Sodium Channel Contratoxins Derived from Cone Snail Venoms

描述（由申请人提供）：靶向离子通道的神经毒素是NIH确定的神经科学研究和反恐联系的化学物质之一。这方面的典型例子是生物碱河豚毒素（TTX）和蛤蚌毒素（SIX），这两种有效的麻痹剂都是通过结合到相同的位点上并阻断电压门控钠通道的功能而起作用的。我们建议开发一种概念新颖的这些和相关神经毒素的拮抗剂。这个概念可以用术语“抗毒素”来概括，定义如下：一种结合毒素目标并保护目标免受毒素侵害的物质。为了提供概念验证的对照毒素数据，我们将利用螺毒素，一种由有毒的锥体蜗牛产生的基于肽的神经毒素。它们对单个离子通道或受体的优良效力和选择性已在许多实例中得到证明。在本探索性提案中，我们的实验策略包括三种平行的方法来获得针对TTX/STX的对照毒素：(1)合理设计，(2)分子生物学/化学合成和(3)筛选天然产物库。在每个目标中，候选的对照毒素将被测试它们一方面无法阻断钠通道，另一方面能够阻断TTX/STX的作用。在Specific Aim I中，我们将合理设计基于giia的concontoxin类似物，然后进行化学合成和功能评价。这些所谓的“泄漏”类似物将针对钠通道上的TTX/STX结合位点，但没有麻痹活性。此外，我们建议筛选通过分子克隆或/或从无脊椎狩猎锥螺的分离毒液成分中发现的选定的螺毒素中的抗毒素活性（分别为特定目的II和III）。我们的长期目标是开发对抗毒素作为跨多个领域的新工具，包括神经科学，神经药理学和针对神经毒性药物的医学对策。该研究也有望为研究其他抗毒素指明道路，例如针对TTX/STX的神经元钠通道以及针对其他离子通道靶点的其他神经毒素的研究。

DESCRIPTION (provided by applicant): Neurotoxins that target ion channels are among the chemicals that NIH has identified as at the nexus of neuroscience research and counterterrorism. Archetypal examples in this regard are the alkaloids tetrodotoxin (TTX) and saxitoxin (SIX), both potent paralytic agents that act by binding to the same site on, and blocking the function of voltage-gated sodium channels. We propose to develop a conceptually novel antagonist of these and related neurotoxins. The concept is encapsulated by the term contratoxin, defined as follows: an agent that binds the target of a toxin and shields the target from the toxin. To provide proof-of-concept contratoxin data, we will utilize conotoxins, peptide-based neurotoxins produced by venomous cone snails. Their exquisite potency and selectivity toward individual ion channels or receptors have been shown in many instances. In this exploratory proposal, our experimental strategy consists of three parallel approaches to obtain contratoxins against TTX/STX: (1) rational design, (2) molecular biology/chemical synthesis and (3) screening natural product libraries. In each aim, candidate contratoxins will be tested for their inability to block sodium channels on one hand and their ability to block the action of TTX/STX on the other. In the Specific Aim I, we will rationally design conotoxin GIIIA-based analogs, followed by a chemical synthesis and functional evaluation. These so-called "leaky" analogs will target the TTX/STX binding site on sodium channels, but void paralytic activity. In addition, we propose to screen for a contratoxin activity among selected conotoxins discovered by molecular cloning or/and from fractionated venom components of invertebrate-hunting cone snails (Specific Aims II and III, respectively). Our long- term goal is to develop contratoxins as novel tools that will be utilized across multiple fields, including neuroscience, neuropharmacology and medical countermeasures against neurotoxic agents. This research is also expected to illuminate the way for investigations of other contratoxins, such as those against TTX/STX on neuronal sodium channels as well as against other neurotoxins with other ion channel targets.