cIAP-1 (Ki = 0.31 nM); cIAP-2 (Ki = 1.1 nM); cIAP
XIAP (X-linked inhibitor of apoptosis protein), specifically targeting both the BIR2 and BIR3 domains concurrently. The binding affinity (IC50) to XIAP protein containing both BIR2 and BIR3 domains (residues 120-356) is 1.39 nM. [1]

SM-164 binds to XIAP containing both BIR domains with IC50 of 1.39 nM, being 300 and 7000 times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. At concentrations as low as 1 nM, SM-164 effectively induces apoptosis in the HL-60 leukemia cell line by targeting cellular XIAP.[1]
SM-164 causes caspase-8 and caspase-3-dependent apoptosis in cancer cells. SM-164 induces TNFα-dependent apoptosis and cIAP-1 degradation.[2]
In vitro, SM-164 and TRAIL work very well together against breast, prostate, and colon cancer cell lines that are both TRAIL-sensitive and TRAIL-resistant. Through amplification of the caspase-8-mediated extrinsic apoptosis pathway, SM-164 increases TRAIL-induced apoptosis in cancer cells. [3]

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SM-164 (compound 4) effectively inhibits cell growth in the HL-60 human leukemia cell line with an IC50 value of 1 nM.
It potently and effectively induces apoptosis in the HL-60 cell line in a dose-dependent manner. At 10 nM and 100 nM for 24 hours, it induces 57% and 69% of HL-60 cells to undergo apoptosis, respectively. At as low as 1 nM for 24 hours, it induces 17% of HL-60 cells to undergo apoptosis.
In HL-60 cells, SM-164 at concentrations as low as 10 nM induces robust activation of caspase-9 and caspase-3 and cleavage of PARP (poly (ADP-ribose)-polymerase) within 24 hours.
In a co-immunoprecipitation assay using HL-60 cell lysates, SM-164 at 10 nM competes off more than 50% of XIAP bound to a biotinylated monovalent Smac mimetic (BL-SM-122 at 10 μM), and at 100 nM completely blocks the binding, indicating it binds to cellular XIAP with much higher affinity. [1]

In MDA-MB-231 xenograft tumor tissues, SM-164 causes strong apoptosis and rapid cIAP-1 degradation in addition to tumor regression, but it has no toxic effects on normal mouse tissues.[2]
The combination of SM-164 and TRAIL causes tumor regression without being toxic to animals because SM-164 induces cIAP1 degradation in tumor tissues and significantly increases TRAIL's in vivo antitumor activity. [3]

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In a MDA-MB-231 xenograft model in SCID mice, a single intravenous dose of SM-164 at 5 mg/kg markedly decreased cIAP-1 protein levels within 1 hour, and the effect lasted for at least 24 hours.Robust activation of caspase-8, caspase-9 and caspase-3, as well as PARP cleavage, were observed at 3 hours and persisted for 24 hours in tumor tissues.[2]
TUNEL assay showed strong apoptosis in tumor tissues as early as 3 hours after treatment; more than 50% of tumor cells were TUNEL positive at the 6-hour time point.[2]
H&E staining revealed profound damage to tumor tissues (cell shrinkage, nuclear pyknosis, chromatin condensation) after SM-164 treatment.[2]
In the same xenograft model, treatment with SM-164 at 1 mg/kg (intravenous, daily, 5 days/week for 2 weeks) completely inhibited tumor growth during the treatment period.[2]
Treatment with SM-164 at 5 mg/kg (same schedule) reduced tumor volume from 147±54 mm³ to 54±32 mm³ (65% reduction) and achieved tumor regression.[2]
The antitumor activity of SM-164 was long-lasting and not transient.[2]
SM-164 at 5 mg/kg was statistically more effective than Taxotere at 7.5 mg/kg at the end of treatment (P<0.01).No significant weight loss or other signs of toxicity were observed in mice treated with SM-164 at 1 or 5 mg/kg.In contrast to tumor tissues, SM-164 had no effect on normal mouse tissues examined, including small intestine, stomach, liver and spleen. [2]

