XIAP (Kd = 45 nM); cIAP1 (Kd <1 nM)

Birinapant binds to both cIAP1 and XIAP with Kd values of 45 and 1 nM, respectively. Birinapant significantly increases the potency of TNF-α-induced apoptosis in SUM149 (triple-negative, EGFR-activated) cells, which are TRAIL-sensitive but TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells. Rapid cIAP1 degradation, activation of caspases, cleavage of PARP, and activation of NF-B are all brought about by birinapant.[1] In vitro, birinapant and TNF- exhibit an effective antimelanoma effect. While neither substance is effective alone, birinapant combined with TNF-(1 ng/mL) inhibits the growth of the human melanoma cell lines WTH202, WM793B, WM1366, and WM164 with IC50s of 1.8, 2.5, 7.9, and 9 nM, respectively. With an IC50 of 2.4 nM, birinapant alone inhibits the proliferation of WM9 cells. In these cell lines, birinapant significantly inhibits the target proteins cIAP1 and cIAP2.[2]

Birinapant (30 mg/kg) treatment significantly induces abrogation of tumor growth in melanoma xenotransplantation models 451Lu with. [2]

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Birinapant inhibits tumor growth in melanoma xenotransplantation models as a single agent [2]
To investigate whether birinapant could inhibit melanoma tumor growth in an in vivo setting as a single agent, two cell lines were selected for xenotransplantation experiments: both were in vitro birinapant single agent resistant, but 451Lu did respond in vitro to the combination of birinapant with TNF-α, whereas 1205Lu did not respond to the combination treatment in vitro. Both cell lines were inoculated s.c. in NUDE mice and allowed to form palpable tumors before being randomized into vehicle control and birinapant treatment groups. During three weeks of dosing, birinapant showed an antitumor effect in both models, although the effect in the in vitro combination sensitive cell line was more sustained with abrogation of tumor growth in the birinapant treated animals. In contrast, 1205Lu tumors showed a marked slowing of tumor growth, but not abrogation of tumors (Fig 5A).
In a subsequent in vivo experiment, we then went on to confirm birinapant target inhibition in both models by immunoblot of tumor lysates. Animals were again inoculated with both xenograft models and tumors allowed to from. Animals were then pre-treated twice in an interval of 48h and tumors were harvested 3, 6, 12, and 24 hours after the second dosing. Compared to vehicle control, cIAP1 protein was reduced to low levels at 3h post and this effect was sustained for 24 hours in both models (Fig 5B). Staining for activated caspase-3 in biopsies of the same tumors showed a modest increase in apoptotic cells in the birinapant treated animals compared to vehicle control, 24h post treatment (Fig 5C).

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Drug treatment increased the mean [(18)F]ICMT-11 tumor uptake with a peak at 24 hours for CPA (40 mg/kg; AUC40-60: 8.04 ± 1.33 and 16.05 ± 3.35 %ID/mL × min at baseline and 24 hours, respectively) and 6 hours for birinapant (15 mg/kg; AUC40-60: 20.29 ± 0.82 and 31.07 ± 5.66 %ID/mL × min, at baseline and 6 hours, respectively). Voxel-based spatiotemporal analysis of tumor-intrinsic heterogeneity suggested that discrete pockets of caspase-3 activation could be detected by [(18)F]ICMT-11. Increased tumor [(18)F]ICMT-11 uptake was associated with caspase-3 activation measured ex vivo, and early radiotracer uptake predicted apoptosis, distinct from the glucose metabolism with [(18)F]fluorodeoxyglucose-PET, which depicted continuous loss of cell viability.
Conclusion: The proapoptotic effects of CPA and birinapant resulted in a time-dependent increase in [(18)F]ICMT-11 uptake detected by PET. [(18)F]ICMT-11-PET holds promise as a noninvasive pharmacodynamic biomarker of caspase-3-associated apoptosis in tumors. [3]

A fluorogenic substrate is used to ascertain the binding affinities of substances to XIAP and cIAP1, and the Kd values are then reported. The fluorescently labeled modified Smac peptide (AbuRPF-K(5-Fam)-NH2; FP pep-tide) dissociation constant (Kd) is first calculated by titrating varying protein concentrations (0.075–5 μM in half log dilutions) with a fixed concentration of peptide (5 nM). With 5 nM of FP peptide and 50 nM of XIAP used in the assay, the nonlinear least squares fit to a single-site binding model using GraphPad Prism produced the dose-response curves. The FP peptide:protein binary complex is mixed with various concentrations of Smac mimetics (100-0.001 μM in half log dilutions) for 15 min at room temperature in 100 mL of 0.1 M potassium phosphate buffer, pH 7.5, containing 100 mg/mL bovine c-globulin. After incubation, the polarization values are determined using a 520 nm emission filter and a 485 nm excitation filter on a multi-label plate reader.

