Positive allosteric modulation of the A2aR for treatment of acute inflammation

A2aR 的正变构调节治疗急性炎症

• 批准号: 8637857
• 负责人: EDWARD P AMENTO
• 金额: $ 29.25万
• 依托单位: MOLECULAR MEDICINE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8637857
• 关键词: Acute; Adenosine; Adjuvant Arthritis; Adverse effects; Affinity; Agonist; Animal Disease Models; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Arthritis; Attenuated; Binding; Biological Assay; Cell Line; Cells; Chronic; Clinical; Cyclic AMP; Disease; Disease Progression; Disorder by Site; Enhancers; Equilibrium; Functional disorder; G Protein-Coupled Receptor Genes; Gases; Goals; Guanosine Triphosphate; Histology; Homeostasis; Human; Immune System Diseases; Immunoprecipitation; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Inositol Phosphates; Interleukin-10; Kinetics; Left; Measures; Mediating; Modeling; Mus; Organ; Pathway interactions; Pattern; Plasma; Play; Process; Production; Purinergic P1 Receptors; Radiolabeled; Rattus; Receptor Activation; Relative (related person); Research; Research Proposals; Resolution; Rheumatoid Arthritis; Rodent Model; Role; Signal Transduction; Site; Splenocyte; System; Therapeutic; Tissues; analog; cellular engineering; chemokine; cytokine; design; in vivo; inhibitor/antagonist; macrophage; monocyte; novel; public health relevance; radiotracer; receptor; receptor binding; receptor coupling; research study; response to injury

DESCRIPTION (provided by applicant): The adenosine A2a receptor (A2aR) plays a critical role in controlling inflammation. Endogenous adenosine is elevated at inflamed sites, where it engages the A2aR to down-regulate the inflammatory response and limit tissue damage. While activation of the A2aR with synthetic orthosteric agonists attenuates inflammation in animal models of disease, this therapeutic approach is limited by adverse effects due to unintended activation of the A2aR outside the target tissue. The current research is designed to explore an alternative approach: the targeted enhancement of the A2aR via positive allosteric modulation, which is expected to have a focused effect at disease sites where endogenous adenosine is elevated. The goal of this proposal is to demonstrate that the A2aR is amenable to positive allosteric modulation, and that such modulation will mount a discernible anti-inflammatory response. These studies will make use of a recently identified positive allosteric modulator of the A2aR, AEA061, as well as human and mouse primary cells and an engineered cell line (CHO-hA2aR) stably expressing the human A2aR (hA2aR). Specific Aim 1 is to demonstrate that the A2aR is amenable to positive allosteric modulation. First, to determine whether the increased cAMP production upon positive allosteric modulation of the A2aR in CHO-hA2aR cells is a direct consequence of A2aR activation, AEA061-induced A2aR-dependent Gas activation will be measured by quantifying GTPg35S incorporation into the Gas subunit. Second, to investigate whether functional enhancement of the A2aR by AEA061 is due to altered orthosteric agonist binding kinetics, equilibrium-binding experiments will be performed to evaluate the adenosine affinity and Bmax at the hA2aR in the presence and absence of AEA061. Third, to examine allosteric modulator-mediated pathway-biased signaling, we will compare the effect of AEA061 on Gas-driven cAMP and Gaq/11-driven inositol phosphate production in CHO-hA2bR cells. Specific Aim 2 is to determine whether positive allosteric modulation of the A2aR alters inflammatory cytokine and chemokine production/release in vitro and in vivo. To evaluate the in vitro effects of positive allosteric modulation, cytokine productio by LPS-stimulated human and mouse monocytes will be quantified in the presence and absence of AEA061. To assess in vivo effects, plasma inflammatory cytokine and chemokine levels of LPS-challenged control and A2aR-deficient mice with and without AEA061 treatment will be determined. Additional in vivo studies will assess the influence of positive allosteric modulation on disease progression in a rodent model of chronic inflammatory arthritis. If successful, we will have validated that increasing A2aR responsiveness to endogenous adenosine with the administration of a positive allosteric modulator, that has no intrinsic abilityto activate the A2aR, is an effective strategy to reduce progression of disease characterized by inflammation.

描述（由申请人提供）：腺苷A2a受体（A2aR）在控制炎症中发挥着关键作用。内源性腺苷在发炎部位升高，与 A2aR 结合，下调炎症反应并限制组织损伤。虽然用合成正位激动剂激活 A2aR 可以减轻疾病动物模型中的炎症，但这种治疗方法受到由于靶组织外 A2aR 意外激活而产生的不利影响的限制。目前的研究旨在探索另一种方法：通过正变构调节有针对性地增强 A2aR，预计这将在内源性腺苷升高的疾病部位产生集中效应。该提案的目的是证明 A2aR 适合正向变构调节，并且这种调节将产生明显的抗炎反应。这些研究将利用最近鉴定的 A2aR 正变构调节剂 AEA061，以及稳定表达人 A2aR (hA2aR) 的人和小鼠原代细胞和工程细胞系 (CHO-hA2aR)。具体目标 1 是证明 A2aR 适合正向变构调节。首先，为了确定 CHO-hA2aR 细胞中 A2aR 正向变构调节后 cAMP 产量的增加是否是 A2aR 激活的直接结果，将通过量化掺入 Gas 亚基的 GTPg35S 来测量 AEA061 诱导的 A2aR 依赖性 Gas 激活。其次，为了研究 AEA061 对 A2aR 的功能增强是否是由于正位激动剂结合动力学改变所致，将进行平衡结合实验以评估在 AEA061 存在和不存在的情况下 hA2aR 的腺苷亲和力和 Bmax。第三，为了检查变构调节剂介导的通路偏向信号传导，我们将比较 AEA061 对 CHO-hA2bR 细胞中气体驱动的 cAMP 和 Gaq/11 驱动的磷酸肌醇产生的影响。具体目标 2 是确定 A2aR 的正变构调节是否会改变体外和体内炎症细胞因子和趋化因子的产生/释放。为了评估正变构调节的体外效果，将在 AEA061 存在和不存在的情况下对 LPS 刺激的人和小鼠单核细胞产生的细胞因子进行定量。为了评估体内效果，将测定接受和未接受 AEA061 治疗的 LPS 攻击对照小鼠和 A2aR 缺陷小鼠的血浆炎症细胞因子和趋化因子水平。其他体内研究将评估正变构调节对慢性炎症关节炎啮齿动物模型疾病进展的影响。如果成功，我们将验证通过施用不具有激活 A2aR 内在能力的正变构调节剂来增加 A2aR 对内源性腺苷的反应性，是减少以炎症为特征的疾病进展的有效策略。