The FP-based assay for the XIAP BIR3 protein is described. In a nutshell, 5-carboxyfluorescein is coupled to the lysine side chain of a mutated Smac peptide with the sequence, and this fluorescently tagged peptide (named SM5F) is used as the fluorescent tracer in FP-based binding assay to XIAP BIR3. This fluorescent tracer has a 17.9 nM Kd value to XIAP BIR3. In experiments involving competitive binding, a tested substance is incubated with 30 nM of XIAP BIR3 protein and 5 nM of SM5F in the assay buffer ((100 mM potassium phosphate, pH 7.5; 100 μg/ml bovine gamma globulin; 0.02 % sodium azide). The Kd value of SM5F to cIAP-1 BIR3 protein is determined to be 4.1 nM. In competitive binding experiments, 10 nM of cIAP-1 BIR3 protein and 2 nM of SM5F tracer are used. The Kd value of SM5F for the cIAP-2 BIR3 protein is found to be 6.6 nM. 25 nM of the cIAP-2 BIR3 protein and 2 nM of the SM5F tracer are used in competitive binding experiments. A bivalent fluorescently tagged tracer known as Smac-1F is used in an FP-based competitive binding assay to measure the binding affinities of Smac mimetics to XIAP that contains both BIR2 and BIR3 domains. The bivalent tagged tracer to XIAP containing BIR2 and BIR3 domains has a Kd value of 2.3 nM. In competitive binding tests, a test substance is incubated with 3 nM of XIAP protein containing both BIR2 and BIR3 domains (residues 120–356) and 1 nM of in the same assay buffer.

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The binding affinity of SM-164 to XIAP protein containing both BIR2 and BIR3 domains was determined using a competitive fluorescence-polarization (FP) assay. A fluorescently tagged, bivalent Smac-based peptide named Smac-1F was used as the tracer. The assay was performed by incubating the test compound with XIAP protein (residues 120–356, 3 nM) and Smac-1F (0.5 nM) in an assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/ml bovine gamma globulin, 0.02% sodium azide) in 96-well black round-bottom plates. Controls included XIAP and Smac-1F (0% inhibition) and Smac-1F only (100% inhibition). Polarization values were measured after 2 hours incubation using an Ultra plate reader. The IC50 value, the concentration at which 50% of bound tracer was displaced, was determined from a plot using nonlinear least-squares analysis. [1]
The binding mode of SM-164 to XIAP was probed using analytical gel filtration experiments with recombinant XIAP BIR3-only (residues 241–356) and BIR2-BIR3 (residues 156–356) proteins. Experiments were performed on a Superdex 75 column in Tris-HCl (pH 7.5, 20 mM), NaCl (200 mM), zinc acetate (50 μM), and DTT (1 mM). Recombinant protein (1 mg/ml) was run alone or after incubation with a 1:1 molar ratio of Smac mimetic. With BIR3-only protein, SM-164 induced dimerization of the protein, indicating a 1:2 complex. With BIR2-BIR3 protein, it did not induce dimerization but made the protein more compact. Using mutated proteins (BIR2(E219R)-BIR3 and BIR2-BIR3(E314S,W323E)) in the same gel filtration experiments confirmed that both BIR2 and BIR3 domains are directly involved in the binding. [1]
Heteronuclear single quantum correlation (HSQC) NMR spectroscopy was employed to investigate the binding mode. 15N HSQC spectra were recorded on a 500MHz NMR spectrometer with samples containing 100 μM of the 15N labeled BIR2-BIR3 protein (residues 156-356) in 50 mM Tris (pH 7.2), 50 μM ZnCl2, 1 mM DTT at 25°C with or without test compound at a final concentration between 10 - 150 μM. The spectra showed that many residues in the protein are affected by SM-164. Line width analysis indicated the protein has the same molecular size with or without SM-164, confirming no dimerization. SM-164 affected more protein residues than the monovalent compound 1, with changes apparent at the lowest concentration tested (10 μM, a 1:10 ratio to protein). Analysis of BIR3 residues (e.g., V298) and BIR2 residues showed that SM-164 binds to both domains with high affinities. [1]
Cell-free caspase functional assays were used to evaluate the ability of SM-164 to antagonize XIAP. MDA-MB-231 cell lysates were prepared and caspase-9 and -3/-7 were activated by adding cytochrome c and dATP. Recombinant XIAP L-BIR2-BIR3 protein (50 nM) was added to completely suppress caspase activity. Different concentrations of SM-164 (1 nM - 100 μM) were added to determine the restoration of caspase activity. 25 μM of caspase-9 substrate (Z-LEHD-AFC) or caspase-3/-7 substrate (Z-DEVD-AFC) was added. Fluorescence detection was carried out using an excitation wavelength of 400 nm and an emission wavelength of 505 nm. SM-164 antagonized XIAP in a dose-dependent manner and at an equal molar concentration of XIAP, it completely overcame the inhibition and fully restored the activity of caspase-9 and -3/-7. [1]

