Bursicon Receptor Antagonists: Templates for Developing Novel Insecticides

描述（由申请人提供）：目前用于控制传播疾病的昆虫媒介种群的杀虫剂作用于一组高度受限的目标。鉴于日益增长的耐药性，迫切需要确定替代的杀虫剂靶点。潜在的候选药物包括G蛋白偶联受体（gpcr），这是一种已知的高度“可药物化”的蛋白质。本提案的重点是鉴定阻断囊体受体的化合物，囊体受体是昆虫生存所必需的一种GPCR。先前对果蝇和甲虫的研究表明，滑囊蛋白介导的信号传导基因下调会导致角质层硬化缺陷、翅膀扩张抑制和生存能力降低。基于这些发现，我们假设小分子滑囊受体拮抗剂将概括这些表型，从而为开发一类新型杀虫剂提供模板结构。我们的研究将利用果蝇这一已被证明在揭示昆虫病媒相关分子机制方面非常有用的模型系统。考虑到果蝇工具（RNAi蝇，克隆cdna）的广泛收集，以及实验室维护的便利性，果蝇为拟议的研究提供了一个实用的起点。在Aim 1中，我们将与BIPDeC合作，实施一种经过验证的高通量筛选，以鉴定小分子滑囊受体拮抗剂。2000种化合物的中试筛选测量荧光素酶活性，作为与该GPCR相关的G¿s介导信号的读数，表明我们的分析具有高度的鲁棒性，敏感性和可用于HTS。在Aim 2中，将使用一系列先前验证的二级/三级分析来评估“命中”的药理学特征。这些包括对不相关的G¿s偶联受体的计数器筛选，使用替代活性指数（直接测量cAMP）评估配体功能，以及评估假定的拮抗剂的效力和功效。最有希望的化合物将在体内进行测试，以确定它们对果蝇的影响（例如翅膀形态和生存能力）。跟随翅膀扩张的能力作为滑囊受体阻断的视觉指标提供了一个独特的优势，这将有助于加快在体内有效的拮抗剂的发展。在Aim 3中，最有希望的化合物将在体外和体内测试的指导下进行结构优化。未来的工作将利用该项目中确定的化学探针作为开发新型杀虫剂的模板。

DESCRIPTION (provided by applicant): The insecticides that are currently used to control populations of disease-transmitting insect vectors act on a highly restricted set of targets. In liht of growing drug resistance, there is an urgent need to identify alternative insecticide targets. Potential candidates include G protein-coupled receptors (GPCRs) which are known to be highly "druggable" proteins. The focus of this proposal is to identify compounds that block the bursicon receptor, a GPCR that is essential for insect survival. Prior studies using both Drosophila and beetles revealed that genetic down-regulation of bursicon-mediated signaling results in defective hardening of the cuticle, inhibition of wing expansion, and compromised viability. Based on these findings, we postulate that small-molecule bursicon receptor antagonists will recapitulate these phenotypes and thus provide template structures for the development of a novel class of insecticides. Our studies will utilize Drosophila, a model system which has proven highly useful in revealing molecular mechanisms relevant to insect disease vectors. Given the extensive collections of Drosophila tools (RNAi flies, cloned cDNAs), as well as the ease of laboratory maintenance, fruit flies offer a practical starting point for the propose investigations. In Aim 1, in collaboration with BIPDeC, we will implement a validated high-throughput screen to identify small molecule bursicon receptor antagonists. A pilot screen of 2000 compounds measuring luciferase activity as a read-out of G¿s-mediated signaling linked to this GPCR revealed that our assay is highly robust, sensitive and ready for HTS. In Aim 2, the pharmacological characteristics of "hits" will be assessed using a series of previously validated secondary/tertiary assays. These include a counter screen on an unrelated G¿s coupled receptor, assessment of ligand function using an alternative index of activity (direct measurement of cAMP), as well as evaluation of potency and efficacy of putative antagonists. The most promising compounds will be tested in vivo to determine their effects on Drosophila (e.g. wing morphology and viability). The ability to follow wing expansion as a visual index of bursicon receptor blockade provides a unique advantage which will help expedite the development of antagonists that are effective in vivo. In Aim 3, the most promising compounds will undergo structural optimization guided by both in vitro and in vivo testing. Future efforts wil utilize the chemical probes identified in this project as templates for the development of novel insecticides.