Antidote for botulism

肉毒杆菌中毒的解毒剂

• 批准号: 7862592
• 负责人: DAVID Owen BEENHOUWER
• 金额: $ 20.64万
• 依托单位: BRENTWOOD BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-10 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862592
• 关键词: Acetylcholine; Acute; Address; Affect; Amino Acids; Antibodies; Antidotes; Antitoxins; Attenuated; Binding; Binding Sites; Bioterrorism; Bontoxilysin; Botulinum Toxin Type A; Botulinum Toxins; Botulism; C-terminal; Categories; Cell membrane; Cells; Centers for Disease Control and Prevention (U.S.); Cleaved cell; Clostridium botulinum; Complex; Confocal Microscopy; Culture Media; Cytoplasm; Disease Progression; Drug Kinetics; Exocytosis; Exposure to; Foundations; Goals; Grant; Hydrolysis; Intensive Care; Interphase Cell; Intoxication; Kinetics; Length; Life; Light; Link; Longevity; Membrane; Motor Neurons; Mus; Mutate; Mutation; Neurons; Neurotransmitters; PC12 Cells; Paralysed; Patients; Peptide Hydrolases; Persons; Positioning Attribute; Production; Proteins; Recombinants; Respiratory Paralysis; Serotyping; Sterile coverings; Structure; Time; Toxin; Ventilator; Western Blotting; Work; Zinc; botulinum; cholinergic neuron; disulfide bond; effective therapy; inhibitor/antagonist; mutant; neuromuscular; presynaptic; prevent; public health relevance; receptor; reconstitution; research study; small molecule; time use; trafficking

DESCRIPTION (provided by applicant): Background. The CDC category A bioterrorism agent, Clostridium botulinum neurotoxin (BoNT), is a zinc protease that cleaves proteins involved in presynaptic acetylcholine release thereby causing paralysis. The proteolytically active light chain of BoNT serotype A (BoNT/A) hydrolyzes SNAP-25. Paralysis caused by BoNT serotype A (BoNT/A) can last several months. Any effective treatment must not only gain entry to the affected neurons and inactivate toxin, but must address the fact that BoNT/A has an extremely long duration of action. Hypothesis. We propose that toxin lacking protease activity (iBoNT/A) will be an effective antidote to acute botulism because it should compete for the same substrate as BoNT/A (SNAP-25) without cleaving it and should have the same longevity as BoNT/A. iBoNT/A should enter neurons, localize to same structure on the cell membrane that appears to stabilize the light chain to give its extraordinary long duration of activity, and at high enough intracellular concentrations, displace active light chain, thereby promoting its degradation and re- constituting exocytosis. Preliminary Studies. In recent experiments, we have demonstrated that recombinant BoNT/A light chain inactivated by the introduction of three mutations in the zinc coordination region (iBoNT/A-L) inhibits cleavage of SNAP-25 by native BoNT/A light chain. Specific Aims. We propose to determine whether iBoNT/A can 1) block BoNT/A protease activity in neuronal cells, 2) shorten the duration of action of BoNT/A , and 3) attenuate effects of BoNT/A in mice. Work Proposed. We will determine the inhibition kinetics of native BoNT/A-L activity by iBoNT/A-L and if SNAP-25 cleavage can be inhibited by addition of full length, nicked iBoNT/A to the culture medium of PC-12 cells transfected with BoNT/A-L. We will determine whether iBoNT/A fused to fluorescent protein localizes to PC-12 plasma membrane and colocalizes with SNAP-25 using confocal microscopy. We will examine whether iBoNT/A can inhibit SNAP-25 cleavage in cultured motor neurons given native BoNT/A. The duration of inhibi- tion in these non-dividing cells will be determined. We will determine whether iBoNT/A can attenuate lethality or paralysis by native botulinum serotype A in mice. PUBLIC HEALTH RELEVANCE: Clostridium botulinum neurotoxin (BoNT) is a CDC category A bioterrorism agent. A patient with BoNT sero- type A (BoNT/A) intoxication may spend several months on a ventilator due to respiratory muscle paralysis. Release of a single gram of BoNT could affect several thousand people, thereby crippling local ICUs. There is currently no treatment available that reverses BoNT-induced paralysis. The ultimate goal of these studies is to generate an effective antidote to BoNT/A intoxication.

描述（由申请人提供）：背景。 CDC类别的生物恐怖剂，肉毒杆菌神经毒素（BONT）是一种锌蛋白酶，可裂解参与突触前乙酰胆碱释放的蛋白质，从而导致麻痹。 BONT血清型A（BONT/A）的蛋白水解活性轻链水解SNAP-25。由BONT血清型A（BONT/A）引起的瘫痪可以持续几个月。任何有效的治疗方法不仅必须进入受影响的神经元并灭活毒素，而且还必须解决BONT/A的作用时间非常长的事实。假设。我们建议缺乏蛋白酶活性的毒素（IBONT/A）将是急性肉毒杆菌症的有效解毒剂，因为它应该与BONT/A（SNAP-25）相同的底物竞争，而不会切断它，并且应具有与BONT/A相同的寿命。 ibont/a应输入神经元，位于细胞膜上的相同结构，该结构似乎可以稳定光链以使其具有非凡的长期活性持续时间，并且在足够高的细胞内浓度下，置换了活跃的轻链，从而促进了其降解和重新组成的胞吞作用。 初步研究。在最近的实验中，我们证明了重组BONT/A轻链通过在锌配位区（IBONT/A-L）引入三个突变而灭活的轻链抑制了天然Bont/A轻链抑制SNAP-25的裂解。 具体目标。我们建议确定IBONT/A CAN是否可以1）阻断神经元细胞中的BONT/A蛋白酶活性，2）缩短BONT/A的作用持续时间，以及3）减弱BONT/A对小鼠的影响。 提议的工作。我们将确定Ibont/A-L对天然BONT/A-L活性的抑制动力学，如果可以通过添加全长添加SNAP-25裂解，则将Ibont/A划分为用BONT/A-L转染的PC-12细胞的培养基。我们将确定Ibont/A融合到荧光蛋白融合到PC-12质膜上是否定位于PC-12，并使用共聚焦显微镜与SNAP-25共定位。我们将检查Ibont/A是否可以抑制鉴于天然BONT/A的培养的运动神经元中的SNAP-25裂解。这些非分散细胞的抑制作用将被确定。我们将确定Ibont/A是否可以减弱小鼠中肉毒杆菌血清型A的致死性或瘫痪。公共卫生相关性：肉毒乳梭菌神经毒素（BONT）是疾病预防控制中的生物恐怖剂。 A型A型（BONT/A）中毒患者可能由于呼吸肌瘫痪而在呼吸机上花费几个月。释放一克骨可能会影响数千人，从而使当地的ICU瘫痪。目前尚无可用的治疗方法可以逆转BONT诱发的麻痹。这些研究的最终目的是产生有效的解毒剂，以散发出BONT/A中毒。