靶向钠离子通道 Nav1.7毒素多肽的筛选系统的构建和应用

Nature venom peptides represent a rich source of pharmacologically active peptides that interact with ion channels.Nav1.7 is expressed in the peripheral nervous system and is an important target for treating pain. The known Nav1.7 inhibitors can not inhibit Nav1.7 specifically due to the high homology among sodium channels, resulting in adverse effects during treatment. Thus it is critical to develop an efficient system to screen specific antagonism of Nav1.7. In the previous study, we have designed an efficient autocrine-based high-throughput selection to discover and refine venom peptide, which is demonstrated by the discovery of novel Kv1.3 channel blockers and engineering of a Kv1.3 blocker peptide (ShK). In the project, we will use the latest CRISPR/Cas9 genome editing tool to construct Nav1.7 reporter cell line by replacing Kv1.3 with Nav1.7 in Kv1.3 reporter cell line and optimize the reporter cell line by mutiple methods. And we will perform positive selection with Nav1.7 reporter cell line followed by negative selection with Nav1.4 and Nav1.5 reporter cell lines to identify Nav1.7 specific inhibitory peptide from nature venom peptides library. Finally, the effects and specificity of selected venom peptides will be tested with path clamp experiments and their therapeutic efficacy will be evaluated by animal pain model. The results of the project will expand the autocrine-based selection system into targeting sodium channels and further other ion channels. In addition, the identification of novel specific venom peptides targeting Nav1.7 will advance the development of drugs for treating pain.

天然毒素多肽数量庞大，是筛选离子通道靶向药物的丰富来源。钠离子通道Nav1.7表达于外围神经系统，是治疗疼痛的重要靶点。由于钠离子通道蛋白高度的同源性，已有的抑制剂都不能完全专一地抑制Nav1.7。在前期研究过程中，我们设计了一套基于细胞自分泌形式的筛选离子通道抑制剂的系统，其不仅可以用来筛选钾离子通道Kv1.3新的靶向毒素多肽，也可以对已知Kv1.3抑制多肽进行改造。但钠离子通道结构比钾离子通道复杂而且家族成员间同源性极高，将基于自分泌形式的筛选推广到钠离子通道上需要大幅改进和优化。为此在本项目中，我们将利用最新的CRISPR/Cas9基因编辑技术在Kv1.3报告细胞系的基础上构建Nav1.7报告细胞系并利用多种方式进行优化；筛选时引入正向筛选联合负向筛选的理念，提高获得Nav1.7特异性毒素多肽的概率；最后鉴定毒素多肽对Nav1.7的抑制效果和特异性并分析其在动物模型中的治疗效果。

天然毒素多肽数量庞大，是筛选离子通道靶向药物的丰富来源。Nav1.7作为钠离子通道家族的一员，由SCN9A基因编码，临床和遗传学证据表明，Nav1.7在人类感知疼痛的过程中扮演了重要角色，是治疗疼痛的重要靶点。在前期研究过程中，我们设计了一套基于细胞自分泌形式的筛选离子通道抑制剂的系统。其不仅可以用来筛选钾离子通道Kv1.3新的靶向毒素多肽，也可以对已知Kv1.3抑制多肽进行改造。但钠离子通道结构比钾离子通道复杂而且家族成员间同源性极高，将基于自分泌形式的筛选推广到钠离子通道上难度较大。在本项目中，我们计划利用CRISPR/Cas9基因编辑技术在Kv1.3报告细胞系的基础上构建Nav1.7报告细胞系以提高构建效率，但实验时发现SCN9A基因非常大，很难正确地替换Kv1.3报告细胞系中的KCNA3基因（Kv1.3编码基因），因此我们利用CRISPR/Cas9技术将Kv1.3筛选系统中各元件克隆进稳定表达SCN9A的商业细胞系CHO-SCN9A中从而建立一套基于细胞自分泌形式筛选靶向离子通道Nav1.7毒素多肽的筛选系统。PCR扩增和测序显示各元件插入正确；TEV酶质粒转染初步验证Nav1.7报告细胞系成功；电生理膜片钳实验显示新建细胞系Nav1.7电流大于1000pA可用于药理学研究；包装有Protx毒素多肽的慢病毒感染新建细胞系后亦可见Nav1.7电流被抑制。其次，深度测序分析毒素多肽Protx或Huwentoxin慢病毒刺激新建细胞系或Nav1.7敲除鼠背根节组织中的差异基因显示存在共同异常调节的细胞通路。此外，我们构建了新的自然毒素多肽库，并用本课题组成熟的Kv1.3报告细胞系进行验证且改进了筛选流程以提高筛选成功率，结果成功筛出新的靶向Kv1.3的毒素多肽。本项目成功构建出了Nav1.7报告细胞系和新的自然毒素多肽库，但囿于时间关系，针对Nav1.7的毒素多肽筛选还在进行中。