SM-164

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. SM-164 targets cellular XIAP and effectively induces apoptosis at concentrations as low as 1 nM in the HL-60 leukemia cell line. ^[1]

SM-164 induces caspase-8– and caspase-3–dependent apoptosis in cancer cells. SM-164 induces TNFα-dependent apoptosis and cIAP-1 degradation. ^[2]

SM-164 is highly synergistic with TRAIL in vitro in both TRAIL-sensitive and TRAIL-resistant cancer cell lines of breast, prostate, and colon cancer. SM-164 enhances TRAIL-induced apoptosis in cancer cells through amplification of the caspase-8–mediated extrinsic apoptosis pathway ^[3]

SM-164 induces rapid cIAP-1 degradation and strong apoptosis in the MDA-MB-231 xenograft tumor tissues and achieves tumor regression, but has no toxicity in normal mouse tissues. ^[2]

SM-164 induces cIAP1 degradation in tumor tissues and dramatically enhances the in vivoantitumor activity of TRAIL, and the combination of SM-164 and TRAIL achieves tumor regression without toxicity to animals. ^[3]

• Binding assays.

  The FP-based assay for XIAP BIR3 protein is described. Briefly, 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. The Kd value of this fluorescent tracer is determined to be 17.9 nM to XIAP BIR3. In competitive binding experiments, a tested compound is incubated with 30 nM ofXIAP 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 to cIAP-2 BIR3 protein is determined to be 6.6 nM. In competitive binding experiments, 25 nM of cIAP-2 BIR3 proteinand 2 nM of SM5F tracer are used. To determine the binding affinities of Smac mimetics to XIAP containing both BIR2 and BIR3 domains, an FP-based competitive binding assay is established using a bivalentfluorescently tagged tracer, named Smac-1F. The Kd value of the bivalent tagged tracer to XIAP containing BIR2 and BIR3 domains is determined to be 2.3 nM. In competitive binding experiments, a tested compound is incubated with 3 nM of XIAP protein containing both BIR2 and BIR3 domain (residues 120-356) and 1 nM of in the sameassay buffer.

• Cell lines

  HT-29 cells

• Concentrations

  1 μM

• Incubation Time

  6 or 12 h

• Method

  Cells pre-treated with HG (5 μM) for 30 min were treatment with SM-164 (1 μM) for 6 or 12 h.

• Animal Models

  MDA-MB-231 xenografted SCID mice

• Dosages

  1 and 5 mg/kg

• Administration

  i.v.