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 I期SBIR PI：Sid A. labed 项目摘要 阿片类药物的高度成瘾性，加上常规的过度处方，导致了 国家危机。在过去的15年里，阿片类药物的意外过量增加了400%以上，从大约8,000阿片类药物- 2015年有超过30，000人服用过量药物。阿片类药物健康危机给美国人带来的惊人成本 每年公共支出超过500亿美元，这凸显了对更安全的疼痛管理疗法的需求。基里尔博士 佛罗里达斯克里普斯研究所的Martemyanov和Brock Grill已经设计了一个模型系统， 功能性哺乳动物阿片受体（莫尔）在模式线虫秀丽隐杆线虫中的发现， 阿片类调节剂使用这个系统，他们确定了一个孤儿G蛋白偶联受体，GPR 139，作为一个孤儿。 莫尔信号的负调节因子。在哺乳动物中，GPR 139与莫尔在阿片敏感的脑中共表达 区域并影响莫尔运输和信号传导。小鼠中GPR 139的缺失增强阿片样物质诱导的 抑制神经元放电，增加吗啡的镇痛和奖励作用，并减少戒断。 以前针对GPR 139的努力主要集中在鉴定小分子激动剂， 未能产生具有有利药理学性质的治疗候选拮抗剂。解决 这一未满足的需求，EvoDenovo将筛选新的GPR 139的环肽（CP）的创新库 拮抗剂通过结合两种最先进的C. EvoDenovo独家的elegans技术：1）InVivo 显示：EvoDenovo发明的高通量筛选技术，可用于基于肽的 通过直接饲喂活的重组E.杆菌 将每个表达不同环肽的克隆直接与表达哺乳动物莫尔的线虫连接， 人GP 139。这大大降低了筛选的成本和时间。2)抗阿片类药物行为平台 由Martemyanov和Gril博士在Scripps开发，评估阿片类药物的行为影响，并确定 不同药物的药理学结果。这两个平台的结合，由一个自动化的C。 elegans运动跟踪系统，将产生一个强大的高通量筛选引擎，能够 在几天之内，我们就能对数千种重组肽进行分析，所有这些都是在一种独特的体内装置中完成的。GPR139 使用该测定鉴定的拮抗剂可能被常规筛选方案遗漏。埃沃德诺沃 使用CP而不是线性肽。肽的环化通过消除易受伤害的细胞而增加肠道稳定性。 N-和C-末端，降低对蛋白水解的敏感性，并增强膜渗透性。 有2个具体目的：目的1：在小鼠中进行GPR 139的环肽拮抗剂的行为筛选。 C.优雅目标2：从C. elegans平台在基于哺乳动物细胞的测定中的应用。这 第一阶段提案将为第二阶段SBIR奠定基础，该阶段将包括一个更大规模的屏幕， 跟进CP命中、哺乳动物试验和IND赋能研究的优先顺序。 EvoDenovo RFA-DA-19-019/ PI：Sid A。标签项目摘要-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