Reversing botulism with agents that accelerate intraneuronal toxin degradation

使用加速神经元内毒素降解的药物逆转肉毒杆菌中毒

• 批准号: 7875009
• 负责人: Charles Bix Shoemaker
• 金额: $ 21.01万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7875009
• 关键词: Animals; Antidotes; Bacillus megaterium; Binding; Bontoxilysin; Botulinum Toxin Type A; Botulinum Toxins; Botulism; Categories; Cell Culture Techniques; Cell Death; Cells; Centers for Disease Control and Prevention (U.S.); Cleaved cell; Clostridium; Clostridium botulinum; Clostridium difficile; Cytosol; Development; Dose; Endocytosis; Engineering; Enzymes; F Box Domain; F-Box Proteins; Glucosyltransferase; Glucosyltransferases; Glutathione S-Transferase; Intoxication; Lead; Ligase; Limb structure; Mediating; Microbe; Modeling; Modification; Mus; Muscle function; Neurons; Paralysed; Paresis; Pathology; Patients; Peptide Hydrolases; Polyubiquitination; Proteins; Recombinants; Research; Serotyping; Specificity; Surface; Symptoms; Testing; Therapeutic; Therapeutic Agents; Toxin; Ubiquitin; Variant; base; biodefense; botulinum; botulinum toxin type B; cell type; design; efficacy testing; functional restoration; killings; microbial host; nanomachine; polypeptide; public health relevance; receptor; receptor binding; rho GTP-Binding Proteins; ubiquitin-protein ligase; vector

DESCRIPTION (provided by applicant): Botulism is caused by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists. We have developed two distinct "designer E3-ligases" that target either the intracellular proteases of botulinum neurotoxin serotype A or B (BoNT/A, BoNT/B) for accelerated ubiquitin- mediated degradation. Our agents consist of the F-box domain of TrCP fused to a came lid VHH domain with binding specificity for the BoNT protease. These polypeptide agents, with a size less than 30 kD, lead to the rapid, intraneuronal destruction of the intoxicating BoNT proteases. Here we propose to develop a Clostridium difficile toxin B (TcdB) based vehicle for delivery of the BoNT designer E3-ligases to the cytosol of intoxicated neurons. We propose to test the engineered TcdB agents for reversal of botulism symptoms within cultured neurons and then in mice, likely with a single dose therapy. TcdB has highly evolved features that make it ideal for delivery of BoNT directed designer E3-ligases to the cytosol of botulism intoxicated neurons within patients. TcdB naturally binds to a surface receptor that is broadly expressed in cells, then is internalized by endocytosis and delivers a glucosyltransferase (GT) that inactivates Rho GTPases leading to cell death. This toxin also contains a protease activity that cleaves its enzymatic "cargo" from the "delivery vehicle", and releases it into the cytosol. TcdB has been shown to deliver significant quantities of functional glutathione-S-transferase (GST) to the cell cytosol when the GST is fused to the amino terminus of the toxin GT domain. We have successfully expressed large amounts of full-size, bioactive, recombinant TcdB, and an atoxic variant TcdB, in microbial host cells. In this proposed project, we will develop and test a neuron-specific TcdB by replacing the TcdB receptor binding domain (RBD) with the equivalent RBD domain from BoNT/A. Secondly; we will test whether BoNT/A protease turnover can be accelerated within intoxicated neurons by treatment with a fusion of BoNT/A designer E3-ligase to atoxic TcdB. Finally, an engineered TcdB targeting the BoNT designer ligase to neurons will be tested in the mouse hind limb paresis model for the ability to reverse the symptoms of botulinum toxin induced paralysis. If successful, similar TcdB agents should be readily possible and rapidly available for all botulinum serotypes by simply switching the VHH domain to those having specificity for other BoNT proteases. Secondly, we will have demonstrated that TcdB is a general vector for the delivery of biomolecules to the cytosol of cells- targeted by the specificity of the RBD. Finally, the biomolecular cargo we are delivering to these cells, designer E3-ligases, are simple fusions between a camelid VHH targeting domain and the TrCP F-box, and it is expected that similar agents could be developed to target accelerated turnover of virtually any cytosolic protein for research or therapeutic applications. PUBLIC HEALTH RELEVANCE: Through this proposal, we are directly seeking to develop a single-dose therapeutic cure for botulism, which is caused by several CDC Category A Clostridium botulinum toxins for which no antidote is currently available. If successful, similar agents can be quickly developed for all botulinum serotypes. The same general strategy could be used to deliver therapeutic agents to specific cells to target other pathogenic cytosolic proteins for rapid proteasomal destruction.

