Identification of Cyclic Peptide Antagonists of an Anti-Opioid G Protein-Coupled Receptor

抗阿片G蛋白偶联受体环肽拮抗剂的鉴定 RFA-DA-19-019 R43第一阶段SBIR 少年派：希德·A·拉贝德 项目摘要 阿片类药物的高度成瘾特性，再加上常规的过度处方，导致了 国家危机。在过去的15年里，阿片类药物意外过量增加了400%以上，从大约8,000个阿片类药物- 2015年与吸毒过量有关的人数超过3万人。阿片类药物健康危机给美国人带来的惊人代价 公众每年超过500亿美元，突显了疼痛管理需要更安全的治疗方法。基里尔博士 佛罗里达州斯克里普斯研究所的Martemyanov和Brock Grill设计了一个模型系统，表达了 在线虫模型线虫中发现功能性哺乳动物阿片受体(MOR) 阿片调节剂。使用这个系统，他们发现一个孤立的G蛋白偶联受体GPR139是一种 MOR信号的负调节因子。在哺乳动物中，GPR139与MOR在阿片类药物敏感的脑中共同表达 地区和影响铁道部贩运和信令。增强阿片类药物诱导的小鼠GPR139缺失 抑制神经元放电，增加吗啡的止痛和奖赏作用，并减少戒断。 以前针对GPR139的努力主要集中在识别小分子激动剂上， 未能产生具有良好药理特性的治疗候选拮抗剂。致信地址 这一未得到满足的需求，EvoDenovo将为新的GPR139筛选一个创新的环肽文库(CP) 通过结合EvoDenovo独有的两项最先进的线虫技术来对抗对手：1)InVivo Display：EvoDenovo发明的高通量筛选技术，可用于基于多肽的筛选 通过直接饲喂活的重组大肠杆菌绕过多肽纯化的药物发现 克隆，每个表达不同的环肽，直接到表达哺乳动物MOR和 人类GP139。这大大减少了筛查的成本和时间。2)非阿片类药物行为平台 由Martemyanov和Gril博士在斯克里普斯开发，用于评估阿片类药物的行为影响并识别 不同药物的药理结果。两个平台的组合，由自动化的C。 优雅的移动跟踪系统，将产生一个强大的高通量筛选引擎，能够 在几天内审问数千个重组多肽，所有这些都在一个独特的体内设置中。GPR139 用这种方法鉴定出的拮抗剂很可能会被常规的筛选方法遗漏。EvoDenovo 使用CPS而不是线性多肽。多肽的环化通过消除易损性来增加肠道的稳定性 N-和C-末端，降低了对蛋白水解性的敏感性，并提高了膜的通透性。 有两个具体目标：目标1：对GPR139的环肽拮抗剂进行行为筛查 线虫。目的2：在哺乳动物细胞检测中验证线虫平台产生的HITS。这 第一阶段提案将为第二阶段SBIR奠定基础，该阶段将包括更大尺寸的屏幕和 对CP命中、哺乳动物测试和IND使能研究进行优先排序的后续行动。 EvoDenovo RFA-DA-19-019/PI：SID A.Labed项目摘要-第1页，共1页

Identification of Cyclic Peptide Antagonists of an Anti-Opioid G Protein-Coupled Receptor RFA-DA-19-019 R43 Phase I SBIR PI: Sid A. labed Project Summary The highly addictive properties of opioid drugs, coupled with routine over-prescription, have resulted in a national crisis. Accidental overdose by opioids has increased over 400% in the last 15 years, from ~8,000 opioid- related overdoses to more than 30,000 in 2015. The staggering cost of the opioid health crisis to the American public exceeds $50 billion annually, highlighting the need for safer therapeutics for pain management. Drs. Kirill Martemyanov and Brock Grill at the Scripps Institute, Florida, have engineered a model system expressing a functional mammalian opioid receptor (MOR) in the model nematode Caenorhabditis elegans for discovery of opioid modulators. Using this system, they identified an orphan G Protein Coupled Receptor, GPR139, as a negative regulator of MOR signaling. In mammals, GPR139 is co-expressed with MOR in opioid-sensitive brain regions and influences MOR trafficking and signaling. Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing, increased the analgesic and rewarding effects of morphine, and reduced withdrawal. Previous efforts to target GPR139, which have largely focused on identification of small molecule agonists, has failed to produce a therapeutic candidate antagonist with favorable pharmacological properties. To address this unmet need, EvoDenovo will screen an innovative library of cyclic peptides (CPs) for new GPR139 antagonists by combining two state-of-the-art C. elegans technologies exclusive to EvoDenovo: 1) InVivo Display: a high-throughput screening technology invented by EvoDenovo that can be used for peptide-based drug discovery that bypasses the necessity of peptide purification by directly feeding live recombinant E. coli clones, each expressing a different cyclic peptide, directly to nematodes expressing mammalian MOR and human GP139. This drastically reduces the cost and time for screening. 2) The anti-opioid behavior platform developed at Scripps by Drs. Martemyanov and Gril that assesses the behavioral effects of opioids and identifies pharmacological outcomes of different drugs. The combination of both platforms, assisted by an automated C. elegans movement tracking system, will yield a powerful high throughput screening engine capable of interrogating thousands of recombinant peptides within a few days, all in a unique in vivo setup. GPR139 antagonists identified using this assay would likely be missed by conventional screening protocols. EvoDenovo uses CPs instead of linear peptides. The cyclization of peptides increases gut stability by eliminating vulnerable N- and C-termini, reduces susceptibility to proteolytic hydrolysis, and enhances membrane permeability. There are 2 specific aims: Aim 1: Perform a behavioral screen for cyclic peptide antagonists of GPR139 in C. elegans. Aim 2: Validate hits generated from the C. elegans platform in mammalian cell-based assays. This Phase I proposal will provide the foundation for a Phase II SBIR which will include a larger-scale screen and follow-up on prioritizing CP hits, mammalian testing, and IND enabling studies. EvoDenovo RFA-DA-19-019/ PI: Sid A. Labed Project Summary - Page 1 of 1