The lipid hydrolase NAAA as a target for non-addictive analgesic medications

安全有效的止痛药的发现是一项紧迫的社会需求，也是美国国立卫生研究院治疗计划的核心内容。在这里，我们测试了一种假设，即N-乙酰乙醇胺酸酰胺酶(NAAA)为无滥用潜力的止痛药提供了一个新的靶点。NAAA水解棕榈酰乙醇胺(PEA)，这是一种脂质信使，通过与核受体PPAR-α结合来抑制伤害感受。最近，国际疼痛研究协会(IASP)对临床前文献进行的荟萃分析已经认识到NAAA抑制剂具有显著的抗伤害性，但这种特性背后的细胞底物仍不清楚。我们发现，与野生型相比，NAAA基因敲除(KO)小鼠(I)对福尔马林的致痛反应显著减少；(Ii)在慢性收缩损伤(CCI)模型中，未能出现持续性感觉异常。相反，NAAA过度表达和Ppara-Ko小鼠即使在给予阈值下福尔马林剂量时也表现出强大的致病行为。NAAA-KO小鼠对疼痛敏感性的降低不是发育代偿的结果，因为它可以通过给予NAAA抑制剂ARN19702来表现出来。重要的是，ARN19702在小鼠条件性位置偏爱(CPP)测试中没有发挥积极的激励作用，这表明缺乏奖励属性。这些数据表明，NAAA是发现非成瘾止痛药的有希望的目标。我们将在两个特定的目标中检验这一假设：目的1.验证NAAA作为止痛药物发现的分子靶点。将解决三个问题：(1)NAAA抑制剂直接针对哪些细胞类型？我们将建立细胞特异性的NAAA-KO小鼠系，并在福尔马林和CCI模型中评估疼痛相关行为和对NAAA阻断的敏感性。同时，我们将确定病理性疼痛对野生型小鼠背根神经节(DRG)和脊髓(SC)中NAAA调节信号的影响。(2)NAAA调节的伤害性信号转导有哪些细胞类型？我们将通过建立细胞特异性的PPAR-α-Ko小鼠品系，并在福尔马林和CCI测试中评估它们的疼痛相关反应和它们对NAAA抑制剂的敏感性，来鉴定参与NAAA依赖信号转导的PPAR-α表达细胞。(3)NAAA抑制剂的药效学特征是否与疼痛治疗中安全有效的使用相适应？我们将确定ARN19702(I)是否减轻自发的伤害性感觉，(Ii)在反复给药后产生耐受性，和(Iii)表现出奖励和/或成瘾的潜力。目的2.开发改良的NAAA抑制剂，并检测其作为镇痛剂的疗效。需要更好的NAAA抑制剂来评估NAAA在疼痛治疗中的可能作用。我们将结合基于计算结构的药物和构效关系研究来发现具有更好的药效学和药代动力学特性、强大的止痛活性和无成瘾潜力的新型药物。

The discovery of safe and effective analgesics is an urgent societal need and a centerpiece of NIH’s HEAL initiative. Here, we test the hypothesis that the enzyme N-Acylethanolamine Acid Amidase (NAAA) offers a novel target for analgesic medications devoid of abuse potential. NAAA hydrolyzes palmitoylethanolamide (PEA), a lipid messenger that suppresses nociception by engaging the nuclear receptor PPAR-α. The substantial antinociceptive properties of NAAA inhibitors have been recently recognized by a meta-analysis of the preclinical literature conducted by the International Association for the Study of Pain (IASP), but the cellular substrates underlying such properties are still unknown. We found that Naaa-knock out (ko) mice (i) have markedly reduced nocifensive responses to formalin, compared to wild-type littermates; and (ii) fail to develop persistent sensory abnormalities in the chronic constriction injury (CCI) model. Conversely, Naaa- overexpressing and Ppara-ko mice exhibit robust nocifensive behavior even when given a subthreshold formalin dose. The reduced sensitivity of Naaa-ko mice to pain does not result from developmental compensation, because it can be phenocopied by administration of the NAAA inhibitor ARN19702. Importantly, ARN19702 does not exert positive motivational effects in the mouse conditioned place preference (CPP) test, which is suggestive of a lack of rewarding properties. These data point to NAAA as a promising target for the discovery of non-addictive analgesics. We will test this hypothesis in two specific aims: Aim 1. Validate NAAA as a molecular target for analgesic drug discovery. Three questions will be addressed: (1) What cell types are directly targeted by NAAA inhibitors? We will generate cell-specific NAAA-ko mouse lines and evaluate, in the formalin and CCI models, pain-related behaviors and sensitivity to NAAA blockade. In parallel, we will determine the impact of pathological pain on NAAA-regulated signaling in dorsal root ganglia (DRG) and spinal cord (SC) of wild-type mice. (2) What cell types mediate NAAA-regulated antinociceptive signaling? We will identify PPAR-α-expressing cells involved in NAAA-dependent signaling by creating cell-specific PPAR-α-ko mouse lines and evaluating their pain-related responses and their sensitivity to NAAA inhibitors in the formalin and CCI tests. (3) Is the pharmacodynamic profile of NAAA inhibitors compatible with safe and effective use in pain therapy? We will determine whether ARN19702 (i) alleviates spontaneous nociception, (ii) produces tolerance after repeated administration, and (iii) exhibits rewarding and/or addicting potential. Aim 2. Develop improved NAAA inhibitors and examine their efficacy as analgesic agents. Better NAAA inhibitors are needed to assess NAAA’s possible role in pain therapy. We will combine computational structure-based drug and structure-activity relationship studies to discover novel agents with improved pharmacodynamic and pharmacokinetic properties, robust analgesic activity, and no addictive potential.