After 24 hours of cell attachment, the cells are incubated with birinapant and/or TNF- for another 24 or 72 hours. The MTS assay is then carried out.

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Cell viability [1]
Trypan blue exclusion assay was performed as described previously [2]. Cells were seeded in 6 well plates at 7.5 × 104 (SUM149) or 1.5 × 105 (SUM190) cells per well and allowed to adhere overnight. Cells were treated with TRAIL (0–100 ng mL−1), Birinapant (0–10,000 nM), GT13402 (0–10,000 nM), TNF-α (50 ng mL−1), TNF-α neutralizing antibody (10 μg mL−1), pan-caspase inhibitor Q-VD-OPh (20 μM), or a combination as indicated. All treatments were applied for 24 h, and then the cells were trypsinized and resuspended in PBS. Next, 10 μL of cell suspension was added to 10 μL 0.4 % trypan blue, and 10 μL of the mixture was loaded onto a hemocytometer; cells were counted, and live and dead cell numbers were recorded.

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Clonogenic growth assay [1]
Cells were plated in triplicate in 6 well plates at 250–500 cells per well (SUM149) or 500–1,000 cells per well (SUM190) and allowed to adhere overnight. Cells were treated with TRAIL (0–100 ng mL−1), Birinapant (0–10,000 nM), GT13402 (0–10,000 nM), TNF-α (50 ng mL−1), TNF-α neutralizing antibody (10 μg mL−1), pan-caspase inhibitor Q-VD-OPh (20 μM), or a combination as indicated. After 24 h treatments, the cells were washed twice with PBS, and regular growth media was added. The cells were then allowed to grow for 5–14 days, changing the media every 4–5 days. Once colonies of at least 50 cells were observed, the cells were washed with PBS, fixed, stained with 0.4 % crystal violet, then rinsed in cold water and left to dry overnight. Colonies were counted and imaged using a ColCount, and colonies formed per cells plated was calculated. Numbers were normalized to untreated.

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Annexin-V staining [1]
Cells were seeded in 6 well plates at 7.5 × 104 (SUM149) or 1.5 × 105 (SUM190) cells per well and allowed to adhere overnight. Cells were treated with TRAIL (0–100 ng mL−1), Birinapant (0–1,000 nM), GT13402 (0–1,000 nM), or a combination as indicated for 12 h. Cells were harvested with 0.25 % trypsin (-EDTA), washed with PBS, and resuspended in biotin-conjugated Annexin-V for 5 min at RT. Cells were washed again with PBS and resuspended in streptavidin-conjugated FITC and 7-AAD dyes for 15 min on ice. Cells were washed and resuspended in PBS, and then at least 25,000 events were collected on a BD FACSCalibur flow cytometer. Results were analyzed using FlowJo software.

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TNFR1 knockdown [1]
Cells were seeded in 6 well plates at 1.5 × 105 cells per well and allowed to adhere overnight. After 24 h, either scramble control siRNA or TNFR1 targeting siRNA at 100 nM was applied in the presence of Dharmafect transfection reagent. Birinapant (0–1,000 nM) was added the day after transfection, and cells were harvested after 24 h for trypan blue viability staining and western immunoblotting to confirm knockdown.

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In vitro drug sensitivity assays [2]
For monolayer cell culture assays, cells were allowed to attach for 24h and subsequently incubated with Birinapant and/or TNF-α for 24 or 72h. CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) assay was performed according to the manufacturer’s description. For cell cycle analysis, melanoma cells were fixed in 70% ethyl alcohol and stained with propidium iodide. Samples were subsequently analyzed with an EPICS XL apparatus. Annexin V staining was performed with an annexin V allophycocyanin conjugate according to the manufacturer’s description. Briefly, cells were treated with DMSO control, Birinapant and/or TNF-α for 24h. Resuspended cells were washed and incubated with the conjugate for 15min and annexin-binding buffer was added. Samples were subsequently analyzed with an EPICS XL apparatus.
451Lu and WM1366 melanoma cells were treated with Birinapant 1uM in combination with TNF-α 1ng/ml. Cells were then incubated for 72h in the presence or absence of Z-VAD-FMK a pan caspase inhibitor. Proliferation was assessed using the MTS assay.
451Lu and WM1366 melanoma cells were treated with Birinapant 1uM in combination with TNF-α 1ng/ml. Cells were then incubated for 72h in the presence or absence of Necrostatin-1 a RIP1 kinase inhibitor. Proliferation was assessed using the MTS assay. [2]

