{"title":"用于提高神经元BDNF表达的化合物的高通量测定。","authors":"Guey-Ying Liao , Haifei Xu , Justin Shumate , Louis Scampavia , Timothy Spicer , Baoji Xu","doi":"10.1016/j.slasd.2023.02.005","DOIUrl":null,"url":null,"abstract":"<div><p>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 <em>Bdnf</em> allele, <em>Bdnf<sup>NLuc</sup></em>, in which the sequence encoding nano luciferase (NLuc) is inserted into the <em>Bdnf</em> 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 <em>Bdnf<sup>NLuc/NLuc</sup></em> homozygous mice grew and behaved almost normally. We were able to establish and optimize cultures of cortical and hippocampal <em>Bdnf<sup>NLuc/+</sup></em> 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.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"28 3","pages":"Pages 88-94"},"PeriodicalIF":2.7000,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"High throughput assay for compounds that boost BDNF expression in neurons\",\"authors\":\"Guey-Ying Liao , Haifei Xu , Justin Shumate , Louis Scampavia , Timothy Spicer , Baoji Xu\",\"doi\":\"10.1016/j.slasd.2023.02.005\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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 <em>Bdnf</em> allele, <em>Bdnf<sup>NLuc</sup></em>, in which the sequence encoding nano luciferase (NLuc) is inserted into the <em>Bdnf</em> 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 <em>Bdnf<sup>NLuc/NLuc</sup></em> homozygous mice grew and behaved almost normally. We were able to establish and optimize cultures of cortical and hippocampal <em>Bdnf<sup>NLuc/+</sup></em> 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.</p></div>\",\"PeriodicalId\":21764,\"journal\":{\"name\":\"SLAS Discovery\",\"volume\":\"28 3\",\"pages\":\"Pages 88-94\"},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2023-04-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"SLAS Discovery\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2472555223000187\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"SLAS Discovery","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2472555223000187","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
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.
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).