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

J Med Chem. 2001 Dec 6;44(25):4313-24. doi: 10.1021/jm010016f.


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-X(L), 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 (IC(50) value) from 1.6 to 14.0 microM. 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 IC(50) value of 4 microM 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-X(L). 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antineoplastic Agents / chemical synthesis*
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology
  • Apoptosis
  • Azocines / chemical synthesis*
  • Azocines / chemistry
  • Azocines / pharmacology
  • Cell Division / drug effects
  • Cell Survival / drug effects
  • Cyclic N-Oxides / chemical synthesis*
  • Cyclic N-Oxides / chemistry
  • Cyclic N-Oxides / pharmacology
  • Databases, Factual
  • Drug Screening Assays, Antitumor
  • Humans
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Proto-Oncogene Proteins c-bcl-2 / antagonists & inhibitors*
  • Structure-Activity Relationship
  • Tumor Cells, Cultured


  • 2,9-dimethoxy-11,12-dihydrodibenzo(e,g)(1,2)diazocine 5,6-dioxide
  • Antineoplastic Agents
  • Azocines
  • Cyclic N-Oxides
  • Proto-Oncogene Proteins c-bcl-2