Efficacious Screening of Peptidic Natural Products Using Drosophila

描述(申请人提供)：这份R21提案旨在开发一种以果蝇为基础的生物测定引导的分级分离方法，用于发现以中枢神经系统为靶点的多肽天然产物。我们将从两种锥形蜗牛的毒液中筛选出组分，作为以果蝇为基础的分析的样本。锥螺毒液的成分可引起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。这种毒液的生物化学多样性是这些动物在海洋环境中有利地争夺猎物的神经化学策略的一部分。注册弧菌和布氏弧菌的毒液是这种分子多样性的经典例子，特别适合于发现，因为它们是近缘物种，对于其中一种(注册弧菌)，我们已经从基于生化的分级中产生了一个重要的新芋螺毒素文库。该项目的主要贡献将是调查以果蝇为基础的筛选对发现新的芋螺毒素的适用性。这种方法可以应用于发现其他供应有限的天然产品，这些产品可以引起神经调节作用。这将在一个从海洋科学到果蝇神经生物学的多学科环境中完成，这是由Mari和Godenchwege实验室共同努力完成的。这些筛选可以提供一套具有独特良好药用性能的新型神经元探针或药理制剂。作为我们研究计划的探索/开发阶段和R21计划目标的一部分，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，我们准备发现新的芋螺毒素，评估它们的生物靶标，并评估它们作为神经元探针或药物先导的潜在应用。具体地说，我们计划完成以下目标：1)利用对果蝇的电生理测量，对东太平洋捕虫锥螺物种布氏华支睾吸虫毒液的部分组分进行体内功能筛选。在筛选过程中观察到的特定神经调节作用有助于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在筛选过程中显示出生物活性的化合物的一级结构3)建立一个基于果蝇的芋螺毒素活性成分文库，并评估它们作为神经生物学应用探针的潜力。一旦这个文库被编译，结构-活性关系就可以用来确定这些化合物的确切生物学作用。公共卫生相关性：多肽类天然产品是强大的神经药理制剂，可用于广泛的应用。锥螺的毒液是捕食性海洋软体动物的一种，是多肽天然产物的极其丰富的来源。我们的实验室(致力于研究锥形蜗牛的毒液)已与Godenchwege实验室联手开发新的方法，以增强我们发现新类别多肽天然产品的能力。更具体地说，我们将在微小的果蝇身上进行快速电生理测量，这些测量消耗从自然来源中分离出来的最少量的珍贵材料。我们选择了两种相关锥形蜗牛的毒液作为测试案例，以开发这些基于果蝇的方法。我们将从分子上鉴定这些筛选的生物活性成分，并建立结构-活性关系，这将导致进一步的研究，例如基于哺乳动物的分析。由于我们已经研究了这些蜗牛毒液的生化成分，可以得出活性和结构之间的直接关联，因此我们正准备开发这一巨大的“药物发现”潜力。我们的最终目标是用已知的活性扩大目前的芋螺毒素资料库，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.