AT-406是有效的，拟Smac的，IAP(通过E3泛素连接酶起作用的凋亡蛋白抑制剂)拮抗剂，与XIAP-BIR3、cIAP1-BIR3和cIAP2-BIR3 结合， Ki为66.4nM、1.9nM和5.1nM，比作用于Smac AVPI肽亲和力高50到100倍。Phase 1。

Apoptosis

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 1μM) completely restores the activity of caspase-9, which is suppressed by 500nM 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 142nM 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.

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 100mg/kg with well toleration even at 200mg/kg. AT-406 induces significant tumor growth inhibition with p of 0.0012 at 100mg/kg.

AT-406 is currently in Phase I clinical trial in patients with advanced solid tumors and lymphomas.

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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 1nM 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 125μL in the assay buffer (100mM potassium phosphate, pH 7.5, 100μg/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 485nM and an emission wavelength of 530nM. 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 20nM XIAP BIR3 protein and 2nM FL-AT-406 in the assay buffer (100mM potassium phosphate, pH 7.5; 100μg/ml bovine γ-globulin; 0.02% sodium azide). In competitive binding experiments for cIAP1 BIR3 protein, 3nM protein and 1nM FL-AT-406 are used. In competitive binding experiments for cIAP2 BIR3, 5nM protein and 1nM 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.

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