Sodium Channel Contratoxins Derived from Cone Snail Venoms

描述(申请人提供)：针对离子通道的神经毒素是NIH确定的与神经科学研究和反恐相关的化学物质之一。这方面的典型例子是生物碱河豚毒素(TTX)和岩藻毒素(SIX)，这两种有效的麻风剂都通过结合到同一部位并阻断电压门控钠通道的功能而起作用。我们建议开发一种概念上新颖的这些和相关神经毒素的拮抗剂。这一概念由反毒素一词概括，其定义如下：一种结合毒素目标并保护目标免受毒素伤害的制剂。为了提供概念验证的抗毒素数据，我们将利用椰子毒素，一种由有毒的锥形蜗牛产生的基于多肽的神经毒素。它们对单个离子通道或受体的精致效力和选择性已在许多情况下显示出来。在这个探索性的方案中，我们的实验策略包括三个平行的方法来获得针对TTX/STX的抗毒素：(1)合理的设计，(2)分子生物学/化学合成和(3)筛选天然产物文库。在每个目标中，候选抗毒素将被测试它们一方面不能阻断钠通道，另一方面它们能阻止TTX/STX的作用。在具体的目标一中，我们将合理地设计基于芋螺毒素GIIIA的类似物，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结合部位，但使麻痹活性失效。此外，我们建议在通过分子克隆或/和从无脊椎动物狩猎锥螺的分级毒液成分中发现的精选椰螺毒素中筛选出抗毒素活性(分别为特异性AIMS II和特异性AIIS III)。我们的长期目标是开发抗毒素作为新的工具，将在多个领域使用，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。这项研究也有望为其他抗毒素的研究指明道路，如针对神经元钠通道上的TTX/STX以及针对其他离子通道靶标的其他神经毒素。

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.