Discovery of small-molecule inhibitors of bcl-2 through structure-based computer screening
Discovery of small-molecule inhibitors of bcl-2 through structure-based computer screening
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DOI：
10.1021/jm010016f
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发表时间：
2001-12-06
影响因子：
7.3
通讯作者：
Wang, SM
中科院分区：
文献类型：
--
作者：
Enyedy, IJ;Ling, Y;Wang, SM
Bcl-2属于调节程序性细胞死亡（凋亡）的蛋白质的不断增长的家族。在70%的乳腺癌、30-60%的前列腺癌、80%的B细胞淋巴瘤、90%的结肠直肠腺癌和许多其他形式的癌症中观察到Bcl-2的过表达。因此，Bcl-2是一个有吸引力的新的抗癌靶点。在此，我们描述了新的小分子抑制剂类的发现，靶向在Bcl-2的BH 3结合口袋。Bcl-2的三维（3D）结构已经基于与Bcl-2共享高序列同源性的Bcl-X-L的高分辨率NMR溶液结构建模。一种基于结构的计算机筛选方法已被用于搜索国家癌症研究所3D数据库的206876种有机化合物，以确定潜在的Bcl-2小分子抑制剂，结合到Bcl-2的BH 3结合位点。首先在体外结合测定中测试这些潜在的Bcl-2小分子抑制剂抑制巴克BH 3肽与Bcl-2结合的效力。在该结合试验中测试了35种潜在抑制剂，发现其中7种的结合亲和力（IC 50值）为1.6至14.0 μ M。这七种活性化合物的抗增殖活性已经使用人髓性白血病细胞系HL-60进行了测试，HL-60在所有检测的癌细胞系中表达最高水平的Bcl-2蛋白。化合物6是最有效的化合物，使用3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑鎓溴化物测定法，其抑制细胞生长的IC 50值为4 μ M。其他五个化合物具有中等活性的抑制细胞生长。进一步评价化合物6诱导癌细胞凋亡的能力。发现6在具有高Bcl-2表达的癌细胞中诱导凋亡，并且其效力与癌细胞中Bcl-2表达水平相关。此外，使用NMR方法，我们最终证明6结合Bcl-XL中的BH 3结合位点。我们的研究结果表明，Bcl-2的小分子抑制剂如6调节Bcl-2的生物学功能，并诱导Bcl-2高表达的癌细胞凋亡，而它们对Bcl-2低表达或未检测到表达水平的癌细胞几乎没有影响。因此，化合物6可以作为一个有价值的药理学工具，以阐明Bcl-2的功能，也可以作为一个新的先导化合物，为进一步的设计和优化。我们的研究结果表明，在研究中采用的基于结构的计算机筛选策略是有效的识别新的，结构多样的，非肽类小分子抑制剂的目标BH 3的Bcl-2结合位点。
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 (IC50 value) from 1.6 to 14.0 muM. 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 muM 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.