Development of Real-Time Cellular Screening Systems for BoNT Intoxication

BoNT 中毒实时细胞筛查系统的开发

基本信息

批准号：
8761600
负责人：
TOBIN J DICKERSON
金额：
$ 44.97万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-16 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8761600
关键词：
Address Affect Automation Biological Assay Biological Models Bioterrorism Bontoxilysin Botulism Cell Count Cell Line Cell model Cells Cellular Assay Cessation of life Clinical Trials Clostridium botulinum Clover Data Detection Development Drug Kinetics Eligibility Determination Equipment Evaluation Event Exhibits Exposure to Fluorescence Resonance Energy Transfer Gene Targeting Gram-Positive Bacteria Health Human In Vitro Intoxication Lead Lethal Dose 50 Libraries Link Methods Modeling Monitor Motor Neurons Muscle Contraction Neurons Paralysed Patients Peptides Permeability Phase Phenotype Population Predictive Value Production Proliferating Property Proteins Proteolysis Protocols documentation Regulation Reporter Reporting Research S-nitro-N-acetylpenicillamine SNAP receptor Safety Screening procedure Signal Transduction Solubility Source Staging Stem cells System Testing Therapeutic Time Toxin Western Blotting aqueous base cellular engineering cost cytotoxicity drug discovery high throughput screening human disease human stem cells improved in vitro Assay in vivo in vivo Bioassay induced pluripotent stem cell inhibitor/antagonist interest man member neurotransmitter release pre-clinical progenitor promoter public health relevance relating to nervous system screening therapeutic development treatment strategy weapons

项目摘要

DESCRIPTION (provided by applicant): This proposal addresses the urgent need for new cellular models of botulinum neurotoxin (BoNT) intoxication for application in high-throughput screening campaigns. Specifically, we will develop cellular model systems that possess high analytical sensitivity and the ability to non-invasively report on intracellular BoNT activity in ral time. Botulinum neurotoxin (BoNT) is the most toxic protein known to man (LD50 ~ 1 ng/kg), where exposure to the toxin ultimately results in host paralysis and death. Despite the broad clinically utility of this toxin, it also can pose a safety threat if misused in the event of a bioterrorist attack. As a result, much effort has been put into the discovery and development of therapeutic strategies for the treatment of botulism. Traditionally, molecules with anti-BoNT activities have been identified through in vitro screening campaigns using fluorescent peptide substrates, or by using cellular models and low-throughput immunological detection of SNARE protein cleavage. Although these efforts have resulted in leads with good in vitro potency, all have failed when advanced to in vivo testing due to poor pharmacokinetic properties such as low aqueous solubility, high cytotoxicity and low cell permeability. In addition, cellular models that rely on secondary cell lines have been shown to have poor predictive value in lead advancement. These limitations, therefore, dictate the need for new cellular models that faithfully recapitulate the phenotype of BoNT intoxication, are better predictors of in vivo efficacy, and are compatible with existing automation and high-throughput screening procedures. In this proposal, we will take advantage of human stem cell-derived motor neurons that have been demonstrated to exhibit high BoNT sensitivities and represent a feasible approach for expansion to cell numbers required for screening applications. Two different approaches will be used to engineer the cells to express a FRET-based reporter previously shown to report intracellular BoNT activity in real time. In the first approach, cells will be prepared to constitutively express the BoNT reporter that will allow for the direct comparison of BoNT sensitivity between differentiation stages. The second prong to our approach is to develop stem cell-derived motor neurons that express the BoNT reporter only in a specific differentiation stage. Stage-specific expression of the reporter protein will allow monitoring of intracellular BoNT activity in real time as well as enable the evaluation of the efficiency of the differentiatio protocol and provide a means to purify cells of interest. Finally, the potential of the developed cellular assays will be evaluated by screening a set of compounds using high content screening equipment with the aim of identifying new leads with anti-BoNT activities that will subsequently be validated using secondary and tertiary screening methods. Through these studies, we propose that viable cellular systems can be produced that will have broad applicability to BoNT antagonist discovery, providing a needed predictive link between in vitro screening models and in vivo bioassays.

描述（由申请人提供）：该提案涉及对肉毒杆菌神经毒素（BONT）中毒的新细胞模型的迫切需求，以在高通量筛查活动中应用。具体而言，我们将开发具有较高分析灵敏度的细胞模型系统，并且能够在RAL时间内无创地报告细胞内BONT活性。肉毒杆菌神经毒素（BONT）是人类已知的最毒性蛋白（LD50〜1 ng/kg），其中暴露于毒素中最终导致宿主麻痹和死亡。尽管这种毒素在临床上有广泛的效用，但如果遭到生物恐怖主义袭击，它也可能构成安全威胁。结果，为治疗肉毒杆菌主义的治疗策略的发现和发展已经付出了很多努力。传统上，通过使用荧光肽底物或使用细胞模型和低通量免疫学检测SNARE蛋白裂解的体外筛选运动，已经通过体外筛选运动来鉴定具有抗BONT活性的分子。尽管这些努力导致了具有良好体外效力的铅，但由于药代动力学特性不佳，例如低水溶性，高细胞毒性和低细胞通透性，所有这些都失败了体内测试。此外，依赖二级细胞系的细胞模型在铅进步中的预测值较差。因此，这些局限性决定了对忠实地概括BONT中毒表型的新细胞模型的需求，它是体内功效的更好预测指标，并且与现有的自动化和高通量筛选程序兼容。在此提案中，我们将利用人类干细胞衍生的运动神经元，这些神经元已被证明表现出很高的骨头敏感性，并代表了一种可行的方法，可以扩展到筛查应用所需的细胞数量。将使用两种不同的方法来设计细胞以表达基于FRET的报告基因，以前显示以实时报告细胞内BONT活性。在第一种方法中，将准备好细胞来组成性地表达BONT报告基因，该报告将允许直接比较分化阶段之间的BONT灵敏度。我们方法的第二阶段是开发干细胞衍生的运动神经元，这些神经元仅在特定的分化阶段表达BONT记者。报告蛋白的阶段特异性表达将允许实时监测细胞内BONT活性，并能够评估区分方案的效率，并提供一种净化感兴趣细胞的方法。最后，将通过使用高内容筛选设备筛选一组化合物来评估开发的细胞测定的潜力，以识别具有抗BONT活动的新导线，随后将使用次级和第三级筛选方法对其进行验证。通过这些研究，我们建议可以生产可行的细胞系统，这些系统将在构造拮抗剂发现中具有广泛的适用性，从而在体外筛查模型和体内生物测定之间提供了所需的预测联系。