SM-164 (1 μM) was applied to cells that had previously received HG (5 μM) treatment for 30 min.

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The effect of SM-164 on HL-60 cell growth was evaluated by a WST-8 assay. Cells (3000-4000 cells per well) were cultured in 96-well plates in medium containing various concentrations of SM-164 for 4 days. At the end of incubation, WST-8 dye was added and incubated for 1-3 hours, then the absorbance was measured at 450 nm. Cell growth inhibition was evaluated as the ratio of the absorbance of the sample to that of the control. The IC50 was determined to be 1 nM. [1]
Apoptosis assays were performed with an annexin-V/propidium iodide (PI) apoptosis detection kit. HL-60 cells were treated with SM-164 for 24 hours. Cells were harvested, washed with ice-cold PBS, and stained with annexin-V-FITC and PI for 15 minutes at room temperature in the dark. Stained cells were analyzed by flow cytometry. Annexin-V (+) cells were measured as apoptotic cells. SM-164 at 10 nM and 100 nM induced 57% and 69% apoptosis, respectively. At 1 nM, it induced 17% apoptosis. [1]
For Western blotting, HL-60 cells were treated with SM-164 for 24 hours. Cell pellets were lysed in double lysis buffer (50 mmol/L Tris, 150 mmol/L sodium chloride, 1 mmol/L EDTA, 0.1% SDS and 1% NP-40). Proteins were electrophoresed onto 4-20% gradient SDS-PAGE, transferred to PVDF membranes, and incubated with specific primary antibodies against cleaved caspase-9, cleaved caspase-3, cleaved PARP, and β-actin, followed by HRP-linked secondary antibody. SM-164 at 10 nM induced robust activation of caspase-9 and -3 and cleavage of PARP. [1]
A biotin-streptavidin pull-down assay was used to investigate the interaction of SM-164 with cellular XIAP. HL-60 cell lysates were precleared with streptavidin-agarose beads, then incubated with biotinylated SM-122 (BL-SM-122) alone or pre-incubated with SM-164 for 5 min followed by co-incubation with BL-SM-122. Complexes were recovered by incubation with streptavidin-agarose beads for 2 h at 4°C, washed, and eluted by boiling in SDS loading buffer. The eluted proteins were detected by Western blotting using a monoclonal XIAP antibody. SM-164 at 10 nM competed off more than 50% of XIAP bound to BL-SM-122, and at 100 nM completely blocked the binding. [1]

For in vivo pharmacodynamic studies, female SCID mice were subcutaneously injected with 5×10⁶ MDA-MB-231 cancer cells mixed with Matrigel on the dorsal side (one tumor per mouse). Mice bearing established xenograft tumors were treated with a single intravenous dose of SM-164 at 5 mg/kg, Taxotere at 7.5 mg/kg, or vehicle control. Tumor tissues and normal mouse tissues were harvested at indicated time points (1, 3, 6, 12, 24 hours) for Western blotting, TUNEL staining and H&E staining.[2]
For in vivo antitumor efficacy studies, SCID mice (8-10 per group) bearing MDA-MB-231 xenograft tumors were treated intravenously with SM-164 at 1 mg/kg or 5 mg/kg, or Taxotere at 7.5 mg/kg, or vehicle control, daily, 5 days per week for 2 weeks. Tumor sizes and animal weights were measured three times per week. Tumor volumes were calculated and represented as mean ± SE.[2]
All animal experiments were performed under the guidelines of the institutional Committee for Use and Care of Animals. [2]

