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）在控制炎症中起关键作用。内源性腺苷在发炎的部位升高，在该部位使A2AR与炎症反应下调并限制组织损伤。虽然与合成的直角激动剂激活A2AR在动物疾病模型中减弱了炎症，但由于目标组织以外的A2AR的意外激活，这种治疗方法受到不利影响的限制。当前的研究旨在探索一种替代方法：通过阳性变构调节对A2AR的有针对性增强，预计在内源性腺苷升高的疾病部位将产生重点作用。该提案的目的是证明A2AR适合阳性变构调节，并且这种调节将具有可辨别的抗炎反应。这些研究将利用A2AR AEA061的最近确定的阳性变构调节剂，以及人类和小鼠原代细胞以及工程细胞系（CHO-HA2AR）稳定地表达人A2AR（HA2AR）。具体目的1是证明A2AR适合阳性变构调节。首先，为了确定CHA2AR细胞中A2AR阳性变构调节的cAMP产生增加是A2AR激活的直接结果，将通过量化GTPG35S掺入气体亚基中AEA061诱导的A2AR诱导的A2AR依赖性气体激活。其次，为了研究AEA061对A2AR的功能增强，是否是由于正前置激动剂结合动力学的改变，将进行平衡结合实验，以评估HA2AR的腺苷亲和力和BMAX在HA2AR的存在和不存在AEA061。第三，为了检查变构调节介导的途径偏向信号传导，我们将比较AEA061对CHO-HA2BR细胞中气体驱动的CAMP和GAQ/11驱动的肌醇磷酸盐产生的影响。具体目的2是确定A2AR的阳性变构调节是否改变了炎症细胞因子和趋化因子的产生/体外和体内释放。为了评估阳性变构调节的体外作用，将在存在和不存在AEA061的情况下量化LPS刺激的人和小鼠单核细胞的细胞因子产物。为了评估体内效应，将确定有或没有AEA061治疗的LPS挑战者对照的血浆炎症细胞因子和趋化因子水平和A2AR缺陷型小鼠。其他体内研究将评估慢性炎性关节炎模型中阳性变构调节对疾病进展的影响。如果成功的话，我们将验证，通过给予阳性变构调节剂，没有固有能力激活A2AR的A2AR对内源性腺苷的反应能力是减少炎症特征的疾病进展的有效策略。