Discovery of Adenosine Receptor Allosteric Modulators for Cardiovascular Disease and Inflammation

项目摘要 心血管疾病是美国男性和女性死亡的主要原因， 3512亿美元，每年造成84万多人死亡。A3腺苷受体（A3 AR）是一种腺苷受体。 Gi蛋白偶联受体，在包括中性粒细胞在内的几种类型的炎性细胞中高度表达。 组织损伤/炎症后腺苷水平的增加导致A3 AR信号传导的激活，这有助于 以限制炎症并促进修复，部分是通过削弱免疫细胞的趋化性和活化。激动剂 A3 AR的表达正在被研究用于多种炎性疾病，包括心肌缺血和 化疗引起的心脏毒性。不幸的是，剂量限制性副作用已在早期报告 临床试验潜在地限制了它们的有用性。增强内源性配体信号传导的化合物， 称为正变构调节剂（PAM），允许时空特异性和降低的脱靶效应。 预计靶向A3 AR的PAM将提供一种更好的上级治疗方法。在这些拟议的研究中， 我的目标是表征和进一步开发A3 AR PAM，其将作为小分子探针和有用的治疗剂，用于治疗以下疾病： 心脏损伤和炎性疾病。先前的构效关系研究（SAR）确定了1H- 咪唑并[4，5]喹啉-4-胺，LUF 6000和2，4-二取代的喹啉，LUF 6096， 在A3 AR处的活性，其中这些调节剂将正构激动剂功效提高2倍以上。 不幸的是，它们也具有降低激动剂效力的不希望的趋势。此外， 迄今为止，我们研究的PAM表现出对啮齿动物受体的PAM活性，使我们无法评估 在临床前啮齿动物疾病模型中的生物活性。在目标1中，我将通过以下方式扩展先前的SAR研究： 基于LUF 6000和LUF 6096的结构表征两个新系列的衍生物，目标是 鉴定双重增强正构激动剂功效和效力的改进的A3 AR PAM。作为其中的一部分 目的，我将研究我们的PAM配体是否支持偏倚（G蛋白依赖vs G蛋白非依赖） 信号传导并评估相对于小鼠A3 AR的活性。在目标2中，我将利用物种差异并生成 人/小鼠嵌合和突变A3 AR，以促进LUF 6000的结合口袋的鉴定， LUF6096。最后，在目标3中，我将研究LUF 6000和LUF 6096对两个关键细胞的生物学效应。 中性粒细胞功能-超氧化物产生和趋化性-利用人嗜中性粒细胞细胞系（HL 60细胞）。 在完成这些拟议的研究后，在我的导师的指导下，一个多元化的合作者团队， 和我的论文委员会，我将获得经验，实验设计，执行生化分析， 分子克隆，以及合理药物设计的基础知识，这将产生可靠的数据，并为我准备 成功竞争一家生物制药公司的研究科学家职位。完成本 这项工作将促进我们对A3 AR变构药理学的理解，并可能导致开发 用于治疗缺血性心脏病和其他炎症性疾病的新疗法。

PROJECT SUMMARY Cardiovascular disease is the leading cause of death in the United States for both men and women, costing $351.2 billion dollars and accounting for over 840,000 deaths annually. The A3 adenosine receptor (A3AR) is a Gi protein-coupled receptor that is highly expressed in several types of inflammatory cells, including neutrophils. Increased adenosine levels following tissue injury/inflammation leads to activation of A3AR signaling, which aids to limit inflammation and to promote repair, in part by impairing immune cell chemotaxis and activation. Agonists of the A3AR are being investigated for a multitude of inflammatory diseases including cardiac ischemia and chemotherapy-induced cardiotoxicity. Unfortunately, dose-limiting side effects have been reported in early clinical trials potentially limiting their usefulness. Compounds that potentiate signaling of endogenous ligands, termed positive allosteric modulators (PAMs), allow for spatiotemporal specificity, and reduced off-target effects. It is anticipated that PAMs targeting the A3AR will offer a superior treatment approach. In these proposed studies, I aim to characterize and further develop A3AR PAMs that will serve as small molecule probes and useful therapeutics for cardiac injury and inflammatory disorders. Prior structure-activity-relationship studies (SAR) identified the 1H- imidazo[4,5]quinoline-4-amine, LUF6000, and the 2,4-disubstituted quinoline, LUF6096, as exhibiting PAM activity at the A3AR, where these modulators enhance orthosteric agonist efficacy upwards of 2-fold. Unfortunately, they also have the undesirable tendency to decrease agonist potency. In addition, none of the PAMs we have investigated thus far exhibit PAM activity versus rodent receptors, preventing us from assessing biological activity in preclinical rodent models of disease. In Aim 1, I will expand on prior SAR studies by characterizing two new series of derivatives, based off the structures of LUF6000 and LUF6096 with the goal of identifying improved A3AR PAMs that dually enhance orthosteric agonist efficacy and potency. As part of this aim, I will investigate whether our PAM ligands support biased (G protein-dependent vs G protein-independent) signaling and assess for activity versus the mouse A3AR. In Aim 2, I will exploit species differences and generate human/mouse chimeric and mutant A3ARs to facilitate identification of the binding pocket for LUF6000 and LUF6096. Lastly, in Aim 3 I will investigate the biological effects of LUF6000 and LUF6096 on two critical neutrophil functions - superoxide production and chemotaxis - utilizing a human neutrophilic cell line (HL60 cells). Upon completion of these proposed studies, under the guidance of my mentor, a diverse team of collaborators, and my dissertation committee, I will gain experience in experimental design, execution of biochemical assays, molecular cloning, and basics of rational drug design, which will result in publishable data and prepare me to successfully compete for a position as a research scientist at a biopharmaceutical company. Completion of this work will advance our understanding of A3AR allosteric pharmacology and potentially lead to the development of a new therapeutic for the treatment of ischemic heart disease and other inflammatory diseases.