Antidote for botulism

描述（由申请人提供）：背景。CDC A类生物恐怖剂，肉毒梭菌神经毒素（BoNT），是一种锌蛋白酶，其切割参与突触前乙酰胆碱释放的蛋白质，从而引起麻痹。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）引起的瘫痪可持续数月。任何有效的治疗不仅必须进入受影响的神经元和肉毒毒素，而且必须解决BoNT/A具有极长的作用持续时间的事实。假说.我们提出缺乏蛋白酶活性的毒素（iBoNT/A）将是急性肉毒中毒的有效解毒剂，因为它应该与BoNT/A（SNAP-25）竞争相同的底物而不切割它，并且应该具有与BoNT/A相同的寿命。iBoNT/A应该进入神经元，定位于细胞膜上的相同结构，其似乎稳定轻链以赋予其非常长的活性持续时间，并且在足够高的细胞内浓度下，置换活性轻链，从而促进其降解并重建胞吐作用。 初步研究。在最近的实验中，我们已经证明了通过在锌配位区中引入三个突变而失活的重组BoNT/A轻链（iBoNT/A-L）抑制天然BoNT/A轻链对SNAP-25的切割。 具体目标。我们建议确定iBoNT/A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在小鼠中的作用。 工作建议。我们将确定iBoNT/A-L对天然BoNT/A-L活性的抑制动力学，以及SNAP-25切割是否可以通过向用BoNT/A-L转染的PC-12细胞的培养基中添加全长、有切口的iBoNT/A来抑制。我们将使用共聚焦显微镜确定与荧光蛋白融合的iBoNT/A是否定位于PC-12质膜并与SNAP-25共定位。我们将检查iBoNT/A是否可以抑制给予天然BoNT/A的培养运动神经元中的SNAP-25切割。将确定这些非分裂细胞中的增殖持续时间。我们将确定iBoNT/A是否可以减弱小鼠中天然肉毒杆菌血清型A的致死性或麻痹。公共卫生相关性：肉毒梭菌神经毒素（BoNT）是CDC A类生物恐怖主义制剂。由于呼吸肌麻痹，BoNT血清型A（BoNT/A）中毒的患者可能在呼吸机上花费数月。释放一克BoNT可能会影响数千人，从而使当地的ICU瘫痪。目前没有可用的治疗方法可以逆转BoNT诱导的瘫痪。这些研究的最终目标是产生BoNT/A中毒的有效解毒剂。

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.