Web Release Date: November 1,
Discovery of Small-Molecule Inhibitors of Bcl-2 through Structure-Based Computer Screening
and
Structural Biology and Cancer Drug Discovery Program, Lombardi Cancer Center and Department of Oncology, Georgetown University Medical Center, 3970 Reservoir Road, Washington, D.C. 20007, and Laboratory of Medicinal Chemistry, National Cancer Institute, FCRDC, Building 376, Room 207, Fredrick, Maryland 21702
Received January 10, 2001
Abstract:
Bcl-2 belongs to a growing family of proteins which regulates programmed cell death (apoptosis).
Overexpression of Bcl-2 has been observed in 70% of breast cancer, 30-60% of prostate cancer,
80% of B-cell lymphomas, 90% of colorectal 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 pocket in Bcl-2. The
three-dimensional (3D) structure of Bcl-2 has been modeled on the basis of a high-resolution
NMR solution structure of Bcl-XL, which shares a high sequence homology with Bcl-2. A
structure-based computer screening 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 potential Bcl-2 small-molecule
inhibitors were first tested in an in vitro binding assay for their potency in inhibition of the
binding of a Bak BH3 peptide to Bcl-2. Thirty-five potential inhibitors were tested in this binding
assay, and seven of them were found to have a binding affinity (IC50 value) from 1.6 to 14.0
M. 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 cell lines examined. Compound 6 was the most potent compound and
had an IC50 value of 4 M in inhibition of cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Five other compounds had moderate activity in
inhibition of cell growth. Compound 6 was further evaluated 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 potency correlates with the Bcl-2 expression level in cancer cells. Furthermore, using
NMR methods, we conclusively demonstrated that 6 binds to the BH3 binding site in Bcl-XL.
Our results showed that small-molecule inhibitors of Bcl-2 such as 6 modulate the biological
function of Bcl-2, and induce apoptosis in cancer cells with high Bcl-2 expression, while they
have little effect on cancer cells with low or undetectable levels of Bcl-2 expression. Therefore,
compound 6 can be used as a valuable pharmacological tool to elucidate the function of Bcl-2
and also serves as a novel lead compound for further design and optimization. Our results
suggest that the structure-based computer screening strategy employed in the study is effective
for identifying novel, structurally diverse, nonpeptide small-molecule inhibitors that target
the BH3 binding site of Bcl-2.
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