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

与慢性疼痛治疗相关的电压门控钠通道 1.7 抑制的从头肽设计和电生理学测试

• 批准号: 10389511
• 负责人: Brandon John Harris
• 金额: $ 3.92万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-03 至 2024-12-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389511
• 关键词: Action Potentials; Adult; Affinity; American; Benchmarking; Binding; Biological Assay; Biological Products; Cardiac; Cells; Charge; Clinical; Communication; Complex; Computational Science; Cryoelectron Microscopy; Data; Development; Disulfides; Drug Design; Electrophysiology (science); Family; Genetic; Heart Arrest; Human; Human Genetics; Hydrophobicity; Image; Industry Collaboration; Integral Membrane Protein; Investigation; Ion Channel; Knowledge; Lead; Learning; Libraries; Manuals; Methodology; Methods; Modification; Mutagenesis; Mutate; Neurobiology; Neurosciences; Neurotoxins; Nociception; Pain management; Paralysed; Pathology; Peptides; Peripheral Nervous System; Peruvian; Pharmaceutical Preparations; Positioning Attribute; Protein Engineering; Proteins; Publishing; Research; Resolution; Safety; Scorpions; Seizures; Side; Sodium; Sodium Channel; Software Design; Spiders; Structure; Testing; Therapeutic; Time; Toxin; Training; Transferable Skills; Translational Research; Validation; Variant; Work; addiction liability; career; chemical stability; chronic pain; chronic pain management; clinical efficacy; cytokine; design; drug candidate; drug discovery; effective therapy; experience; experimental study; falls; improved; knowledgebase; mimetics; nanomolar; neuronal excitability; non-opioid analgesic; novel; pain signal; patch clamp; peptidomimetics; pre-clinical; prescription opioid; preservation; protein structure prediction; rational design; screening; skeletal; skills; small molecule; targeted treatment; thermostability; validation studies; virtual screening; voltage

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.

项目概要/摘要 当前用于非成瘾性慢性疼痛管理的药物发现工作正在针对人类电压门控 钠 (hNaV) 通道：成孔跨膜蛋白，可激发兴奋性快速动作电位 神经元、心脏和骨骼细胞。遗传和临床前目标验证研究已确定 hNaV1.7、 hNaV1.8、hNaV1.9 通道亚型作为疼痛信号传导的关键蛋白，重点是 hNaV1.7 主要表达于周围神经系统。尝试选择性地针对 hNaV1.7 进行预 由于与其他 hNaV 通道亚型和其他通道的非选择性结合，临床小分子药物存在不足。 离子通道家族；非选择性结合可导致心脏骤停、瘫痪和癫痫发作。肽毒素 源自狼蛛、蜘蛛和蝎子的病毒已被鉴定为有效的 hNaV 抑制生物制剂。 值得注意的是，Protoxin-II，一种来自秘鲁绿丝绒狼蛛的神经毒素，具有大约 1 nM half- hNav1.7 的最大抑制浓度 (IC50)。然而，这些肽毒素也具有非选择性 绑定到 hNav1.7。最近，Protoxin-II 与电压传感域 II 结合的冷冻电镜结构图像 hNaV1.7，现在可以设计对 hNaV1.7 具有选择性的模拟 Protoxin-II 的肽。进展 使用 Rosetta（蛋白质结构预测和设计软件套件）的从头蛋白质设计方法 – 在实验验证之前开辟药物设计和虚拟筛选的新途径。因此，该项目 旨在创造受 Protoxin-II 启发的新肽，选择性靶向 hNaV1.7。方法论设计 新的肽拓扑包含原毒素-II 基序，结构显示可与 hNaV1.7 结合。这 肽经过进一步优化，可与 hNaV1.7 特有的残基结合：天然不靶向的残基 肽毒素。候选肽合成后，将使用以下方法验证它们是否选择性靶向 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结合使用 Rosetta de novo 蛋白质设计方法可创建针对 hNaV1.7 的独特肽选择性。申请人于 完成该项目将拥有计算蛋白质设计方法的精炼专业知识，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人的神经科学和药物发现知识，同时 扩大他们的协作网络，并提高他们的科学沟通技巧。这个计划是 量身定制，使申请人为学术生涯做好准备，为以下领域设计肽生物制剂 离子通道病理学的治疗和研究。