SM-164 at 5 mg/kg (single intravenous dose) caused no detectable toxicity in normal mouse tissues examined, including highly proliferative tissues such as small intestine, stomach, liver and spleen, as determined by H&E staining.[2]
In a panel of normal human cells (fibroblasts, epithelial cells, endothelial cells), SM-164 alone or in combination with TNFα showed no or minimal toxicity.[2]
In the 2-week efficacy study (daily intravenous dosing, 5 days/week), mice treated with SM-164 at 1 or 5 mg/kg showed no significant weight loss or other signs of toxicity.[2]
All normal human cells examined had very low expression of cIAP-1 compared to tumor cell lines, suggesting that normal cells do not depend on cIAP-1 for blocking apoptotic signals.[2]

[1]. J Am Chem Soc . 2007 Dec 12;129(49):15279-94.

[2]. Cancer Res . 2008 Nov 15;68(22):9384-93.

[3]. Mol Cancer Ther . 2011 May;10(5):902-14.

SM-164 is a potent, cell-permeable bivalent Smac mimic that binds to XIAP, cIAP-1, and cIAP-2 proteins (Ki values of 0.56 nM, 0.31 nM, and 1.1 nM, respectively). It induces apoptosis and tumor regression in tumor xenograft models. SM-164 exhibits multiple activities as a radiosensitizer, antitumor agent, and apoptosis inducer. It belongs to the triazole, benzene, secondary amide, and organic heterobicyclic compounds.

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XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting caspase-3/-7 and caspase-9. Smac protein antagonizes XIAP. SM-164 mimics this by concurrently targeting both BIR2 and BIR3 domains in XIAP. The potency of bivalent SM-164 in binding, functional and cellular assays is 2-3 orders of magnitude higher than its corresponding monovalent Smac mimetics. It is a powerful tool for elucidating the cellular functions of XIAP and a very promising lead compound for developing new anticancer therapies aimed at overcoming apoptosis resistance of cancer cells. [1]

C62H84N14O6

1121.45

1120.67

C, 66.40; H, 7.55; N, 17.49; O, 8.56

957135-43-2

SM-164 Hydrochloride

17756618

White to light yellow solid powder

7.6

6

12

24

82

1930

10

C([C@@H]1CC[C@@H]2CCCC[C@@H](C(N12)=O)NC(=O)[C@H](C)NC)(=O)N[C@@H](C1C=CC=CC=1)C1N=NN(CCCCC2C=CC(CCCCN3N=NC([C@H](C4C=CC=CC=4)NC([C@@H]4CC[C@@H]5CCCC[C@@H](C(N45)=O)NC(=O)[C@H](C)NC)=O)=C3)=CC=2)C=1

LGYDZXNSSLRFJS-IOQQVAQYSA-N

InChI=1S/C62H84N14O6/c1-41(63-3)57(77)65-49-27-13-11-25-47-33-35-53(75(47)61(49)81)59(79)67-55(45-21-7-5-8-22-45)51-39-73(71-69-51)37-17-15-19-43-29-31-44(32-30-43)20-16-18-38-74-40-52(70-72-74)56(46-23-9-6-10-24-46)68-60(80)54-36-34-48-26-12-14-28-50(62(82)76(48)54)66-58(78)42(2)64-4/h5-10,21-24,29-32,39-42,47-50,53-56,63-64H,11-20,25-28,33-38H2,1-4H3,(H,65,77)(H,66,78)(H,67,79)(H,68,80)/t41-,42-,47-,48-,49-,50-,53-,54-,55-,56-/m0/s1

(3S,6S,10aS)-N-[(S)-[1-[4-[4-[4-[4-[(S)-[[(3S,6S,10aS)-6-[[(2S)-2-(methylamino)propanoyl]amino]-5-oxo-2,3,6,7,8,9,10,10a-octahydro-1H-pyrrolo[1,2-a]azocine-3-carbonyl]amino]-phenylmethyl]triazol-1-yl]butyl]phenyl]butyl]triazol-4-yl]-phenylmethyl]-6-[[(2S)-2-(methylamino)propanoyl]amino]-5-oxo-2,3,6,7,8,9,10,10a-octahydro-1H-pyrrolo[1,2-a]azocine-3-carboxamide

SM 164; SM-164; SM164

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)

DMSO: 3~25 mg/mL (2.7~22.3 mM)

Solubility in Formulation 1: 0.83 mg/mL (0.74 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.

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