新型NAAA抑制剂的分子构建及非依赖性抗神经痛作用研究

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is an attractive new drug target involved in biological deactivation of N-palmitoylethanolamide (PEA), which exerts analgesic effects through the activation of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-α). However, the molecular mechanism undergone the interaction between NAAA inhibitors and NAAA target are still unclear as current classic NAAA inhibitors showed low-activity and instability in vivo, which limited their medical applications. Therefore, it is important to develop the new compounds targeted human NAAA protein and evaluate their pharmacologic properties. We reported that oxazolidinone derivative, showed a significant inhibition on NAAA in vivo as well as in vitro. Base on our previous data, we will design and synthesize a series of oxazolidinone derivatives according structure-activity relationship (SAR) studies, in which interpret the structural modifications of hydrogen bond acceptor, hydrophobic fragment and carbon chain of oxazolidinone compound. Assistant by computer-aided molecular docking methodology, we will obtain the selective, potent NAAA inhibitors that will be evaluated their pharmacology properties by using enzymatic screening, cell-base activity screening, and in vivo pain/inflammation animal models. This project crosses the multiple disciplines including computational chemistry, synthetic chemistry, molecular biology, and pharmacology. The aim of project is going to develop the promising analgesic drug targeted human NAAA and provide effective tools for drug discovery platform.

NAAA是一种选择性作用于外周神经系统的新型镇痛类药物靶标，极具关注力。然而现有的NAAA抑制剂存在活性低、体内稳定性差等缺点，无法应用于系统给药，对全面了解NAAA-PEA途径的镇痛机制亦缺乏帮助。在前期研究中，我们发现以噁唑烷酮为亲水性头部的一类化学结构对NAAA抑制活性高、选择性好且在生物环境中稳定。更多构效研究表明，噁唑烷酮2位增加亲水性取代基团、调整疏水区相对空间位置、提高连接桥刚性可大幅度提高其活性及选择性。本项目拟采用合成生物学手段建立高通量体外筛选模型、化学空间分析法构建药效团及其连接方式、片段药物设计法理性设计匹配性能良好的疏水区，以蛋白、细胞、整体动物多层次药效及副作用评价筛选体系结合柔性分子模拟对接实验总结构效关系，对噁唑烷酮酰亚胺结构进行进一步优化，发现新型抗神经性疼痛活性强、稳定性好，且克服中枢依赖性的先导化合物。

机械性外伤、炎症、缺血、机体代谢异常等因素都可以直接或间接地诱发一系列由神经系统损伤介导的感觉、运动功能异常，其中神经性疼痛是最容易觉察和最难以忍受的病理现象，迄今为止仍缺乏良好的治疗手段。在本课题资助下，经过4年的研究较好地完成了计划书预期设定的研究目标。通过计算机模拟药物设计及基于片段组学的药物发现，我们共合成了170余个NAAA抑制剂，主要为恶唑烷酮酰亚胺类结构及吡咯烷环结构两类，在分子、细胞及动物水平对上述结构进行了成药性评价，结果显示，上述两类结构化学及生物学稳定性良好，均克服了四元内酯环类NAAA抑制剂半衰期极短的问题，且有数十个化合物IC50值低至50-200 nM，一个化合物的IC50值达到单纳摩尔级别。体内实验显示，代表性NAAA抑制剂F96可以很好地抑制神经病理性疼痛，且未表现出任何毒副作用，目前正在进行临床前开发。本项研究通过大量构效关系研究初步阐明了NAAA酶催化中心的空间结构，为开发NAAA抑制剂提供了重要理论指导和线索。同时阐明了此类抑制剂的作用机制以及初步的分子信号通路，结合毒理学研究显示相对于目前常用的镇痛类药物，NAAA抑制剂不产生精神依赖性，不产生胃肠溃疡，为非常有开发价值的新型治疗神经病理性疼痛药物靶点。在本项目的资助下，已发表SCI论文11篇，获得国家发明专利授权2项。