摘要
目的筛选上调骨形态发生蛋白2（BMP-2）表达的新型小分子活性化合物，为研发抗骨质疏松药物提供先导化合物。方法应用国家新药（微生物）筛选实验室前期建立的BMP-2表达上调剂的高通量筛选模型，对化合物库中10000余个化合物进行筛选，以染料木素作为阳性对照；应用实时定量 PCR 和 Western blot 方法检测活性化合物对 U-2OS 细胞 BMP-2 mRNA 和蛋白表达水平的影响。在mRNA 水平考察了活性化合物对 Runx2表达的影响， Western blot 法检测活性化合物对 Smad1/5/8蛋白磷酸化水平的影响；用 PNPP 法检测碱性磷酸酶活性，验证活性化合物体外促进成骨细胞分化的作用。结果在 BMP-2表达上调剂模型细胞上筛选得到活性化合物 E19773，其在模型细胞上的 EC50为46.2μmol/L；实时定量 PCR 结果显示，化合物 E19773能够提高 U-2OS细胞 BMP-2和 Runx2的 mRNA 表达水平；Western blot结果显示，Smad1/5/8蛋白磷酸化水平明显升高；碱性磷酸酶结果表明 E19773能够在体外促进成骨细胞的分化。结论化合物 E19773在0.75~50μmol/L 浓度范围内能明显上调 BMP-2的表达，主要通过 Smads 通路来调控细胞成骨分化，是活性较好的 BMP-2上调剂。
Objective To discover potential small-molecular compounds with the anti-osteoporosis functions by up-regulating the expression of bone morphogenetic protein-2 （BMP-2）. Methods The process of screening was performed using a cell-based high-throughput screening model for BMP-2 with genistein as a positive control. Runx2 is a major osteogenic transcription factor in BMP-2-mediated pathway, and Smad1/5/8 is a very important signaling molecule in this pathway. The mRNA and protein expression levels of BMP-2, Runx2 and Smad1/5/8 phosphorylation were measured by real time PCR and Western blot, respectively. The activity of ALP was measured by the method of PNPP. Results Compound E19773 with the activity of up-regulating the expression of BMP-2 was found by PMB models, and the EC50 value of E19773 was 46.2μmol/L. E19773 was found to significantly increase the mRNA expression levels of BMP-2 and Runx2, as well as increase the protein expression levels of BMP-2, Runx2 and Smad1/5/8 phosphorylation in U-2OS cells. In vitro results of ALP activity indicated that E19773 was able to promote osteoblast differentiation. Conclusion Compound of E19773 can up-regulate the expression of BMP-2 through BMP-Smad pathway at the concentration of 0.75-50μmol/L. Thus the compound can be regarded as a lead compound for the development of new drugs to prevent and treat osteoporosis in the future.
出处
《中国医药生物技术》
2015年第3期236-241,共6页
Chinese Medicinal Biotechnology
关键词
骨质疏松
高通量筛选
骨形态发生蛋
白质2
Osteoporosis
High-throughput screening
Bone morphogenetic protein 2