Human melanoma xenografts 451Lu
30 mg/kg
3 times per week intraperitoneally

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All animal experiments were performed in accordance with Wistar IACUC protocol 111954 in NUDE mice. Ten animals each were inoculated s.c. with 1×106 451Lu or 1205Lu human melanoma cells in a suspension of matrigel / complete media at a ratio of 1:1. After formation of palpable tumors, mice from both tumor models were randomized into two groups. Both groups were treated intra-peritoneal three times/ week with either vehicle control or Birinapant 30mg/kg for 21 days. Birinapant was dissolved in 12.5% Captisol in distilled water. Tumor size was assessed twice weekly by caliper measurement. Subsequently, satellite groups of ten and fifteen mice were inoculated in the same fashion with 451Lu and 1205Lu tumor cells respectively. After tumors had reached a mean volume of 200mm3 animals were dosed with either Birinapant or vehicle control as described above. After 48 hours and two doses, animals were sacrificed and tumors were harvested at four time points after the last treatment. Tumor samples were snap frozen in liquid nitrogen for subsequent protein analysis and preserved as FFPE blocks for immune-histochemistry. [2]

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Small animal experimental models [3]
The in vivo experimental xenograft models were established by subcutaneous injection of 5 × 103 38C13 cells in C3H mice and 5 × 106 HCT116 or 2 × 106 MDA-MB-231 cells in BALB/c nude mice. All mice were 6- to 8-week-old females from Charles River. When xenografts reached approximately 80 mm3 [tumor dimensions were measured using a caliper and tumor volumes were calculated using the ellipsoid formula that is best for estimating tumor mass; volume mm3 = (π/6) × a × b × c, where a, b, and c represent 3 orthogonal axes of the tumor], mice were injected a single dose of CPA (40 mg/kg i.p.) or Birinapant (15 m/kg i.p.) and subjected to positron emission tomography-computed tomography (PET-CT) imaging in a longitudinal setting where the same animal serves as its own control.

[1]. Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-α-independent mechanism. Breast Cancer Res Treat. 2013 Jan;137(2):359-71.

[2]. The novel SMAC mimetic birinapant exhibits potent activity against human melanoma cells. Clin Cancer Res. 2013 Apr 1;19(7):1784-94.

[3]. Temporal and spatial evolution of therapy-induced tumor apoptosis detected by caspase-3-selective molecular imaging. Clin Cancer Res. 2013 Jul 15;19(14):3914-24.

[4]. Birinapant (TL32711), a bivalent SMAC mimetic, targets TRAF2-associated cIAPs, abrogates TNF-induced NF-κB activation, and is active in patient-derived xenograft models. Mol Cancer Ther. 2014 Apr;13(4):867-79.

Birinapant is a dipeptide.
Birinapant has been investigated for the treatment of Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukemia (CMML).
Birinapant is a synthetic small molecule that is both a peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) and inhibitor of IAP (Inhibitor of Apoptosis Protein) family proteins, with potential antineoplastic activity. As a SMAC mimetic and IAP antagonist, birinapant selectively binds to and inhibits the activity of IAPs, such as X chromosome-linked IAP (XIAP) and cellular IAPs 1 (cIAP1) and 2 (cIAP2), with a greater effect on cIAP1 than cIAP2. Since IAPs shield cancer cells from the apoptosis process, this agent may restore and promote the induction of apoptosis through apoptotic signaling pathways in cancer cells and inactivate the nuclear factor-kappa B (NF-kB)-mediated survival pathway. IAPs are overexpressed by many cancer cell types. They are able to suppress apoptosis by binding to, via their baculoviral lAP repeat (BIR) domains, and inhibiting active caspases-3, -7 and -9. IAP overexpression promotes both cancer cell survival and chemotherapy resistance.

