Next generation of botulinum neurotoxins with enhanced binding to human receptors

项目摘要 肉毒神经毒素（Botanicneurotoxins，BoNTs）是一类能阻断突触囊泡的细菌毒素 胞吐作用两种类型的BoNT（BoNT/A和B）现在被广泛用于治疗越来越多的 医疗条件。随着BoNT使用的增长，主要的局限性和不利影响 已确定的原因包括：（1）注入的毒素扩散到其他地区，这是 频繁发生各种非危及生命的不良反应，以及严重 后果包括在罕见情况下死亡;（2）中和抗体的产生 使未来的治疗无效的患者。这两个问题都与注射直接相关 剂量因此，增强BoNT的功效和特异性将降低免疫原性。 治疗中所需的毒素剂量，并减少数百万人中不良事件的发生。 患者 事实上，正如我们最近发现的那样， BoNT/B不能与主要的人类受体突触结合蛋白II（Syt II）结合，这是由于单一的 人Syt II序列中的残基变化。这种人类受体缺陷提高了需要， 为工程化BoNT/B受体结合结构域以恢复其功能提供了机会。 与人Syt II结合。这种修饰的BoNT/B受体结合结构域将显著地 提高BoNT/B在人体中的功效并减少毒素扩散/免疫应答。这里 我们建议在BoNT/B受体结合结构域中进行合理设计诱变， 使用BoNT/B-Syt II复合物的共晶体结构作为指导，以鉴定特异性的 恢复BoNT/B与人Syt II结合的突变。一旦发现这种突变，我们 将进一步表征它们在试管中和神经元表面上与人Syt II的结合。 这些研究将产生修饰的BoNT/B受体结合结构域， 提高了结合人类神经元的功效，这将直接导致创造一种新的 产生具有改进的功效、特异性和安全性的治疗性毒素。

Project Summary Botulinum neurotoxins (BoNTs) are a family of bacterial toxins that block synaptic vesicle exocytosis. Two types of BoNTs (BoNT/A and B) are now widely used to treat a growing list of medical conditions. As the use of BoNTs grows, major limitations and adverse effects have been identified including (1) diffusion of injected toxins to other regions, which is the cause of frequent occurrences of a wide range of non-life-threatening adverse effects, as well as serious consequences including death in rare occasions; (2) generation of neutralizing antibodies in patients that renders future treatment ineffective. Both issues are directly related to injection doses. Accordingly, enhancing the efficacy and specificity of BoNTs would decrease the required toxin doses in treatment and reduce the occurrence of adverse events in millions of patients. Indeed, the binding of BoNTs to human neurons can be improved, as we recently discovered that BoNT/B cannot bind to the major human receptor synaptotagmin II (Syt II) due to a single residue change in human Syt II sequence. This human receptor defect raises the need and also presents an opportunity for engineering the BoNT/B receptor binding domain to restore its binding to human Syt II. Such a modified BoNT/B receptor binding domain will significantly improve the efficacy of BoNT/B in humans and reduce toxin diffusion/immune response. Here we propose to carry out rational design mutagenesis in the BoNT/B receptor binding domain, using the co-crystal structure of BoNT/B-Syt II complexes as a guide, to identify specific mutations that restore BoNT/B binding to human Syt II. Once such mutations are identified, we will further characterize their binding to human Syt II in test tubes and on neuronal surfaces. These studies will generate modified BoNT/B receptor binding domains with significantly improved efficacy for binding human neurons, which will directly lead to the creation of a new generation of therapeutic toxins with improved efficacy, specificity, and safety.