Positive allosteric modulation of the A2aR for treatment of acute inflammation

描述（由申请人提供）：腺苷A2 a受体（A2 aR）在控制炎症中起关键作用。内源性腺苷在发炎部位升高，在那里它与A2 aR结合以下调炎症反应并限制组织损伤。虽然用合成的正构激动剂激活A2 aR减弱了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A2 aR的非预期激活引起的不良反应的限制。目前的研究旨在探索一种替代方法：通过正变构调节靶向增强A2 aR，预计这将在内源性腺苷升高的疾病部位产生集中效应。该提议的目标是证明A2 aR服从正变构调节，并且这种调节将产生可辨别的抗炎反应。这些研究将利用最近鉴定的A2 aR的正变构调节剂AEA 061，以及人和小鼠原代细胞和稳定表达人A2 aR（hA 2aR）的工程化细胞系（CHO-hA 2aR）。具体目标1是证明A2 aR服从正变构调节。首先，为了确定CHO-hA 2aR细胞中A2 aR的正变构调节后cAMP产生的增加是否是A2 aR活化的直接结果，将通过定量GTPg 35 S掺入Gas亚基来测量AEA 061诱导的A2 aR依赖性Gas活化。其次，为了研究AEA 061对A2 aR的功能增强是否是由于改变的正构激动剂结合动力学，将进行平衡结合实验以评价在存在和不存在AEA 061的情况下hA 2aR处的腺苷亲和力和Bmax。第三，为了检查变构调节剂介导的通路偏倚信号传导，我们将比较AEA 061对CHO-hA 2bR细胞中Gas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A2 aR的正变构调节是否在体外和体内改变炎性细胞因子和趋化因子的产生/释放。为了评价正变构调节的体外作用，将在存在和不存在AEA 061的情况下定量LPS刺激的人和小鼠单核细胞的细胞因子产生。为了评估体内效应，将测定LPS攻击的对照小鼠和A2 aR缺陷小鼠在有和没有AEA 061治疗的情况下的血浆炎性细胞因子和趋化因子水平。另外的体内研究将在慢性炎性关节炎的啮齿动物模型中评估正变构调节对疾病进展的影响。如果成功的话，我们将证实，通过给予没有激活A2 aR内在能力的正变构调节剂来增加A2 aR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.