Bioymifi (DR5 Activator) is a novel and potent small-molecule activator of the TRAIL receptor DR5 in human cancer cells.

Bioymifi is able to promote cell death without the need for the Smac mimetic in T98 g cells. At a 10 μM concentration, bioymifi induces processing of caspase-3 into smaller fragments. Caspase-8 and the related extrinsic apoptotic pathway are essential for bioymifi-induced cell death. Bioymifi induces DR5-dependent death pathways and is independent of TRAIL. Bioymifi binds the ECD of DR5 with a Kd of 1.2 μM but shows little binding affinity to the DR4 ECD. It has poor solubility in buffer solutions. Bioymifi promotes apoptosis by directly binding to and facilitating aggregation of DR5. When bioymifi reaches micromolar concentration, its capability to aggregate DR5 is strong enough to induce apoptosis in various cancer cells[1].

Solubilized membranes from 3T3 Flk-1 cells are added to polystyrene ELISA plates that has been precoated with a monoclonal antibody that recognizes Flk-1. After an overnight incubation with lysate at 4°C, serial dilutions of SU5416 are added to the immunolocalized receptor. To induce autophosphorylation of the receptor, various concentrations of ATP are added to the ELISA plate wells containing serially diluted solutions of SU5416. The autophosphorylation is allowed to proceed for 60 min at room temperature and then stopped with EDTA. The amount of phosphotyrosine present on the Flk-1 receptors in the individual wells is determined by incubating the immunolocalized receptor with a biotinylated monoclonal antibody directed against phosphotyrosine. After removal of the unbound anti-phosphotyrosine antibody, avidin-conjugated horseradish peroxidase H is added to the wells. A stabilized form of 3,3′,5,5′-tetramethyl benzidine dihydrochloride and Water2 is added to the wells. The color readout of the assay is allowed to develop for 30 min, and the reaction is stopped with H2SO4. Parallel biochemical kinase assays are performed to measure autophosphorylation on EGFR and fibroblast growth factor receptor[1].

Dose-response curves of bioymifi and A2C2 in T98 g cells are plotted as a function of A2C2 or bioymifi concentration. Human glioblastoma (T98 g) cells are treated with various concentrations of A2C2 or bioymifi alone or in combination with 1 μM Smac mimetic (SM) for 48 h. The corresponding cell survival is normalized to the treatment without A2C2 or bioymifi.(Only for Reference)