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BMP2 target of new anti-osteoporosis drug screening model and applied research

Author: LiXue
Tutor: SiShuYi；JiangWei；HeXiaoBo
School: Peking Union Medical College , China
Course: Microbial and Biochemical Pharmacy
Keywords: BMP2 On swap High Throughput Screening Anti-osteoporosis Lead compounds
CLC: R96
Type: Master's thesis
Year: 2009
Downloads: 222
Quote: 1
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Abstract

Bone morphogenetic protein Ⅱ (BMP2) is one of the members of the TGF-β superfamily, it is highly expressed in osteoblasts, an important regulatory factor in vivo and osteoblast differentiation. Numerous studies show that of BMP2 to efficiently induce bone formation, bone loss is expected to be accompanied by disease, especially in menopausal and post-menopausal women, osteoporosis and other diseases, new therapeutic targets. In order to find BMP2 expression levels of the active compound can be improved, the work build the level of a high-throughput screening can be used for cell stably transfected with the model. The report will contain mouse bmp2 promoter and luciferase reporter gene plasmid stable transfection to the mouse skull cells MC3T3-E1 cells, complete model building, known BMP2 spice model validation and conditions optimization. According to the reported selected BMP2 swap on this model are presented and a higher signal-to-noise ratio of the dose-effect relationship, and prove that the model has good specificity, sensitivity and stability. 3192 natural products and synthetic compounds were screened, the application of the model obtained after screening and rescreening, 7 having bmp2 promoter increases the activity of positive compounds. Stability parameter Z 'factor> 0.5, suitable for high-throughput screening requirements. Active compounds screened, select daidzein, the thorn Formononetin and 2 - acetyl-benzothiophene molecular pharmacology and cellular levels of biological activity study. The results show that the three compounds can be raised in the micromolar concentration BMP2 expression, using real-time quantitative RT-PCR and flow cytometry confirmed at the mRNA and protein levels of three compounds Upregulation of BMP2 expression in human osteosarcoma cells MG63 . 2 - acetyl benzothiophene level of detection of the in vitro cell proliferation inhibition detection and alkaline phosphatase (AKP), the results show that the compound is less toxic to increase bone formation marker proteins AKP activity. Nucleus design synthesis of 2 - acetyl-benzothiophene-76 derivatives activity evaluation, and select one of the exploration of the molecular mechanisms. The present study is the first generation of pGL-4 plasmid construct suitable for high throughput screening BMP2 expression the toner stably transfected screening model, 7 having a raised BMP2 expression of active compound is obtained by screening, and the molecular and cellular conclusive evidence of its biological activity level, 76 benzothiophene derivatives and its analogues were active, laid the foundation for looking for a new anti-osteoporosis drug or lead compound, while the use of small molecule compounds in-depth study BMP2 transcriptional regulation mechanisms become possible, has important theoretical and practical significance.

BMP2 target of new anti-osteoporosis drug screening model and applied research

Abstract

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