A Cell-based Assay for Botulinum Neurotoxin Detection and Development

肉毒杆菌神经毒素检测和开发的细胞测定法

• 批准号: 7602931
• 负责人: WARD C TUCKER
• 金额: $ 29.9万
• 依托单位: BIOSENTINEL, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602931
• 关键词: Accounting; Activity Cycles; Adverse event; Animals; Antibodies; B-Lymphocytes; Bacteria; Bacterial Toxins; Biological; Biological Assay; Bioterrorism; Blepharospasm; Bontoxilysin; Botulinum Toxin Type A; Categories; Cell Line; Cell Separation; Cells; Cervical Dystonia; Cessation of life; Characteristics; Chemicals; Cleaved cell; Clinical; Clostridium; Collaborations; Cosmetics; Cytoplasm; Cytosol; Department of Defense; Detection; Development; Devices; Diagnostic; Disease; Energy Transfer; Engineering; Evaluation; Event; FDA approved; Facial Nerve Diseases; Failure; Fluorescence; Fluorescence Microscopy; Goals; Government; Growth; Half-Life; Hand; Health; Human; In Vitro; Industry; Infection; Intoxication; Lead; Letters; Life; Marketing; Measures; Medical Research; Methods; Monitor; Mus; Muscle Contraction; Muscle Tension; Myopathy; N-ethylmaleimide-sensitive protein; Nervous system structure; Neuroblastoma; Neurologic; Neurons; Neurosecretory Systems; Pain; Peptide Hydrolases; Performance; Persons; Pharmacologic Substance; Phase; Poison; Poisoning; Preparation; Process; Production; Proteins; Quality Control; Reader; Receptor Cell; Reporter; Reporting; Research; Research Institute; Research Personnel; Respiratory Failure; SNAP receptor; Screening procedure; Serotyping; Specificity; System; Technology; Testing; Therapeutic; Time; Toxin; Training; Universities; Validation; Vesicle; Wisconsin; Work; base; botulinum toxin type B; cellular engineering; conventional therapy; emergency service responder; fluorescence imaging; fluorophore; high throughput screening; improved; in vivo; instrument; meetings; neurotoxin receptor; neurotransmitter release; next generation; novel; overexpression; pressure; public health relevance; receptor; receptor binding; receptor expression; receptor function; research and development; stable cell line; success; uptake

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNT) are the most toxic substances known to humans causing respiratory failure upon poisoning. BoNT's lethality and ease of production has led to a category "A" bioterrorism agent designation for BoNT by the Department of Defense (DoD). Developing effective, post-exposure antagonists to BoNT is a top priority of the DoD. Despite their lethality, BoNTs have cosmetic and pharmaceutical applications and are currently FDA-approved for the treatment of glabellar lines (wrinkles), cervical dystonia, blepharospasm, cranial nerve VII disorders, and cosmoses. BoNT provides relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long lasting reductions in pain. BoNT's exquisite specificity for neurons and long time of action make it a lead candidate for the treatment of neurological and muscle disorders where conventional treatments have failed. Development of new BoNT-based therapies is hindered by the lack of assays to measure BoNT activity. Currently, the only widely accepted method of measuring BoNT activity involves injecting BoNT into mice and counting the number of mice that die after one or more days. Because this process takes days to complete, requires special facilities, and is extremely low-throughput, it makes antagonist screening or development of new BoNT-derived therapeutics prohibitively expensive and time-consuming. The overall objective of this Phase I proposal is to develop a unique technology for measuring BoNT activity in living cells. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-a cell-based assay is required to effectively measure BoNT activity. BioSentinel proposes to develop a cell-based assay that measures BoNT activity by using Forster resonance energy transfer (FRET) to monitor BoNT cleavage of SNARE proteins, the target of BoNT. BioSentinel will generate FRET-based reporters for measuring BoNT protease activity, identify cell lines that are highly sensitive to BoNT, generate stable cell lines that express the reporters, and investigate high- throughput instruments for monitoring those reporters in vivo. BioSentinel proposes to develop the assay into a high-throughput platform that will enable researchers and pharmaceutical companies to rapidly quantify BoNT preparations, perform quality control measures, screen for new BoNT-based therapies, and screen for BoNT antagonists. Because of increased pressure for animal-free, high-throughput assays, a cell-based assay for BoNT detection is widely desired by researchers and industry, and would open a gateway to development of new BoNT-based therapies. Public Health Relevance: Botulinum neurotoxins are extremely lethal bacterial toxins that attack the nervous system and are considered a significant bioterrorism threat. Botulinum neurotoxins are also widely used for cosmetic and pharmaceutical applications providing relief of muscle contraction and pain. BioSentinel proposes to develop a high-throughput assay for measuring botulinum neurotoxin activity in living cells, providing a much needed platform for screening antagonists to BoNT and developing new BoNT-based therapies.

描述（由申请方提供）：肉毒神经毒素（BoNT）是已知对人类毒性最大的物质，中毒后可导致呼吸衰竭。BoNT的致命性和易于生产导致了国防部（DoD）将BoNT指定为“A”类生物恐怖主义制剂。开发有效的BoNT暴露后拮抗剂是DoD的首要任务。尽管它们具有致命性，但BoNT具有化妆品和药物应用，并且目前FDA批准用于治疗眉间纹（皱纹）、颈部肌张力障碍、眼睑痉挛、颅神经VII病症和宇宙病。BoNT通过沉默引起肌肉收缩的神经元来缓解肌肉紧张。对于许多疾病，基于BoNT的治疗提供了显着和持久的疼痛减轻。BoNT对神经元的特异性和长时间的作用使其成为治疗常规治疗失败的神经和肌肉疾病的主要候选者。新的基于BoNT的疗法的开发受到缺乏测量BoNT活性的测定的阻碍。目前，唯一被广泛接受的测量BoNT活性的方法是将BoNT注射到小鼠体内，并计算一天或多天后死亡的小鼠数量。因为这个过程需要几天才能完成，需要特殊的设施，并且是极低的通量，它使得拮抗剂筛选或新的BoNT衍生的治疗剂的开发过于昂贵和耗时。该第一阶段提案的总体目标是开发一种用于测量活细胞中BoNT活性的独特技术。由于BoNT具有三种活性-细胞受体结合和摄取、囊泡易位和靶切割-需要基于细胞的测定来有效地测量BoNT活性。BioSentinel提出开发一种基于细胞的检测方法，通过使用Forster共振能量转移（FRET）来监测BoNT对SNARE蛋白（BoNT的靶点）的切割，从而测量BoNT活性。BioSentinel将产生用于测量BoNT蛋白酶活性的基于FRET的报告基因，鉴定对BoNT高度敏感的细胞系，产生表达报告基因的稳定细胞系，并研究用于在体内监测这些报告基因的高通量仪器。BioSentinel建议将该检测方法开发成一个高通量平台，使研究人员和制药公司能够快速定量BoNT制剂，执行质量控制措施，筛选新的BoNT疗法，并筛选BoNT拮抗剂。由于无动物、高通量测定的压力增加，研究人员和行业广泛期望用于BoNT检测的基于细胞的测定，并且将打开开发新的基于BoNT的疗法的大门。公共卫生相关性：植物神经毒素是一种攻击神经系统的致命细菌毒素，被认为是一种重大的生物恐怖主义威胁。植物神经毒素还广泛用于美容和制药应用，以缓解肌肉收缩和疼痛。BioSentinel提出开发一种高通量检测方法，用于测量活细胞中肉毒杆菌神经毒素的活性，为筛选BoNT拮抗剂和开发新的基于BoNT的疗法提供急需的平台。