Rapid development of in vivo models for countermeasure discovery: organophosphate

描述(由申请人提供)： 化学对策发现中的一个主要问题是可能会出现新的制剂，而目前还没有已知的解毒剂或暴露后治疗方法。经典的对策往往只是偶然发现的，或者需要数年时间才能开发出来。随着新的化学威胁的出现(以新的化学战剂或环境污染物的形式出现)，我们能够理解和对抗每一种威胁的速度将决定其社会影响的程度。一种有希望的方法是使用基于表型的化学筛选来快速识别和操纵潜在的对任何化学威胁的反应的分子途径。可以开发特定毒物在细胞或整个生物体中产生可复制的表型的模型。通过对这些模型进行高通量筛选(HTS)，可以通过各种新的机制识别出逆转表型的小分子。这些筛查发现的小分子将是新对策的极佳先导化合物，也是剖析毒性途径的强大工具，这反过来可能指向更多的对策靶点。在这一应用中，我们概述了一个快速开发体内有机磷毒性模型的过程，以及它们在发现新的有机磷对策方面的应用。虽然我们预计拟议的项目将直接导致开发新的有机磷对策，但我们也希望它成为快速制定对策的典范，可以广泛应用于其他现有的和新出现的化学威胁。具体地说，我们提出：目的1.建立和验证斑马鱼体内有机磷毒性的检测方法。我们将寻求识别生理反应，这些反应可以替代已知的人类对有机磷的反应，并可以在活体筛选中进行高通量筛选。目的2.将这些新的分析方法应用于多孔板的自动化高通量筛选。我们将开发可在96孔格式下自动执行的有机磷毒性检测方法。目的3.确定新的化合物，以抵消先前有机磷暴露的影响。高通量筛选将确定有助于从有机磷接触中恢复的化合物

DESCRIPTION (provided by applicant): A major problem in chemical countermeasure discovery is the potential emergence of novel agents for which there are no known antidotes or post exposure therapies. Classic countermeasures have often been discovered only serendipitously or have taken years to develop. As novel chemical threats emerge (in the form of novel chemical warfare agents or environmental pollutants), the speed with which we are able to understand and counteract each threat will determine the magnitude of its societal impact. One promising approach for rapidly identifying and manipulating the molecular pathways underlying the response to any chemical threat is the use of phenotype-based chemical screens. Models can be developed in which specific toxicants result in reproducible phenotypes in cells or whole organisms. By subjecting these models to high- throughput screening (HTS), small molecules could be identified that reverse the phenotype through a variety of novel mechanisms. Small molecules discovered by these screens would be excellent lead compounds for novel countermeasures and powerful tools for dissecting toxicity pathways, which may in turn point to additional countermeasure targets. In this application, we outline a process for rapid development of in vivo organophosphate toxicity models and their use in discovery of novel organophosphate countermeasures. Although we expect the proposed project will lead directly to development of novel organophosphate countermeasures, we also expect it to serve as a model for rapid countermeasure development that can be applied broadly to other existing and emerging chemical threats. Specifically, we propose: Aim 1. To develop and validate in vivo assays for organophosphate toxicity in the zebrafish. We will seek to identify physiological responses that are surrogates for known human responses to organophosphates and that can be scaled for high throughput in vivo screening. Aim 2. To scale these novel assays for automated high-throughput screening in multiwell plates. We will develop assays for organophosphate toxicity that can be performed automatically in 96-well format. Aim 3. To identify novel compounds that counteract the effects of prior organophosphate exposure. High-throughput screening will identify compounds that facilitate recovery from organophosphate exposure