Novel Probes for N-acylethanolamine-hydrolyzing acid amidase function

描述（由申请人提供）：n -酰基乙醇胺水解酸酰胺酶（NAAA）是一种溶酶体酶，在n -酰基乙醇胺（NAEs）失活中起重要作用，NAEs是哺乳动物组织中存在的生物活性脂质介质/信号分子（1,2）。在这个应用中，我们建议开发新的NAAA抑制剂，作为未来设计药理学探针和治疗药物的基础。主要底物是N-棕榈酰乙醇胺（PEA），它是过氧化物酶体增殖体激活受体（ppar配体）的激动剂（3,4）。然而，对其他NAEs有显著的活性，包括大麻素受体CB1和CB2的内源性激动剂，也称为内源性大麻素(5)。NAAA作为一种可药物靶点的潜力已经在临床前被证明用于治疗慢性疼痛和炎症的镇痛，并且可能很少或没有副作用（6,7）。减少药物滥用的增强成瘾性（8-10）也被报道抑制NAAA。这些特性使NAAA成为一个极好的治疗靶点，用于发现新的化合物来治疗疼痛和炎症，而不具有阿片类药物的成瘾性。此外，NAAA抑制剂是潜在的重要药理学探针，可以研究PPAR-a信号对成瘾的影响。目前，可用的NAAA抑制剂很少，发表的最成功的抑制剂表现出非常短的作用时间。我们开发了一种基于荧光的检测方法，通过筛选化合物库，发现了几种具有不同NAAA抑制谱的先导化合物。此外，我们还克隆、表达和纯化了毫克量的NAAA，并获得了该酶的第一个核磁共振谱。有了这些工具，我们将利用我们的先导化合物来探测酶催化位点的分子特征，使用物理/生化相结合的方法来阐述结构细节，为下一代naaa特异性抑制剂的合成提供信息。这些抑制剂将作为探针来开发其作为新的治疗靶点的潜力。

DESCRIPTION (provided by applicant): N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that has an important role in the deactivation of N-acylethanolamines (NAEs), bioactive lipid mediators/signaling molecules present in mammalian tissues (1, 2). In this application, we propose to develop novel NAAA inhibitors to serve as a basis for the future design of pharmacological probes and therapeutic medications. The primary substrate is N- palmitoylethanolamine (PEA), an agonist for the peroxisome proliferator-activated receptor-¿ (PPAR-ligand ¿) (3, 4). However, there is significant activity against other NAEs including the endogenous agonists of the cannabinoid receptors CB1 and CB2, also known as endocannabinoids(5). The potential of NAAA as a druggable target has been demonstrated preclinically for analgesia in treating chronic pain and inflammation with the possibility of few or no side effects (6, 7). A reduction in the reinforcing addictive nature for drugs of abuse (8-10) has also been reported for inhibition of NAAA. These characteristics make NAAA an excellent therapeutic target for discovery of novel compounds to treat pain and inflammation without the addictive properties of opioids. In addition NAAA inhibitors are potentially important pharmacological probes by which the influence of PPAR-a signaling on addiction may be studied. At present, there are few NAAA inhibitors available, with the most successful of those published exhibiting a very short duration of action. We have developed a fluorescence-based assay through which we have screened our library of compounds and found several lead compounds that have distinct NAAA inhibitory profiles. In addition we have cloned, expressed and purified milligram amounts of NAAA and obtained the first NMR spectrum of the enzyme. With these tools in hand, we will utilize our lead compounds to probe the molecular features involved in the catalytic site of the enzyme, using a combined iophysical/biochemical approach that will elaborate structural details to inform the synthesis of next-generation NAAA-specific inhibitors. Such inhibitors will be used as probes to exploit its potential as a novel therapeutic target.