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

项目摘要/摘要 目前用于非上瘾、慢性疼痛治疗的药物发现工作针对的是人类的电压门控 钠(HNaV)通道：在可兴奋状态下激发快速动作电位的成孔跨膜蛋白 神经细胞、心脏细胞和骨骼细胞。遗传学和临床前靶点验证研究已经确定了hNaV1.7， HNaV1.8、hNaV1.9通道亚型作为疼痛信号的关键蛋白，重点是hNaV1.7在其 在外周神经系统中的主要表达。尝试选择性地将hNaV1.7作为靶点 临床小分子药物由于非选择性结合其他hNaV通道亚型和其他 离子通道家族；非选择性结合可导致心脏骤停、瘫痪和癫痫。多肽毒素 来源于狼蛛、蜘蛛和蝎子已被确定为有效的抑制hNaV的生物制品。 值得注意的是，原毒素-II，来自秘鲁绿色天鹅绒狼蛛的神经毒素，大约有1 NM的一半- 对hNav1.7的最大抑菌浓度(IC50)。然而，这些多肽毒素也具有非选择性 绑定到hNav1.7。结合最新的冷冻-EM结构图像，原毒素-II结合到电压敏感结构域II HNaV1.7之后，现在可以设计对hNaV1.7具有选择性的模拟原毒素-II的多肽。最新进展 使用Rosetta的从头蛋白质设计方法-一套蛋白质结构预测和设计软件- 在实验验证之前启用药物设计和虚拟筛选的新途径。因此，该项目 目的是创造受原毒素-II启发的新多肽，以选择性地靶向hNaV1.7。方法论设计 结构上显示与hNaV1.7结合的结合了原毒素-II基序的新的多肽拓扑。这个 进一步优化多肽以结合hNaV1.7独有的残基：天然未靶向的残基 多肽毒素。在合成候选多肽后，它们将被验证为选择性靶向hNaV1.7 全细胞膜片钳电生理学。结果将提高我们对以下方面所需的机械理解 选择性的，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蛋白设计方法创建对hNaV1.7具有选择性的独特多肽。申请人须于 完成这个项目将拥有精湛的计算蛋白质设计方法的专业知识并获得 第一次接受电生理学培训。在整个项目中，赞助商和共同赞助商将 实施培训计划，以加强申请者的神经科学和药物发现知识，同时 拓宽他们的合作网络，提高他们的科学交流技能。这个计划是 量身定做，使申请人为学术生涯做好准备，为设计多肽生物制品 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.