Discovery of small-molecule inhibitors of bcl-2 through structure-based computer screening

Bcl-2 belongs to a growing family of proteins which regulates programmed ce ll death (apoptosis). Overexpression of Bcl-2 has been observed in 70% of b reast cancer, 30-60% of prostate cancer, 80% of B-cell lymphomas, 90% of co lorectal adenocarcinomas, and many other forms of cancer. Thereby, Bcl-2 is an attractive new anti-cancer target. Herein, we describe the discovery of novel classes of small-molecule inhibitors targeted at the BH3 binding poc ket in Bcl-2. The three-dimensional (3D) structure of Bcl-2 has been modele d on the basis of a high-resolution NMR solution structure of Bcl-X-L, whic h shares a high sequence homology with Bcl-2. A structure-based computer sc reening approach has been employed to search the National Cancer Institute 3D database of 206 876 organic compounds to identify potential Bcl-2 small- molecule inhibitors that bind to the BH3 binding site of Bcl-2. These poten tial Bcl-2 small-molecule inhibitors were first tested in an in vitro bindi ng assay for their potency in inhibition of the binding of a Bak BH3 peptid e to Bcl-2. Thirty-five potential inhibitors were tested in this binding as say, and seven of them were found to have a binding affinity (IC50 value) f rom 1.6 to 14.0 muM. The anti-proliferative activity of these seven active compounds has been tested using a human myeloid leukemia cell line, HL-60, which expresses the highest level of Bcl-2 protein among all the cancer cel l lines examined. Compound 6 was the most potent compound and had an IC50 v alue of 4 muM in inhibition of cell growth using the 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide assay. Five other compounds had mode rate activity in inhibition of cell growth. Compound 6 was further evaluate d for its ability to induce apoptosis in cancer cells. It was found that 6 induces apoptosis in cancer cells with high Bcl-2 expression and its potenc y correlates with the Bcl-2 expression level in cancer cells. Furthermore, using NMR methods, we conclusively demonstrated that 6 binds to the BH3 bin ding site in Bcl-XL. Our results showed that small-molecule inhibitors of B cl-2 such as 6 modulate the biological function of Bcl-2, and induce apopto sis in cancer cells with high Bcl-2 expression, while they have little effe ct on cancer cells with low or undetectable levels of Bcl-2 expression. The refore, compound 6 can be used as a valuable pharmacological tool to elucid ate the function of Bcl-2 and also serves as a novel lead compound for furt her design and optimization. Our results suggest that the structure-based c omputer screening strategy employed in the study is effective for identifyi ng novel, structurally diverse, nonpeptide small-molecule inhibitors that t arget the BH3 binding site of Bcl-2.