De novo protein neurotoxins for ion channels

离子通道的从头蛋白神经毒素

• 批准号: 9493056
• 负责人: Steve A N Goldstein
• 金额: $ 13.76万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9493056
• 关键词: De; novo; protein; neurotoxins; ion

 DESCRIPTION (provided by applicant): Protein neurotoxins are among the most powerful tools in the arsenal for investigation of membrane receptors and are now used as well for treatment of disease. This grant seeks, first, to advance our method of creation of de novo toxins by targeting potassium and proton channels that lack these useful ligands; second, to use de novo toxins to assess the potential of Kv1.5 and Kv2.1 channels as targets for treatment of two diseases of broad public impact- Atrial Fibrillation (AF) and Type 2 Diabetes (T2D), respectively. Success on these two fronts will allow wider use of these tools to produce toxins for other targets and motivate analogous work directed towards other types of membrane receptors important to human health and disease. To date, toxins have been derived primarily from limited sources: crude venoms, venom gland gene cloning, and laboratory synthesis of known toxins. Therefore, most receptors of interest lack a recognized, specific, high-affinity ligand. This state-of-affairs is easily understood: neither the purpose of toxins in the wild nor screens of venoms favor target specificity. Here, these problems are avoided by cloning toxins based on desired attributes using a new, high- throughput method we developed to generate a selective inhibitor of Kv1.3 channel pore. Specific Aim 1 seeks to improve the method by creating highly-diverse libraries on native scaffolds that bind to ion channel pores or voltage sensors, first using purified ion channel receptors, and then using channels on cell surfaces so the many receptors that do not retain their structure when purified can also be targeted. A recently identified channel for protons (Hv1) is used here to develop tools for voltage sensors (and to test directly the postulated role of this orphan receptor in male fertility). Specific Aim seeks de novo toxins directed to the Kv1.5 potassium channel. This orphan receptor is widely pursued as a potential therapeutic target for AF and lack of specific blockers has left unclear fundamental questions about its role in the heart and precluded direct tests of its utility as a drg target. Specific Aim 3 seeks de novo toxins specific for the Kv2.1 channel. Kv2.1 inhibition enhances glucose-stimulated insulin secretion from pancreatic -cells in a SUMO-dependent manner and inhibitors that do not cross inhibit are sought as potential drugs for T2D. De novo toxins for Kv1.5 and Kv2.1 are not only useful to validate the channels as drug targets but offer potential as therapeutic leads. Thus, small, disulfide-stabilized peptide toxins in use as drugs (Byetta(r), Integrilin(r), and Prialt(r)) have proven effective, protease resistant and non-immunogenic.

 描述（由申请人提供）：蛋白质神经毒素是研究膜受体的最有力的工具之一，现在也用于治疗疾病。这项资助旨在，首先，通过靶向缺乏这些有用配体的钾和质子通道来推进我们创造从头毒素的方法;其次，使用从头毒素来评估Kv1.5和Kv2.1通道作为治疗两种具有广泛公众影响的疾病的靶点的潜力-房颤（AF）和2型糖尿病（T2 D），分别。在这两个方面的成功将允许更广泛地使用这些工具来为其他目标生产毒素，并激励针对对人类健康和疾病重要的其他类型的膜受体的类似工作。迄今为止，毒素主要来自有限的来源：粗毒液、毒腺基因克隆和已知毒素的实验室合成。因此，大多数感兴趣的受体缺乏识别的、特异性的、高亲和力的配体。这种状况很容易理解：无论是野生毒素的目的，还是毒液的筛选，都不利于目标特异性。在这里，通过使用我们开发的新的高通量方法基于期望的属性克隆毒素来避免这些问题，以产生Kv1.3通道孔的选择性抑制剂。具体目标1试图通过在结合到离子通道孔或电压传感器的天然支架上创建高度多样化的文库来改进该方法，首先使用纯化的离子通道受体，然后使用细胞表面上的通道，因此也可以靶向许多在纯化时不保留其结构的受体。最近确定的质子通道（Hv 1）在这里用于开发电压传感器的工具（并直接测试这种孤儿受体在男性生育力中的假定作用）。Specific Aim寻求针对Kv1.5钾通道的从头毒素。这种孤儿受体被广泛用作AF的潜在治疗靶点，由于缺乏特异性阻断剂，其在心脏中的作用的基本问题尚不清楚，因此无法直接测试其作为drg靶点的效用。特异性目标3寻求对Kv2.1通道特异的从头毒素。Kv2.1抑制以SUMO依赖性方式增强葡萄糖刺激的胰岛素从胰腺β细胞分泌，并且寻找不交叉抑制的抑制剂作为T2 D的潜在药物。Kv1.5和Kv2.1的从头毒素不仅可用于验证通道作为药物靶标，而且可作为治疗先导物提供潜力。因此，用作药物的小的、二硫键稳定的肽毒素（Byetta（r）、Integrilin（r）和Prialt（r））已被证明是有效的、抗蛋白酶的和非免疫原性的。