Birinapant (TL32711) 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.

CIAP1:<1 nM(K_d), XIAP:45 nM(K_d)

Birinapant binds with XIAP and cIAP1 with Kd of 45 and <1 nM, respectively. Birinapant induces cell death as a single agent in TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells (IC₅₀, ~300 nM), and significantly increases potency of TRAIL-induced apoptosis in TRAIL-sensitive SUM149 (triple-negative, EGFR-activated) cells. Birinapant causes rapid cIAP1 degradation, caspase activation, PARP cleavage, and NF-κB activation. ^[1] Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant in combination with TNF-α(1 ng/mL) inhibits the growth of human melanoma cell lines WTH202, WM793B, WM1366 and WM164 with IC₅₀s of 1.8, 2.5, 7.9 and 9 nM, respectively, while neither compound is effective individually. Birinapant singly treatment induces inhibition on proliferation of WM9 cells with IC₅₀ of 2.4 nM. Birinapant significantly inhibits the target protein cIAP1 and cIAP2 in these cell lines.^[2]

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

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

Cells are allowed to attach for 24 hours and subsequently incubated with Birinapant and/or TNF-α for 24 or 72 hours. Then MTS assay is conducted(Only for Reference)

Ethanol: 51.00 mg/mL (63.20 mM), Sonication is recommended.

DMSO: 245.00 mg/mL (303.62 mM), Sonication is recommended.

H2O: < 1 mg/mL (insoluble or slightly soluble)

10% DMSO+90% Saline: < 10 mg/mL (12.39 mM), Lower concentrations may be soluble, but exact solubility limit is unknown.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 10.00 mg/mL (12.39 mM), Solution.