Next generation of botulinum neurotoxins with enhanced binding to human receptors

描述(申请人提供)：肉毒杆菌神经毒素(BoNTs)是一类细菌毒素，可阻断突触小泡的胞吐作用。两种类型的BoNT(BONT/A和B)现在被广泛用于治疗越来越多的疾病。随着BoNTs使用的增加，已经确定了主要的局限性和不良影响，包括：(1)注射的毒素扩散到其他区域，这是各种非危及生命的不良反应频繁发生的原因，以及在极少数情况下包括死亡在内的严重后果；(2)患者产生中和抗体，使未来的治疗无效。这两个问题都与注射剂量直接相关。相应地，增强了疗效和特异性 BoNTs的使用将减少治疗中所需的毒素剂量，并减少数百万患者的不良事件发生。事实上，BoNTs与人类神经元的结合是可以改善的，因为我们最近发现，由于人类SYT II序列的单一残基变化，BoNT/B不能与人类主要受体突触素II(SYT II)结合。这种人类受体缺陷增加了对BONT/B受体结合域进行改造以恢复其与人SYT II结合的需要，这种修饰的BONT/B受体结合域将显著提高人类BONT/B的疗效并减少毒素扩散/免疫反应。在这里，我们建议以BONT/B-SYT II复合体的共晶结构为指导，在BONT/B受体结合域进行合理的设计突变，以确定恢复BONT/B与人SYT II结合的特定突变。一旦鉴定出这些突变，我们将在试管和神经元表面进一步鉴定它们与人SYT II的结合。这些研究将产生改良的BONT/B受体结合域，显著提高与人类神经元的结合效率，这将直接导致创造出效率、特异性和安全性都更高的新一代治疗性毒素。

DESCRIPTION (provided by applicant): 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.