Novel Probes for N-acylethanolamine-hydrolyzing acid amidase function

其他项目信息-第7节-项目概要/摘要 N-酰基乙醇胺水解酸性酰胺酶（NAAA）是一种溶酶体酶，在细胞内的代谢中具有重要作用。 N-酰基乙醇胺（NAE），生物活性脂质介质/信号分子， 哺乳动物组织（1，2）。在本申请中，我们建议开发新的NAAA抑制剂作为基础， 用于未来设计药理学探针和治疗药物。主要底物是N- 棕榈酰乙醇胺（PEA），一种过氧化物酶体增殖物激活受体（PPAR-ligand）的激动剂 （3，4）。然而，对其他NAE有显著的活性，包括内源性激动剂， 大麻素受体CB1和CB2，也称为内源性大麻素（5）。NAAA作为一个 临床前已证明可药用靶点可用于治疗慢性疼痛和炎症的镇痛作用 很少或没有副作用的可能性（6，7）。减少毒品的强化成瘾性， 还报告了滥用（8 - 10）抑制NAAA的情况。这些特点使NAAA成为一个优秀的 用于发现新化合物的治疗靶点，以治疗疼痛和炎症，而无需成瘾性药物 阿片类药物的特性。此外，NAAA抑制剂是潜在的重要药理学探针， 可以研究PPAR-a信号传导对成瘾的影响。 目前，可用的NAAA抑制剂很少，其中最成功的已发表抑制剂表现出 行动的持续时间非常短。我们开发了一种基于荧光的检测方法， 筛选了我们的化合物库，发现了几个具有明显NAAA抑制作用的先导化合物， 数据区.此外，我们还克隆、表达和纯化了毫克量的NAAA，并获得了第一个 酶的NMR光谱。有了这些工具，我们将利用我们的铅化合物来探测 涉及酶催化位点的分子特征，使用组合的生物物理/生物化学 一种方法，将详细说明结构细节，以告知下一代NAAA特定的合成 抑制剂的这种抑制剂将被用作探针，以开发其作为新的治疗靶点的潜力。

OTHER PROJECT INFORMATION - SECTION 7 - PROJECT SUMMARY/ABSTRACT 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 biophysical/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.