Positive allosteric modulation of the A2aR for treatment of acute 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。内源性腺苷在炎症部位升高，与A2aR结合，下调炎症反应，限制组织损伤。虽然在动物疾病模型中，用合成矫形受体激动剂激活A2aR可以减轻炎症，但这种治疗方法受到靶组织外A2aR意外激活的不良反应的限制。目前的研究旨在探索另一种方法：通过正变构调节靶向增强A2aR，这有望在内源性腺苷升高的疾病部位产生集中作用。本研究的目的是证明A2aR可接受正变构调节，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发现的A2aR阳性变构调节剂AEA061，以及人类和小鼠原代细胞和稳定表达人类A2aR （hA2aR）的工程细胞系CHO-hA2aR。具体目的1是证明A2aR可适应正变构调节。首先，为了确定CHO-hA2aR细胞中A2aR阳性变构调节后cAMP产量的增加是否是A2aR激活的直接结果，将通过量化GTPg35S掺入Gas亚基来测量aea061诱导的A2aR依赖性Gas激活。其次，为了研究AEA061对A2aR的功能增强是否由于正位激动剂结合动力学的改变，将进行平衡结合实验，以评估AEA061存在和不存在时hA2aR的腺苷亲和力和Bmax。第三，为了检验变弹性调节剂介导的通路偏倚信号，我们将比较AEA061对CHO-hA2bR细胞中气体驱动cAMP和Gaq/11驱动肌醇磷酸产生的影响。特异性目的2是确定A2aR的阳性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。为了评估体外正变构调节的作用，我们将量化lps刺激的人和小鼠单核细胞在存在和不存在AEA061的情况下产生的细胞因子。为了评估AEA061在体内的作用，将测定lps挑战对照组和a2ar缺陷小鼠在接受和不接受AEA061治疗时的血浆炎症细胞因子和趋化因子水平。另外的体内研究将评估慢性炎性关节炎啮齿动物模型中阳性变构调节对疾病进展的影响。如果成功，我们将证实，通过给予没有内在能力激活A2aR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。

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.