Next generation of botulinum neurotoxins with enhanced binding to human receptors

项目摘要 肉毒杆菌神经毒素(BoNTs)是一类阻断突触小泡的细菌毒素 胞吐。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BoNTs使用的增加，主要限制和不利影响 已查明包括(1)注射的毒素扩散到其他区域，这是 频繁发生各种不危及生命的不良影响，以及严重的 后果包括极少数情况下死亡；(2)产生中和抗体 使未来的治疗无效的患者。这两个问题都直接与注射有关 剂量。因此，提高BoNTs的有效性和特异性将降低 在治疗中所需的毒素剂量和减少不良事件的发生 病人。 事实上，正如我们最近发现的那样，BoNTs与人类神经元的结合可以得到改善 该BONT/B不能与主要的人类受体突触素II(SYT II)结合，因为 人SYT II序列中的残基变化。这种人类受体缺陷增加了人们的需要，也 提供了一个工程BONT/B受体结合域以恢复其 这种修饰的BONT/B受体结合域将显著 提高BONT/B在人类中的疗效，减少毒素扩散/免疫反应。这里 我们建议在BONT/B受体结合域进行合理的设计突变， 以BONT/B-SYT II络合物的共晶结构为指导，鉴定特定的 恢复与人类SYT II结合的BONT/B的突变。一旦鉴定出这样的突变，我们 将在试管和神经元表面进一步鉴定它们与人类SYT II的结合。 这些研究将产生修饰的BONT/B受体结合域，具有显著的 提高了结合人类神经元的效率，这将直接导致创造一个新的 产生的治疗性毒素具有更高的有效性、特异性和安全性。

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.