Discovery of Adenosine Receptor Allosteric Modulators for Cardiovascular Disease and Inflammation

发现用于心血管疾病和炎症的腺苷受体变构调节剂

• 批准号: 10464473
• 负责人: Courtney L Fisher
• 金额: $ 3.95万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-02 至 2023-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10464473
• 关键词: Accounting; Adenosine; Adenosine A3 Receptor; Affinity; Agonist; Amines; Amino Acids; Basic Science; Behavior; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cardiotoxicity; Cardiovascular Diseases; Cause of Death; Cell Line; Cell physiology; Cells; Cellular Stress; Cessation of life; Chemicals; Chemotaxis; Chronic; Clinical Trials; Collaborations; Coupled; Data; Development; Disease; Disease model; Dissociation; Dose-Limiting; Drug Design; Exhibits; Experimental Designs; Future; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; HL-60 Cells; HL60; Heart Injuries; Human; Hypoxia; Immune; Impairment; Inflammation; Inflammatory; Knowledge; Laboratories; Lead; Left; Ligands; Medicine; Mentors; Molecular Cloning; Mus; Mutagenesis; Myocardial Ischemia; NADP; Pharmacology; Positioning Attribute; Production; Proteins; Publishing; Purinergic P1 Receptors; Rattus; Receptor Activation; Receptor Signaling; Reporting; Research; Rodent; Rodent Model; Role; Scientist; Series; Signal Transduction; Signaling Molecule; Site; Specificity; Structure; Structure-Activity Relationship; Superoxides; Testing; Therapeutic; Time; United States; United States National Institutes of Health; Woman; Work; base; biopharmaceutical industry; chemotherapy; cost; drug development; early phase clinical trial; efficacy evaluation; experience; immune activation; improved; men; mutant; neutrophil; novel; novel therapeutics; positive allosteric modulator; pre-clinical; prevent; quinoline; radioligand; receptor; repaired; response; side effect; small molecule; spatiotemporal; species difference; therapeutic target; tissue injury

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.

项目概要 心血管疾病是美国男性和女性死亡的主要原因， 价值 3,512 亿美元，每年造成超过 840,000 人死亡。 A3 腺苷受体 (A3AR) 是一种 Gi 蛋白偶联受体在多种类型的炎症细胞（包括中性粒细胞）中高度表达。 组织损伤/炎症后腺苷水平升高会激活 A3AR 信号传导，从而有助于 限制炎症并促进修复，部分是通过损害免疫细胞趋化性和激活来实现的。激动剂 目前正在研究 A3AR 与多种炎症性疾病的关系，包括心肌缺血和 化疗引起的心脏毒性。不幸的是，早期已经报道了剂量限制性副作用 临床试验可能限制其用途。增强内源配体信号传导的化合物， 称为正变构调节剂（PAM），具有时空特异性，并减少脱靶效应。 预计针对 A3AR 的 PAM 将提供更好的治疗方法。在这些拟议的研究中， 我的目标是表征并进一步开发 A3AR PAM，它将作为小分子探针和有用的治疗方法 心脏损伤和炎症性疾病。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，如 PAM 所示 A3AR 的活性，这些调节剂将正构激动剂的功效提高 2 倍以上。 不幸的是，它们也具有降低激动剂效力的不良倾向。此外，没有一个 迄今为止，我们研究的 PAM 表现出与啮齿动物受体相比的 PAM 活性，这使我们无法评估 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： 表征基于 LUF6000 和 LUF6096 结构的两个新系列衍生物，目标是 鉴定出改进的 A3AR PAM，可双重增强正构激动剂的功效和效力。作为本次活动的一部分 目标，我将研究我们的 PAM 配体是否支持有偏差（G 蛋白依赖性与 G 蛋白独立） 信号传导并评估与小鼠 A3AR 相比的活性。在目标 2 中，我将利用物种差异并生成 人/小鼠嵌合体和突变体 A3AR 有助于识别 LUF6000 和 LUF6096。最后，在目标 3 中，我将研究 LUF6000 和 LUF6096 对两个关键的生物效应 中性粒细胞功能 - 超氧化物产生和趋化性 - 利用人类中性粒细胞系（HL60 细胞）。 完成这些拟议的研究后，在我的导师和多元化合作者团队的指导下， 和我的论文委员会，我将获得实验设计、生化分析执行的经验， 分子克隆和合理药物设计的基础知识，这将产生可发表的数据并让我做好准备 成功竞争一家生物制药公司的研究科学家职位。完成此 这项工作将增进我们对 A3AR 变构药理学的理解，并有可能促进 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。