Discovery of Small-Molecule Inhibitors of Bcl-2 through Structure-Based Computer Screening

• Published in: Journal of medicinal chemistry 2001-12, Vol.44 (25), p.4313-4324
• Main Authors: Enyedy, Istvan J, Ling, Yan, Nacro, Kassoum, Tomita, York, Wu, Xihan, Cao, Yeyu, Guo, Ribo, Li, Bihua, Zhu, Xiaofeng, Huang, Ying, Long, Ya-Qiu, Roller, Peter P, Yang, Dajun, Wang, Shaomeng
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antineoplastic agents; Antineoplastic Agents - chemical synthesis; Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacology; Apoptosis; Azocines - chemical synthesis; Azocines - chemistry; Azocines - pharmacology; Biological and medical sciences; 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; General aspects; Humans; Magnetic Resonance Spectroscopy; Medical sciences; Models, Molecular; Molecular Sequence Data; Pharmacology. Drug treatments; Protein Binding; Proto-Oncogene Proteins c-bcl-2 - antagonists & inhibitors; Structure-Activity Relationship; Tumor Cells, Cultured
• ISSN: 0022-2623; 1520-4804
• 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-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 μM. 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 μM 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.