title: Discovery of small-molecule inhibitors of Bcl-2 through structure-based computer screening.
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-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.
authors.affiliation: Structural Biology and Cancer Drug Discovery Program, Lombardi Cancer Center and Department of Oncology, Georgetown University Medical Center, 3970 Reservoir Road, Washington, DC 20007, USA.
authors.forename: I J
authors.initials: IJ
authors.lastname: Enyedy
authors.forename: Y
authors.initials: Y
authors.lastname: Ling
authors.forename: K
authors.initials: K
authors.lastname: Nacro
authors.forename: Y
authors.initials: Y
authors.lastname: Tomita
authors.forename: X
authors.initials: X
authors.lastname: Wu
authors.forename: Y
authors.initials: Y
authors.lastname: Cao
authors.forename: R
authors.initials: R
authors.lastname: Guo
authors.forename: B
authors.initials: B
authors.lastname: Li
authors.forename: X
authors.initials: X
authors.lastname: Zhu
authors.forename: Y
authors.initials: Y
authors.lastname: Huang
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Long
authors.forename: P P
authors.initials: PP
authors.lastname: Roller
authors.forename: D
authors.initials: D
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D017209:Apoptosis; D001392:Azocines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003497:Cyclic N-Oxides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D016208:Databases, Factual; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: C442800:2,9-dimethoxy-11,12-dihydrodibenzo(e,g)(1,2)diazocine 5,6-dioxide; D000970:Antineoplastic Agents; D001392:Azocines; D003497:Cyclic N-Oxides; D019253:Proto-Oncogene Proteins c-bcl-2