Development of Real-Time Cellular Screening Systems for BoNT Intoxication

BoNT 中毒实时细胞筛查系统的开发

• 批准号: 8892063
• 负责人: TOBIN J DICKERSON
• 金额: $ 44.64万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 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/8892063
• 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; nerve stem cell; neurotransmitter release; pre-clinical; promoter; relating to nervous system; screening; therapeutic development; treatment strategy; weapons

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）：该提案解决了对肉毒杆菌神经毒素（BoNT）中毒新细胞模型的迫切需求，以应用于高通量筛选活动。具体来说，我们将开发具有高分析灵敏度和非侵入性报告细胞内BoNT活性的能力的细胞模型系统。肉毒神经毒素（BoNT）是已知的对人类毒性最大的蛋白质（LD 50 ~ 1 ng/kg），其中暴露于该毒素最终导致宿主麻痹和死亡。尽管这种毒素具有广泛的临床用途，但如果在生物恐怖袭击事件中滥用，它也会构成安全威胁。因此，已经投入了大量的努力来发现和开发用于治疗肉毒杆菌中毒的治疗策略。传统上，具有抗BoNT活性的分子已经通过使用荧光肽底物的体外筛选活动或通过使用细胞模型和SNARE蛋白切割的低通量免疫学检测来鉴定。尽管这些努力已经产生了具有良好体外效力的先导化合物，但由于药代动力学特性较差，例如低水溶解度、高细胞毒性和低细胞渗透性，在进行体内测试时所有这些都失败了。此外，依赖于次级细胞系的细胞模型已被证明在铅进展中具有较差的预测价值。因此，这些局限性决定了需要新的细胞模型，忠实地再现BoNT中毒的表型，是体内疗效的更好预测因子，并且与现有的自动化和高通量筛选程序兼容。在这项提案中，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具有高BoNT敏感性，并代表了一种可行的方法，用于扩增筛选应用所需的细胞数量。将使用两种不同的方法来工程化细胞以表达先前显示为真实的时间报告细胞内BoNT活性的基于FRET的报告子。在第一种方法中，将制备细胞以组成型表达BoNT报告基因，这将允许直接比较分化阶段之间的BoNT敏感性。我们方法的第二个方面是开发干细胞衍生的运动神经元，其仅在特定的分化阶段表达BoNT报告基因。报告蛋白的阶段特异性表达将允许真实的监测细胞内BoNT活性，以及能够评估分化方案的效率，并提供纯化感兴趣细胞的手段。最后，将通过使用高含量筛选设备筛选一组化合物来评估所开发的细胞测定的潜力，目的是鉴定具有抗BoNT活性的新先导化合物，随后将使用二级和三级筛选方法进行验证。通过这些研究，我们提出，可以产生可行的细胞系统，这将具有广泛的适用性BoNT拮抗剂的发现，在体外筛选模型和体内生物测定之间提供所需的预测联系。