描述（由申请人提供）：肉毒杆菌中毒是由肉毒杆菌神经毒素 (BoNT) 引起的，BoNT 是 CDC A 类生物防御威胁剂，目前尚无解毒剂。我们开发了两种不同的“设计 E3 连接酶”，靶向肉毒杆菌神经毒素血清型 A 或 B 的细胞内蛋白酶（BoNT/A、BoNT/B），以加速泛素介导的降解。我们的试剂由 TrCP 的 F-box 结构域与骆驼盖 VHH 结构域融合而成，具有与 BoNT 蛋白酶结合特异性。这些多肽试剂的大小小于 30 kD，可导致神经元内快速破坏令人中毒的 BoNT 蛋白酶。在这里，我们建议开发一种基于艰难梭菌毒素 B (TcdB) 的载体，用于将 BoNT 设计器 E3 连接酶递送到中毒神经元的细胞质中。我们建议在培养的神经元中测试工程化的 TcdB 药物是否可以逆转肉毒杆菌中毒症状，然后在小鼠中进行测试（可能采用单剂量疗法）。 TcdB 具有高度进化的功能，使其成为将 BoNT 定向设计的 E3 连接酶递送至患者体内肉毒杆菌中毒神经元细胞质的理想选择。 TcdB 自然地与细胞中广泛表达的表面受体结合，然后通过内吞作用内化，并传递葡萄糖基转移酶 (GT)，使 Rho GTP 酶失活，导致细胞死亡。这种毒素还含有蛋白酶活性，可将其酶促“货物”从“递送载体”上裂解下来，并将其释放到细胞质中。当 GST 与毒素 GT 结构域的氨基末端融合时，TcdB 已被证明可以将大量的功能性谷胱甘肽-S-转移酶 (GST) 递送至细胞质。我们已经在微生物宿主细胞中成功表达了大量全尺寸、生物活性的重组 TcdB 和无毒变体 TcdB。在这个拟议的项目中，我们将通过用 BoNT/A 中的等效 RBD 结构域替换 TcdB 受体结合结构域 (RBD) 来开发和测试神经元特异性 TcdB。第二;我们将测试是否可以通过 BoNT/A 设计师 E3 连接酶与无毒 TcdB 的融合来加速中毒神经元内的 BoNT/A 蛋白酶周转。最后，将在小鼠后肢麻痹模型中测试一种针对神经元的 BoNT 设计连接酶的工程化 TcdB，以检验其逆转肉毒杆菌毒素引起的麻痹症状的能力。 如果成功，通过简单地将 VHH 结构域转换为对其他 BoNT 蛋白酶具有特异性的结构域，类似的 TcdB 试剂应该可以轻松实现并快速用于所有肉毒杆菌血清型。其次，我们将证明 TcdB 是一种通用载体，用于通过 RBD 的特异性将生物分子递送至细胞的胞质溶胶。最后，我们向这些细胞提供的生物分子货物，即设计的 E3 连接酶，是骆驼科动物 VHH 靶向结构域和 TrCP F-box 之间的简单融合，并且预计可以开发类似的试剂来针对几乎任何细胞的加速周转。用于研究或治疗应用的胞浆蛋白。 公共健康相关性：通过这项提案，我们直接寻求开发一种单剂量治疗肉毒杆菌中毒的方法，肉毒杆菌中毒是由几种 CDC A 类肉毒杆菌毒素引起的，目前尚无解毒剂。如果成功，可以快速开发出适用于所有肉毒杆菌血清型的类似药物。相同的通用策略可用于将治疗剂递送至特定细胞以靶向其他致病性胞质蛋白以快速破坏蛋白酶体。