Bursicon Receptor Antagonists: Templates for Developing Novel Insecticides

描述（由申请方提供）：目前用于控制疾病传播昆虫媒介种群的杀虫剂作用于一组高度受限的靶标。随着抗药性的增加，迫切需要确定替代杀虫剂的靶标。潜在的候选者包括G蛋白偶联受体（GPCR），其已知是高度“可药物化”的蛋白质。该提案的重点是确定阻断囊体激素受体的化合物，囊体激素受体是昆虫生存所必需的。先前使用果蝇和甲虫的研究表明，囊子介导的信号转导的遗传下调导致角质层的缺陷硬化，抑制翅膀扩张和受损的生存能力。基于这些发现，我们假设，小分子bursicon受体拮抗剂将重演这些表型，从而为一类新的杀虫剂的开发提供模板结构。我们的研究将利用果蝇，一个模式系统，已被证明是非常有用的，在揭示相关的昆虫疾病载体的分子机制。由于果蝇工具（RNAi果蝇，克隆cDNA）的广泛收集，以及易于实验室维护，果蝇为拟议的调查提供了一个实际的起点。在目标1中，与BIPDeC合作，我们将实施经验证的高通量筛选以鉴定小分子囊子受体拮抗剂。对2000种化合物进行了中试筛选，测量荧光素酶活性，作为与该GPCR相关的Gs介导的信号传导的读数，表明我们的检测方法非常稳健、灵敏，并已为HTS做好准备。在目标2中，将使用一系列先前验证的二级/三级试验评估“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，以及评估推定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）。作为囊子受体阻断的视觉指标的跟随翼扩张的能力提供了独特的优势，这将有助于加速体内有效的拮抗剂的开发。在目标3中，最有前途的化合物将在体外和体内测试的指导下进行结构优化。未来的工作将利用本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.