinetic (PK) properties and oral bioavailability in mice, rats, non-human primates, and dogs. In the MDA-MB-231 xenograft, AT-406 effectively induces cIAP1 degradation and processing of procaspase-8, cleavage of PARP in tumor tissues at 100 mg/kg with well toleration even at 200 mg/kg. AT-406 induces significant tumor growth inhibition with p of 0.0012 at 100 mg/kg. [1]

Protocol (from reference)

Kinase Assay:

[1]

  • Fluorescence Polarization Based Assays for XIAP, cIAP1, and cIAP2 BIR3 Proteins

    FL-AT-406 (the fluorescently tagged AT-406) is employed to develop a set of new FP assays for determination of the binding affinities of Smac mimetics to XIAP, cIAP-1, and cIAP-2 BIR3 proteins. The Kd value of FL-AT-406 to each IAP protein is determined by titration experiments using a fixed concentration of FL-AT-406 and different concentrations of the protein up to full saturation. Fluorescence polarization values are measured using an Infinite M-1000 plate reader in Microfluor 2 96-well, black, round-bottom plates. To each well, FL-AT-406 (2, 1, and 1 nM for experiments with XIAP BIR3, cIAP-1 BIR3, and cIAP-2 BIR3, respectively) and different concentrations of the protein are added to a final volume of 125 μL in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02% sodium azide, with 4% DMSO). Plates are mixed and incubated at room temperature for 2-3 hours with gentle shaking. The polarization values in millipolarization units (mP) are measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Equilibrium dissociation constants (Kd) are then calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 5.0 software. In competitive binding experiments for XIAP3 BIR3, AT-406 is incubated with 20 nM XIAP BIR3 protein and 2 nM FL-AT-406 in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine γ-globulin; 0.02% sodium azide). In competitive binding experiments for cIAP1 BIR3 protein, 3 nM protein and 1 nM FL-AT-406 are used. In competitive binding experiments for cIAP2 BIR3, 5 nM protein and 1 nM FL-AT-406 are used. For each competitive binding experiment, polarization values are measured after 2-3 hours of incubation using an Infinite M-1000 plate reader. The IC50 value, the inhibitor concentration at which 50% of the bound tracer is displaced, is determined from the plot using nonlinear least-squares analysis. Curve fitting is performed using the PRISM software. A Ki value for AT-406 is calculated.

Cell Assay:

[1]

  • Cell lines

    MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines

  • Concentrations

    ~ 1 μM

  • Incubation Time

    4 days

  • Method

    Cells are seeded in 96-well flat bottom cell culture plates at a density of (3-4) × 103 cells/well with AT-406 and incubated for 4 days. The rate of cell growth inhibition after treatment with different concentrations of AT-406 is determined by assaying with (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8). WST-8 is added to each well to a final concentration of 10%, and then the plates are incubated at 37 °C for 2−3 hours. The absorbance of the samples is measured at 450 nm using a TECAN ULTRA reader. Concentration of AT-406 that inhibited cell growth by 50% (IC50) is calculated by comparing absorbance in the untreated cells and the cells treated with AT-406.

Animal Study:

[1]

  • Animal Models

    MDA-MB-231 xenograft tumors in severe combined immune deficiency (SCID) mice

  • Dosages

    10 mg/kg (i.v.), 10 mg/kg (p.o.), 30 mg/kg (p.o.) and 100 mg/kg (p.o.)

  • Administration

    Administered via intravenously (i.v.) or oral gavage (p.o.)

Customer Product Validation

, , Cancer Res, 2015, 75(8):1736-48.

, , Int J Oncol, 2016, 49(1):153-63.

, , Biochem Biophys Res Commun, 2016, 478(1):293-9.

Data from [Data independently produced by , , Oncotarget, 2017, 8(6):9466-9475]

Selleck's Xevinapant (AT406) has been cited by 37 publications

Complex IIa formation and ABC transporters determine sensitivity of OSCC to Smac mimetics [ Cell Death Dis, 2024, 15(11):855] PubMed: 39578442
The Effect of Xevinapant Combined with Ionizing Radiation on HNSCC and Normal Tissue Cells and the Impact of Xevinapant on Its Targeted Proteins cIAP1 and XIAP [ Cells, 2023, 12(12)1653] PubMed: 37371123
The Effect of Xevinapant Combined with Ionizing Radiation on HNSCC and Normal Tissue Cells and the Impact of Xevinapant on Its Targeted Proteins cIAP1 and XIAP [ Cells, 2023, 12(12)1653] PubMed: 37371123
Mycoplasma hyorhinis infection promotes TNF-α signaling and SMAC mimetic-mediated apoptosis in human prostate cancer [ Heliyon, 2023, 9(10):e20655] PubMed: 37867861
Mycoplasma hyorhinis infection promotes TNF-α signaling and SMAC mimetic-mediated apoptosis in human prostate cancer [ Heliyon, 2023, 9(10):e20655] PubMed: 37867861
CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects [ Sci Adv, 2023, 9(33):eadf6692] PubMed: 37595047
CD4+ T cell-mimicking nanoparticles encapsulating DIABLO/SMAC mimetics broadly neutralize HIV-1 and selectively kill HIV-1-infected cells [ Theranostics, 2021, 11(18):9009-9021] PubMed: 34522224
Targeting c-IAP1, c-IAP2, and Bcl-2 Eliminates Senescent Glioblastoma Cells Following Temozolomide Treatment [ Cancers (Basel), 2021, 13(14)3585] PubMed: 34298797
Smac mimetics and TRAIL cooperate to induce MLKL-dependent necroptosis in Burkitt's lymphoma cell lines [ Neoplasia, 2021, 23(5):539-550] PubMed: 33971465
SMAC mimetics induce autophagy-dependent apoptosis of HIV-1-infected macrophages [ Cell Death Dis, 2020, 11(7):590] PubMed: 32719312

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