De novo protein neurotoxins for ion channels

 描述(申请人提供)：蛋白质神经毒素是研究膜受体的最强大的工具之一，现在也用于疾病的治疗。这笔赠款寻求，首先，通过靶向缺乏这些有用配体的钾和质子通道，推动我们创造从头毒素的方法；其次，利用从头毒素来评估Kv1.5和Kv2.1通道分别作为治疗两种具有广泛公共影响的疾病--房颤(AF)和2型糖尿病(T2D)--的潜力。在这两个方面的成功将使这些工具能够更广泛地用于为其他目标产生毒素，并推动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。到目前为止，毒素主要来自有限的来源：粗毒、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公认的、特异的、高亲和力的配体。这种情况很容易理解：无论是野生毒素的目的还是毒液的筛选都不利于靶标特异性。在这里，通过使用我们开发的一种新的高通量方法来根据所需的属性克隆毒素来避免这些问题，该方法可以产生Kv1.3通道孔的选择性抑制物。特定目的1试图通过在结合到离子通道毛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，然后使用细胞表面的通道，以便许多在纯化时不保留其结构的受体也可以成为靶向。最近发现的质子通道(Hv1)被用来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中的假定作用)。特定的目的是寻找针对Kv1.5钾通道的从头毒素。这种孤儿受体被广泛用作房颤的潜在治疗靶点，缺乏特定的阻滞剂使得它在心脏中的作用留下了不清楚的基本问题，并排除了对其作为DRG靶点的有效性的直接测试。特异性目标3寻找Kv2.1通道特异性的从头毒素。Kv2.1抑制以相扑依赖的方式增强葡萄糖刺激的胰腺细胞的胰岛素分泌，并且寻找不交叉抑制的抑制剂作为治疗T2D的潜在药物。Kv1.5和Kv2.1的从头毒素不仅用于验证作为药物靶点的通道，而且提供潜在的治疗线索。因此，作为药物使用的二硫键稳定的小分子多肽毒素(BYETA(R)、Integrlin(R)和Prialt(R))已被证明是有效的，对蛋白酶具有抵抗力，并且不具有免疫原性。

 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.