Bursicon Receptor Antagonists: Templates for Developing Novel Insecticides

描述(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。在抗药性日益增强的LHT中，迫切需要寻找替代杀虫剂的靶点。潜在的候选者包括G蛋白偶联受体(GPCRs)，这是一种已知的高度“可下药”的蛋白质。这项提议的重点是确定能够阻断滑囊素受体的化合物，滑囊素受体是一种对昆虫生存至关重要的GPCR。之前使用果蝇和甲虫的研究表明，滑囊素介导的信号的遗传下调会导致角质层有缺陷的硬化，抑制翅膀扩张，并损害生存能力。基于这些发现，我们推测小分子囊素受体拮抗剂将概括这些表型，从而为开发一类新的杀虫剂提供模板结构。我们的研究将利用果蝇，这是一个已被证明在揭示与昆虫疾病媒介相关的分子机制方面非常有用的模型系统。鉴于果蝇工具(RNAi Fly，克隆的cDNA)的广泛收集，以及实验室维护的简便性，果蝇为拟议的调查提供了一个实际的起点。在目标1中，我们将与BIPDeC合作，实施一种经过验证的高通量筛选，以识别小分子滑囊素受体拮抗剂。对2000种化合物进行的中试筛选显示，我们的检测方法是高度可靠、灵敏的，并为高温超导做好了准备。在目标2中，将使用一系列先前验证的二级/三级化验来评估“HITS”的药理学特征。这些措施包括对不相关的G？S偶联受体进行计数器筛选，使用另一种活性指标(直接测量cAMP)评估配基功能，以及评估假定的拮抗剂的效力和疗效。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)。能够跟随翅膀扩张作为滑囊素受体阻断的视觉指标提供了独特的优势，这将有助于加速体内有效的拮抗剂的开发。在目标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。未来的努力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.