Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells

Bcl-2 and related proteins are key regulators of apoptosis or programmed ce ll death implicated in human disease including cancer. We recently showed t hat cell-permeable Bcl-2 binding peptides could induce apoptosis of human m yeloid leukemia in vitro and suppress its growth in severe combined immunod eficient mice. Here we report the discovery of HA14-1, a small molecule (mo lecular weight = 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl -2 protein. In vitro binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological func tion. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the dec rease in mitochondrial membrane potential and activation of caspase-9 follo wed by caspase-3, Cytokine response modifier A, a potent inhibitor of Fas-m ediated apoptosis, did not block apoptosis induced by HA14-1, Whereas HA14- 1 strongly induced the death of NIH 3T3 (Apaf-1(+/+)) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1(-/-)) mouse embryonic fibrobla st cells. These data are consistent with a mechanism by which HA14-1 induce s the activation of Apaf-1 and caspases, possibly by binding to Bcl-2 prote in and inhibiting its function. The discovery of this cell-permeable molecu le provides a chemical probe to study Bcl-2-regulated apoptotic pathways in vivo and could lead to the development of new therapeutic agents.