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.

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer

“…Pan-caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (40 mM; Bachem, Bubendorf, Switzerland) or 25 mM necrostatin (Santa Cruz Biotechnology) were added 1 h before ABT-737 and cells were processed after 3 additional days. TNF-a was kindly provided by Dr. TL Haas (see Acknowledgements and studies by Haas et al, 34 Enyedy et al 35 and Ewald et al 36 ). Free radical scavengers used to inhibit ROS (superoxide dismutase and catalase 1 mg/ml each), nitric oxide (carboxy-PTIO 0.1 mM) and peroxynitrite (uric acid 1 mM) were added 24 h before ABT-737 and cell viability was determined after 3 additional days of incubation.…”

Small-Molecule Inhibitors of IL-2/IL-2R: Lessons Learned and Applied

“…Due to their high value and detailed structural characterization, Bcl-xL, MDM2, and IL-2 have all been used for successful validation of virtual screening methods (Betzi et al 2009;Sperandio et al 2010;Casey et al 2009;Fuller et al 2009;Helms 2007, 2009;Enyedy et al 2001;Bowman et al 2007;Mukherjee et al 2010). Bcl-xL and MDM2, in particular, have attracted special attention for in silico screening against molecules in existing chemical libraries.…”

BCL-2 in the crosshairs: tipping the balance of life and death

“…This approach yielded HA14-1 (IC 50 ¼ 9 mM for competing BAK BH3/BCL-2 interaction), 103 several micromolar affinity hits from the National Cancer Institute 3D database including Compound 6, 104 A major hurdle of small molecule library screening approaches, whether virtual or biochemical, is the chemical optimization required to achieve high-potency target-binding activity from hits that are typically in the micromolar range. An alternative strategy developed by Fesik and co-workers 115 circumvents this shortcoming of library screening by chemically linking ligands that bind to adjacent sites within a target interface, effectively converting relatively low affinity interactors into conjoined high-affinity compounds.…”