Design of de novo peptides and electrophysiological testing for voltage-gated sodium channel 1.7 inhibition related to chronic pain treatment

项目总结/摘要 目前，用于非成瘾性慢性疼痛管理的药物发现工作针对人类电压门控性疼痛。 钠（hNaV）通道：在可兴奋的细胞中引起快动作电位的成孔跨膜蛋白 神经元、心脏和骨骼细胞。遗传和临床前靶点验证研究已经鉴定出hNaV1.7， hNaV1.8、hNaV1.9通道亚型作为疼痛信号传导中的关键蛋白，重点是hNaV1.7的 主要在周围神经系统中表达。尝试用pre-DNA选择性靶向hNaV1.7， 临床小分子药物由于与其他hNaV通道亚型的非选择性结合而不足， 离子通道家族;非选择性结合可导致心脏骤停、麻痹和癫痫发作。肽毒素 来源于狼蛛、蜘蛛和蝎子的化合物已被鉴定为有效的hNaV抑制生物制剂。 值得注意的是，来自秘鲁绿色天鹅绒狼蛛的神经毒素原毒素-II具有大约1 nM的半- 对hNav1.7的最大抑制浓度（IC 50）。然而，这些肽毒素也具有非选择性 与hNav1.7结合。结合最近的冷冻电镜结构图像，原毒素-II结合到电压敏感结构域II， hNaV1.7，现在可以设计对hNaV1.7有选择性的模拟原毒素-II的肽。进展 使用Rosetta的从头蛋白质设计方法-蛋白质结构预测和设计软件套件- 在实验验证之前，使药物设计和虚拟筛选的新途径成为可能。因此，该项目 目的是创造新的肽灵感来自Protoxin-II选择性靶向hNaV1.7。方法论设计 新的肽拓扑结构，其包含结构上显示与hNaV1.7结合的原毒素-II基序。的 进一步优化肽以结合hNaV1.7特有的残基：天然靶向的非靶向残基。 肽毒素在合成候选肽后，将使用以下方法验证它们选择性靶向hNaV1.7 全细胞膜片钳电生理学这些结果将提高我们对机械的理解， 选择性但有效的hNaV1.7抑制。此外，这项研究将是首次尝试使用 Rosetta de novo蛋白质设计方法以产生对hNaV1.7具有选择性的独特肽。申请人在 完成这个项目将有一个完善的专业知识计算蛋白质设计方法，并收到 第一次接受电生理学的培训。在整个项目中，赞助商和共同赞助商将 实施培训计划，以加强申请人对神经科学和药物发现的知识，同时 扩大他们的合作网络，提高他们的科学沟通技巧。这个计划是 量身定制，使申请人准备为学术生涯设计肽生物制剂， 离子通道病理的治疗和研究。

Project Summary/Abstract Current drug discovery efforts for non-addictive, chronic pain management are targeting human voltage-gated sodium (hNaV) channels: pore-forming transmembrane proteins that evoke the fast action potential in excitable neuronal, cardiac, and skeletal cells. Genetic and preclinical target validation studies have identified hNaV1.7, hNaV1.8, hNaV1.9 channel subtypes as key proteins in pain signaling with emphasis on hNaV1.7 for its predominant expression in the peripheral nervous system. Attempts at selectively targeting hNaV1.7 with pre- clinical small-molecule drugs fall short due to non-selective binding to other hNaV channel subtypes and other ion channel families; non-selective binding can lead to cardiac arrest, paralysis and seizure. Peptide toxins originating from tarantula, spider, and scorpion have been identified as potent hNaV-inhibiting biologics. Notably, Protoxin-II, the neurotoxin from the Peruvian green velvet tarantula, has approximately 1 nM half- maximal inhibitory concentration (IC50) to hNav1.7. However, these peptide toxins also have non-selective binding to hNav1.7. With recent cryo-EM structural images of Protoxin-II bound to voltage-sensing domain II of hNaV1.7, it is now possible to design peptides mimicking Protoxin-II that are selective to hNaV1.7. Advances in de novo protein design methods using Rosetta – a protein structure prediction and design software suite – enable a new avenue of drug design and virtual screening prior to experimental validation. Thus, the project aims to create new peptides inspired from Protoxin-II to selectively target hNaV1.7. The methodology designs new peptide topologies that incorporate the Protoxin-II motif structurally shown to bind to hNaV1.7. The peptides are further optimized to bind to residues unique to hNaV1.7: residues that are not targeted by natural peptide toxins. Upon synthesis of candidate peptides, they will be validated to selectively target hNaV1.7 using whole-cell patch-clamp electrophysiology. The results will improve our mechanistic understanding needed for selective, yet potent hNaV1.7 inhibition. Further, this research will be the first attempt at using a combination of Rosetta de novo protein design methods to create unique peptides selective for hNaV1.7. The applicant upon completion of this project will have a refined expertise of computational protein design methods and receive training in electrophysiology for the first time. Throughout this project, the sponsor and co-sponsor will implement a training plan to strengthen the applicant’s knowledge of neuroscience and drug discovery, while broadening their collaborative network, and sharpening their scientific communication skills. This plan is tailored such that the applicant is prepared for an academic career designing peptide biologics for the treatment and investigation of ion channel pathologies.