Efficacious Screening of Peptidic Natural Products Using Drosophila

描述（由申请人提供）：该R21提案旨在开发基于果蝇的生物测定引导分离，以发现靶向中枢神经系统的肽类天然产物。我们将筛选两种圆锥蜗牛（Conus regius和Conus brunneus）毒液中的部分成分，作为开发基于果蝇的检测方法的样本案例。锥螺毒液的成分引起了广泛的神经生理反应，并显示出作为神经元探针的巨大潜力。毒液的生化多样性是这些动物在海洋环境中有利地竞争猎物所使用的神经化学策略的一部分。C. regius和C. brunneus的毒液是这种分子多样性的经典例子，特别适合于发现，因为它们是相关物种，对于其中的一种（C. regius），我们已经从生物化学的基础上分离出了一个重要的新型concontoxin文库。这个项目的主要贡献将是研究基于果蝇的筛选对发现新的conotoxins的适用性。这种方法可以应用于发现其他供应有限的天然产品，可以引起神经调节作用。这将在一个多学科的环境中完成，项目范围从海洋科学到果蝇神经生物学，由Mari和Godenschwege实验室共同努力。这些筛选可以提供一套新颖的神经元探针或具有独特有利药性的药理学药物。作为我们研究项目探索/发展阶段的一部分，在R21项目目标范围内，我们寻求资金来开展这些毒液的生物筛选技术的开发，同时，我们准备发现新的concontoxins，评估它们的生物靶点，并评估它们作为神经元探针或药物先导的潜在应用。具体而言，我们拟实现以下目标：1)利用电生理测量对黑腹果蝇（Drosophila melanogaster）的C. brunneus（一种东太平洋捕虫锥螺）毒液进行体内功能筛选。在筛选过程中观察到的特定神经调节作用可以帮助确定其特定的分子靶标类型2)我们将使用各种生物分析技术来分离和确定筛选过程中显示生物活性的化合物的初级结构3)编译基于果蝇的concontoxin活性成分文库并评估其作为神经生物学探针的潜力。一旦这个文库被编译，结构-活性关系可以用来确定这些化合物的精确生物学作用。公共卫生相关性：多肽天然产物是强大的神经药理学制剂，可用于各种各样的应用。锥螺是一种掠食性海洋软体动物，它的毒液是一种极其多产的天然肽类产品。我们的实验室（致力于研究锥体蜗牛的毒液）已经与Godenschwege实验室联手开发新的方法，这将增强我们发现新型肽类天然产物的能力。更具体地说，我们将对微小的果蝇进行快速电生理测量，这些果蝇消耗的珍贵物质从它们的自然来源中分离出来的量最少。我们选择了两种相关的锥螺物种的毒液作为开发这些基于果蝇的方法的测试案例。我们将从这些筛选中分子鉴定生物活性成分，并将建立结构-活性关系，这将导致进一步的研究，如基于哺乳动物的分析。我们在开发“药物发现”的巨大潜力方面处于独特的地位，因为我们已经研究出了这些蜗牛毒液的生化成分，并且可以绘制出活性和结构之间的直接相关性。我们的最终目标是增加目前已知活性的贝壳毒素库，以评估它们作为治疗剂的潜在用途。

DESCRIPTION (provided by applicant): This R21 proposal is aimed at the development of a Drosophila-based bioassay-guided fractionation for the discovery of peptidic natural products that target the CNS. We will screen fractions from the venom of two cone snail species, Conus regius and Conus brunneus, as sample cases for development of the Drosophila-based assay. Components of the venom of cone snails elicit a wide range of neurophysiological responses and show great potential as neuronal probes. The biochemical diversity of the venom is part of a neurochemical strategy used by these animals to compete favorably for prey in the marine environment. The venom of C. regius and C. brunneus are classical examples of this molecular diversity and are particularly suited for discovery, as they are related species, and for one of them (C. regius) we have already produced a significant library of novel conotoxins from biochemical-based fractionations. The main contribution from this project would be to investigate the suitability of Drosophila-based screenings for the discovery of novel conotoxins. This methodology can be applied to the discovery of other supply-limited natural products that can elicit neuromodulatory effects. This is to be done in a multidisciplinary environment for a project that ranges from marine science to Drosophila neurobiology by the collaborative efforts of the Mari and Godenschwege laboratories. These screenings can provide a novel set of neuronal probes or pharmacological agents with unique favorable medicinal properties. As part of the exploratory/developmental phase of our research program and within the R21 program objetives, we seek funding to carry out the development of bioscreening techniques of these venoms and at the same time, we are poised to discover novel conotoxins, assess their biological targets, and evaluate their potential applications as neuronal probes or drug leads. Specifically, we propose to accomplish the following goals: 1) To carry out in vivo functional screening of fractions of the venom of C. brunneus (an Eastern Pacific worm-hunting cone snail species) using electrophysiological measurements on Drosophila melanogaster. The specific neuromodulatory effects observed during the screenings can help to define their specific molecular target type 2) We will use an assortment of bioanalytical techniques to isolate and determine the primary structure of the compounds that show bioactivity during the screenings 3) To compile a Drosophila-based conotoxin library of active components and evaluate their potential as probes for neurobiology applications. Once this library is compiled, structure-activity relationships can be used to established the precise biological role of these compounds. PUBLIC HEALTH RELEVANCE: Peptidic natural products are powerful neuropharmacological agents that can be used for a wide variety of applications. The venom of cone snails, a genus of predatory marine mollusks, are an extremely prolific source of peptidic natural products. Our laboratory (dedicated to study the venom of cone snails) has join forces with the Godenschwege lab to develop new methodologies that will enhance our ability to discover new classes of peptidic natural products. More specifically, we will carry out rapid electrophysiological measurements in tiny Drosophila that consume minimal amounts of precious material isolated from their natural sources. We have chosen the venom of two related cone snail species as a test case for the development of these Drosophila-based methods. We will molecularly identify the bioactive components from these screens and will establish structure-activity relationships that will lead to further studies, such as mammalian-based assays. We are uniquely poised to develop this great potential for "Drug Discovery" as we have already worked-up the biochemical composition of the venom of these snails and immediate correlations between activity and structure can be drawn. Our ultimate goal is to augment the current library of conotoxins with known activity in order to evaluated their potential use as therapeutic agents.