Discovery of Adenosine Receptor Allosteric Modulators for Cardiovascular Disease and Inflammation

项目总结 心血管疾病是美国男性和女性的主要死亡原因， 3512亿美元，每年造成超过840,000人死亡。A3腺苷受体(A3AR)是一个 胃肠道蛋白偶联受体，在包括中性粒细胞在内的几种类型的炎症细胞中高表达。 组织损伤/炎症后腺苷水平升高导致A3AR信号激活，这有助于 抑制炎症和促进修复，部分是通过损害免疫细胞的趋化和激活。激动剂 的A3AR正在接受多种炎症性疾病的调查，包括心脏缺血和 化疗引起的心脏毒性。不幸的是，早期已有剂量限制副作用的报道。 临床试验可能会限制它们的有效性。增强内源性配体信号的化合物， 被称为正变构调节器(PAM)，允许时空特异性，并减少脱靶效应。 预计针对A3AR的PAM将提供一种更好的治疗方法。在这些拟议的研究中， 我的目标是表征和进一步开发A3AR PAM，它将作为小分子探针和有用的治疗药物 心脏损伤和炎症紊乱。先前的结构-活性-关系研究(SAR)确定了1H- 咪唑并[4，5]喹啉-4-胺，LUF6000和2，4-二取代喹啉，LUF6096，作为展示PAM的 A3AR的活性，这些调节剂将正构体激动剂的功效提高了2倍以上。 不幸的是，它们也有降低激动剂效力的不良倾向。此外，没有一个 到目前为止，我们研究的PAM与啮齿动物受体相比显示出PAM活性，这阻止了我们评估 临床前啮齿动物疾病模型的生物学活性。在目标1中，我将通过以下方式扩展以前的SAR研究 基于LUF6000和LUF6096的结构表征了两个新的系列衍生品，目标是 确定改进的A3AR PAM，可双重增强正畸激动剂的疗效和效力。作为这项工作的一部分 目的，我将调查我们的PAM配体是否支持有偏见(G蛋白依赖与G蛋白非依赖) 发出信号并评估活动与小鼠A3AR的对比。在目标2中，我将利用物种差异并产生 人/鼠嵌合和突变型A3AR，以便于识别LUF6000和 LUF6096。最后，在目标3中，我将研究LUF6000和LUF6096对两个关键基因的生物学效应 利用人类中性粒细胞系(HL60细胞)，中性粒细胞的功能--产生超氧化物和趋化作用。 在完成这些拟议的研究后，在我的导师、一个不同的合作者团队的指导下， 而我的论文委员会，我将在实验设计、生化分析执行方面获得经验， 分子克隆，以及合理药物设计的基础，这将导致可发布的数据，并为我 成功竞争一家生物制药公司的研究科学家职位。完成这项工作 这项工作将促进我们对A3AR变构药理学的理解，并有可能导致A3AR变构药理学的发展 一种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.