A platform for therapeutic agents that promote rapid recovery from botulism

描述(申请人提供)：肉毒杆菌中毒是由于接触肉毒梭菌神经毒素(BONT)产生的毒素引起的，BONT是CDC A类生物防御威胁剂，目前尚无解毒剂。到目前为止，已经发现了七种不同的BONT血清型(BONT/A-G)，其中许多还有许多额外的BONT亚型。为了防范所有这些不同的BONT生物恐怖威胁，昂贵的常规小分子药物开发需要分别针对七种不同的药物靶标进行，或许还有其他药物。这一挑战，加上BONT小分子药物开发面临的其他极端障碍，尤其使开发治疗肉毒杆菌中毒的小分子药物的努力复杂化。迫切需要新的治疗模式来应对与这些容易获得、易于生产和极其危险的生物恐怖制剂相关的巨大风险。我们已经开发并广泛优化了两种不同的“设计的E3-连接酶”药物，每种药物都可以加速被两种最危险的A型或B型肉毒神经毒素(BONT/A，BONT/B)之一中毒的神经元的“分子治愈”。这两种先导剂由TrCP的F-box结构域与骆驼状的纳米体结构域融合而成，与其中一种BONT蛋白酶具有结合特异性。这些多肽制剂的大小小于30 kDa，与BONT蛋白酶结合，导致其迅速的神经元内破坏，导致神经元的快速恢复。由于这种解毒剂的模块化性质，开发类似的试剂来治疗所有其他BONT血清型和亚型将是简单和直接的，只需将纳米体域替换为另一个具有适当特异性的结构域。在这项提案中，我们将开发一种通用的递送载体，将我们的两种先导剂递送到肉毒杆菌中毒患者体内中毒神经元的细胞液中，并进行临床前评估。作为载体，我们建议使用一种基于梭状芽胞杆菌毒素的无毒神经元递送载体(TNDV)，因为它具有高度进化的能力，可以进入体内，在血清中存活，并将酶活性递送到靶细胞的胞浆中。我们将开发三种不同的、经过验证的TNDV系统，每个系统都具有独特而引人注目的功能，然后选择最好的运载工具(S)进行进一步开发和动物试验。这三个TNDV系统将被改造，无毒形式：1)CONT C型；2)艰难梭菌毒素B；3)梭状芽胞杆菌C2毒素。如果成功，预计可以开发类似的药物来针对几乎任何用于研究或治疗应用的胞浆神经元蛋白的加速周转。 公共卫生相关性：肉毒杆菌神经毒素是一种极其危险的CDC A类生物防御威胁，可广泛获得，易于生产，具有极强的毒性，并且没有解毒剂可用。此外，至少存在七种不同的毒素类型，每种类型都由不同的药物靶点组成，因此需要不同的、具有挑战性和昂贵的小分子药物开发计划。相比之下，我们建议开发的生物分子疗法的创新新平台有可能迅速为所有BONT血清型带来商业上可行的解毒剂。

DESCRIPTION (provided by applicant): Botulism is caused by exposure to toxins produced by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote currently exists. Seven different BoNT serotypes have been discovered to date (BoNT/A-G), many having numerous additional BoNT subtypes. To protect against all of these diverse BoNT bioterror threats, expensive conventional small molecule drug development would need to be separately performed for each of the seven different drug targets and perhaps others. This challenge, together with other extreme hurdles confronting BoNT small molecule drug development, particularly complicates efforts to develop small molecule drugs to treat botulism. New therapeutic paradigms are urgently needed to counter the enormous risks associated with these easy to obtain, easy to produce and extremely dangerous bioterror agents. We have developed and then extensively optimized two distinct 'designer E3-ligase' agents that each accelerate the 'molecular cure' of neurons intoxicated by one of the two most dangerous Botulinum neurotoxins, serotypes A or B (BoNT/A, BoNT/B). These two lead agents consist of the F-box domain of TrCP fused to a camelid 'nanobody' domain with binding specificity for one of the BoNT proteases. These polypeptide agents, with a size less than 30 kDa, bind to the BoNT protease and cause its rapid, intraneuronal destruction leading to rapid recovery of the neuron. Because of the modular nature of this antidote, it will be simple and straightforward to develop similar agents to treat all other BoNT serotypes and subtypes simply by substituting the nanobody domains with another having the appropriate specificity. In this proposal, we will develop a general delivery vehicle to deliver our two lead agents to the cytosol of intoxicated neurons within botulism patients and perform pre-clinical evaluation. As the vehicle, we propose to use an atoxic Clostridial toxin-based neuronal delivery vehicle (TNDV) due to its highly evolved capability to enter the body, survive in serum and deliver enzyme activities to the cytosol of targeted cells. We will develop three different and proven TNDV systems, each having unique and compelling features, and then select the best vehicle(s) for further development and animal testing. The three TNDV systems will be modified, atoxic forms of: 1) BoNT serotype C; 2) C. difficile toxin B and; 3) Clostridial C2 toxin. If successful, it is expected that similar agents could be developed to target the accelerated turnover of virtually any cytosolic neuronal protein for research or therapeutic applications. Public Health Relevance: Botulinum neurotoxin is an extremely dangerous, CDC Category A biodefense threat that is widely available, easily produced, exceedingly toxic and for which no antidote is available. Furthermore, at least seven different toxin types exist, each constituting of different drug targets and thus requiring distinct, challenging and expensive small molecule drug development programs. In contrast, the innovative new platform for biomolecule therapeutics that we propose to develop has the potential to quickly lead to commercially viable antidotes for all BoNT serotypes.