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X-linked inhibitor of apoptosis protein (XIAP), the most potent mammalian caspase inhibitor, has been associated with acquired therapeutic resistance in inflammatory breast cancer (IBC), an aggressive subset of breast cancer with an extremely poor survival rate. The second mitochondria-derived activator of caspases (Smac) protein is a potent antagonist of IAP proteins and the basis for the development of Smac mimetic drugs. Here, we report for the first time that bivalent Smac mimetic Birinapant induces cell death as a single agent in TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells and significantly increases potency of TRAIL-induced apoptosis in TRAIL-sensitive SUM149 (triple-negative, EGFR-activated) cells, two patient tumor-derived IBC models. Birinapant has high binding affinity (nM range) for cIAP1/2 and XIAP. Using isogenic SUM149- and SUM190-derived cells with differential XIAP expression (SUM149 wtXIAP, SUM190 shXIAP) and another bivalent Smac mimetic (GT13402) with high cIAP1/2 but low XIAP binding affinity (K (d) > 1 μM), we show that XIAP inhibition is necessary for increasing TRAIL potency. In contrast, single agent efficacy of Birinapant is due to pan-IAP antagonism. Birinapant caused rapid cIAP1 degradation, caspase activation, PARP cleavage, and NF-κB activation. A modest increase in TNF-α production was seen in SUM190 cells following Birinapant treatment, but no increase occurred in SUM149 cells. Exogenous TNF-α addition did not increase Birinapant efficacy. Neutralizing antibodies against TNF-α or TNFR1 knockdown did not reverse cell death. However, pan-caspase inhibitor Q-VD-OPh reversed Birinapant-mediated cell death. In addition, Birinapant in combination or as a single agent decreased colony formation and anchorage-independent growth potential of IBC cells. By demonstrating that Birinapant primes cancer cells for death in an IAP-dependent manner, these findings support the development of Smac mimetics for IBC treatment. [1]

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Purpose: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas. Experimental design: Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models. Results: When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line. Conclusions: Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors. [2]

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Purpose: Induction of apoptosis in tumors is considered a desired goal of anticancer therapy. We investigated whether the dynamic temporal and spatial evolution of apoptosis in response to cytotoxic and mechanism-based therapeutics could be detected noninvasively by the caspase-3 radiotracer [(18)F]ICMT-11 and positron emission tomography (PET). Experimental design: The effects of a single dose of the alkylating agent cyclophosphamide (CPA or 4-hydroperoxycyclophosphamide), or the mechanism-based small molecule SMAC mimetic birinapant on caspase-3 activation was assessed in vitro and by [(18)F]ICMT-11-PET in mice bearing 38C13 B-cell lymphoma, HCT116 colon carcinoma, or MDA-MB-231 breast adenocarcinoma tumors. Ex vivo analysis of caspase-3 was compared to the in vivo PET imaging data. [3]

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The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.[4]

C42H56F2N8O6

806.94

806.42909

C, 62.51; H, 6.99; F, 4.71; N, 13.89; O, 11.90

1260251-31-7

49836020

White solid powder

1.3±0.1 g/cm3

1090.5±65.0 °C at 760 mmHg

613.3±34.3 °C

0.0±0.3 mmHg at 25°C

1.628

2.98

8

10

15

58

1350

8

FC1C([H])=C([H])C2=C(C=1[H])N([H])C(C1=C(C3C([H])=C([H])C(=C([H])C=3N1[H])F)C([H])([H])[C@]1([H])C([H])([H])[C@@]([H])(C([H])([H])N1C([C@]([H])(C([H])([H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C([H])([H])[H])=O)=O)O[H])=C2C([H])([H])[C@]1([H])C([H])([H])[C@@]([H])(C([H])([H])N1C([C@]([H])(C([H])([H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C([H])([H])[H])=O)=O)O[H]

PKWRMUKBEYJEIX-DXXQBUJASA-N

InChI=1S/C42H56F2N8O6/c1-7-33(49-39(55)21(3)45-5)41(57)51-19-27(53)15-25(51)17-31-29-11-9-23(43)13-35(29)47-37(31)38-32(30-12-10-24(44)14-36(30)48-38)18-26-16-28(54)20-52(26)42(58)34(8-2)50-40(56)22(4)46-6/h9-14,21-22,25-28,33-34,45-48,53-54H,7-8,15-20H2,1-6H3,(H,49,55)(H,50,56)/t21-,22-,25-,26-,27-,28-,33-,34-/m0/s1

(2S)-N-[(2S)-1-[(2R,4S)-2-[[6-fluoro-2-[6-fluoro-3-[[(2R,4S)-4-hydroxy-1-[(2S)-2-[[(2S)-2-(methylamino)propanoyl]amino]butanoyl]pyrrolidin-2-yl]methyl]-1H-indol-2-yl]-1H-indol-3-yl]methyl]-4-hydroxypyrrolidin-1-yl]-1-oxobutan-2-yl]-2-(methylamino)propanamide

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: ≥ 2.08 mg/mL (2.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: 2.08 mg/mL (2.58 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 20.8 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: ≥ 2.08 mg/mL (2.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 15% Captisol: 15mg/mL

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