In vitro activity: BDA-366 induces robust apoptosis in MM(Multiple myeloma) cell lines and primary MM cells by inducing BCL2 conformational change. BDA-366 induces a conformational change in the BCL2 molecule that converts it to a death protein, and inhibits lung cancer growth in vitro and in vivo. BDA-366 did not bind to other Bcl2 family members, including Bcl-XL, Mcl-1, or Bfl-1/A1, indicating the specificity of its Bcl2 binding. BDA-366 induces apoptotic cell death in a Bax-dependent manner and induces calcium (Ca2+) release via inhibition of Bcl2/IP3R interaction.

Kinase Assay: BDA-366 is a potent and selective small-molecule antagonist of the Bcl2-BH4 domain, it binds to BH4 with high affinity and selectivity. The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. BDA-366 induced conformational change of BCL2 that exposed the BH3 domain, resulting in abrogation of its prosurvival function and conversion of BCL2 to a prodeath protein. In non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells, BDA-366 selectively bound to BCL2 with high affinity. BDA-366 induced apoptosis by BCL2-dependent BAX activation and cytochrome c release. In H460 cells, BDA-366 reduced Bcl2/IP3R binding, which then increased Ca2+ release.

Cell Assay: Human MM cell lines RPMI8226 and U266 were treated with BDA366 at increasing concentrations (0, 0.1, 0.25, 0.5μM) for 48hr. Cells were harvested, stained with Annexin V and propidium iodide (PI), and subjected to FACS analysis. Apoptotic cells were gated on the Annexin V positive population. Annexin V+PI– cells were early apoptotic cells, Annexin+PI+ cells were late apoptotic cells, and Annexin–PI+ cells were necrotic cells.