用于提高神经元BDNF表达的化合物的高通量测定。

IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Guey-Ying Liao , Haifei Xu , Justin Shumate , Louis Scampavia , Timothy Spicer , Baoji Xu
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引用次数: 1

摘要

脑源性神经营养因子(BDNF)的缺陷与几种大脑疾病有关,这使得能够促进神经元BDNF合成的化合物作为潜在的治疗方法具有吸引力。然而,用于高通量筛选(HTS)的敏感和定量BDNF测定法仍然缺失。在这里,我们报道了一种新的小鼠Bdnf等位基因BdnfNLuc的产生,其中编码纳米荧光素酶(NLuc)的序列在终止密码子之前插入到Bdnf基因座中,使得该等位基因将产生Bdnf NLuc融合蛋白。随着BdnfNLuc/NLuc纯合小鼠的生长和行为几乎正常,BDNF NLuc蛋白的功能似乎与BDNF相似。我们能够在384孔板中建立并优化从小鼠胚胎中分离的皮层和海马BdnfNLuc/+神经元的培养物。我们使用培养物作为表型测定来检测10mM KCl刺激BDNF合成的能力,并在该测定中获得了可重复的Z'因子>0.50,这一测量被认为适合HTS。我们成功地扩大了测定规模,以筛选1280化合物LOPAC文库(药理学活性化合物文库)。筛选鉴定了几种BDNF增强化合物,其中一种是Bay K8644,一种L-型电压门控钙通道(L-VGCC)激动剂,以前被证明可以刺激BDNF的合成。这些结果表明,我们的表型神经元测定已为HTS鉴定新的BDNF增强化合物做好了准备。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
High throughput assay for compounds that boost BDNF expression in neurons

Deficiencies in brain-derived neurotrophic factor (BDNF) have been linked to several brain disorders, making compounds that can boost neuronal BDNF synthesis attractive as potential therapeutics. However, a sensitive and quantitative BDNF assay for high-throughput screening (HTS) is still missing. Here we report the generation of a new mouse Bdnf allele, BdnfNLuc, in which the sequence encoding nano luciferase (NLuc) is inserted into the Bdnf locus immediately before the stop codon so that the allele will produce a BDNF-NLuc fusion protein. BDNF-NLuc protein appears to function like BDNF as BdnfNLuc/NLuc homozygous mice grew and behaved almost normally. We were able to establish and optimize cultures of cortical and hippocampal BdnfNLuc/+ neurons isolated from mouse embryos in 384-well plates. We used the cultures as a phenotypic assay to detect the ability of 10 mM KCl to stimulate BDNF synthesis and achieved a reproducible Z′ factor > 0.50 for the assay, a measure considered suitable for HTS. We successfully scaled up the assay to screen the 1280-compound LOPAC library (Library of Pharmacologically Active Compounds). The screen identified several BDNF-boosting compounds, one of which is Bay K8644, a L-type voltage-gated calcium channel (L-VGCC) agonist, which was previously shown to stimulate BDNF synthesis. These results indicate that our phenotypic neuronal assay is ready for HTS to identify novel BDNF-boosting compounds.

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来源期刊
SLAS Discovery
SLAS Discovery Chemistry-Analytical Chemistry
CiteScore
7.00
自引率
3.20%
发文量
58
审稿时长
39 days
期刊介绍: Advancing Life Sciences R&D: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease. SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success. SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies. SLAS Discovery is of particular interest to those involved in analytical chemistry, applied microbiology, automation, biochemistry, bioengineering, biomedical optics, biotechnology, bioinformatics, cell biology, DNA science and technology, genetics, information technology, medicinal chemistry, molecular biology, natural products chemistry, organic chemistry, pharmacology, spectroscopy, and toxicology. SLAS Discovery is a member of the Committee on Publication Ethics (COPE) and was published previously (1996-2016) as the Journal of Biomolecular Screening (JBS).
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