Advancing mGlu1 positive allosteric modulators as therapeutics to facilitate abstinence in cocaine use disorder

项目摘要 治疗可卡因使用障碍的一个主要挑战是对线索诱导的渴望和复发的脆弱性持续存在 即使在长期禁欲之后也是如此。目前还没有FDA批准的药物来减轻可卡因的渴望。 Eleutheria PharmPharmticals LLC的目标是开发代谢性谷氨酸受体1(MGlu1)阳性 变构调节剂(PAM)作为治疗药物，减少饥饿感，从而促进戒断 有可卡因使用障碍的人。目标mGlu1已经由PI和 公司创办人，并通过其他工作。我们的研究采用了药物渴求的孵化模型，在该模型中 在禁欲期间，线索诱导的大鼠的渴求逐渐加剧，然后保持在较高的水平 月份。人类也会产生渴望的潜伏期。我们表明，孵化依赖于加强 在药物渴求的关键脑区(伏隔核)通过掺入非典型高电荷量而形成的谷氨酸突触 电导型AMPA型谷氨酸受体(CP-AMPAR)MGlu1 PAM通过诱导 长期抑郁(LTD)的形式，由CP-AMPAR内化表达，并通过这种mGlu1- 有限公司，减少对可卡因的渴望。2020年，PI获得了NIDA U18拨款，将我们的项目带到了Assay 发展阶段。在这个第一阶段的STTR应用中，我们提出了两个目标，这将导致识别 新的化学类型，并使我们能够申请第二阶段的STTR资金。MGlu1的经典体外筛选 激活依赖于检测细胞系中的钙离子动员。我们已经开发了一种新的检测方法来选择性地检测 激活mGlu1下游介导渴求减少的信号通路，即快速 LTD蛋白OPHN1的翻译。简单地说，HiBiT标记的OPHN1(稳定地转染到 MGlu1过表达细胞系)在mGlu1刺激时产生发光信号。目标1将优化 该方法可用于AIM 2的高通量筛选。 基准信号以实现最佳信号窗口，Z‘≥0.6作为成功的基准。重现性在 规模将在1430种独特化合物的试点筛选中进行评估，这些化合物一次测试三份。在……里面 目的2、将优化后的方法用于4382生物活性成分的高通量初筛 俄勒冈州立大学高通量筛选服务实验室的化合物。来自此屏幕的点击 与我们的mGlu1 PAM工具化合物相似的其他化合物将从新鲜的剂量来源 曲线和细胞毒性评估，并进行药物化学优先排序。如果我们在 实现我们的第一阶段目标(优化和执行主屏幕，识别和验证Hit支架)，我们 将提交第二阶段申请，以评估二次分析中优先考虑的化合物，优化其活性 和PK特性，并进行体内测试(渴望的孵化模型)以 确定先导化合物。为了实现这些目标，公安局组建了一支拥有毒品专业知识的强大团队 筛查、药物化学、业务发展和物质使用障碍患者的需求。

Project Summary A major challenge in treating cocaine use disorder is that vulnerability to cue-induced craving and relapse persist even after long periods of abstinence. There is presently no FDA-approved medication to lessen cocaine craving. The goal of Eleutheria Pharmaceuticals LLC is to develop metabotropic glutamate receptor 1 (mGlu1) positive allosteric modulators (PAMs) as therapeutic agents to reduce craving and thereby facilitate abstinence in persons with cocaine use disorder. The target, mGlu1, has been thoroughly validated by the lab of the PI and Company Founder, and by other work. Our studies have used the ‘incubation of drug craving’ model, in which cue-induced craving in rats progressively intensifies (‘incubates’) during abstinence and then remains high for months. Incubation of craving also occurs in humans. We showed that incubation depends on strengthening of glutamate synapses in a key brain region for drug craving (nucleus accumbens) via incorporation of atypical high conductance AMPA-type glutamate receptors (CP-AMPARs). mGlu1 PAMs reverse this plasticity by eliciting a form of long-term depression (LTD) that is expressed by CP-AMPAR internalization and, through this mGlu1- LTD, reduce cocaine craving. In 2020, the PI obtained a NIDA U18 grant that has taken our project to the Assay Development stage. In this Phase I STTR application, we propose two Aims that will result in the identification of novel chemotypes and position us to apply for Phase II STTR funding. Classic in vitro screens for mGlu1 activation rely on detecting Ca2+ mobilization in cell lines. We have developed a novel assay to selectively detect activation of the signaling pathway downstream of mGlu1 that mediates craving reduction, namely the rapid translation of the LTD protein oligophrenin-1 (OPHN1). Briefly, HiBiT-tagged OPHN1 (stably transfected into an mGlu1 over-expressing cell line) generates a luminescent signal upon mGlu1 stimulation. Aim 1 will optimize this assay so it can be used in high throughput screening in Aim 2. The assay will be optimized by reduction of baseline signal to achieve an optimal signal window and Z’ ≥ 0.6 as a benchmark for success. Reproducibility at scale will be evaluated during a pilot screen of 1430 unique compounds tested in triplicate at a single dose. In Aim 2, the optimized assay will be used for a quantitative high throughput primary screen of 4382 bioactive compounds at Oregon State University’s High-Throughput Screening Services Laboratory. Hits from this screen and additional compounds chosen for similarity to our mGlu1 PAM tool compound will be sourced fresh for dose curves and cytotoxicity assessment, and medicinal chemistry prioritization conducted. If we are successful in achieving our Phase I goals (optimize and execute the primary screen, identify and validate hit scaffolds), we will submit a Phase II application to assess prioritized compounds in secondary assays, optimize their activity and PK properties during iterative SAR studies, and conduct in vivo testing (‘incubation of craving’ model) to identify lead compounds. To achieve these goals, the PI has assembled a strong team with expertise in drug screening, medicinal chemistry, business development, and the needs of substance use disorder patients.