眼镜蛇神经毒蛋白部分水解后所得镇痛多肽的序列优化及镇痛机制研究

.Some three-fingered proteins (TFPs) in snake venom were reported to show long-acting analgesic effects. However, the analgesic effects of TFOs were not related to their neurotoxicity or cytotoxicity. These findings suggested that TFPs might have relatively independent analgesic activity center. In previous studies, cobrotoxin, which is a TFP found in Chinese cobra (Naja atra), showed analgesic effects by oral contraception, although almost 0.5-2 hours were needed to act. These is a possibility that cobrotoxin needs to be partially digested by pepsin before acting. This possibility was ignored for the past decades. Our research results indicated that when TFPs were pre-treated with pepsin, the small peptide mixture showed fast analgesic acting, that is only 10 min after oral administration in mice. Recently, a peptide with analgesic effect has been purified and its sequence has been determined by proteomics methods. A peptide with proposed sequence has been synthesized. The synthesized peptide showed similar analgesic effect and MALDI-TOF, HPLC performance to the isolated analgesic peptide. In this project, the sequence of the peptide will be optimized and functional residues will be screened. Affinity chromatography will be applied to isolate and purify the receptor. And the analgesic mechanism, solution structure NMR of the peptide, and the properties of the receptor will be studied. Based on all these results and previous studies, the structure and function relationship, the analgesic mechanism of TFPs can be discussed.

部分蛇毒三指蛋白被发现具有长效镇痛作用，但与其神经毒性或细胞毒性无直接相关性，提示这类蛋白具有相对独立的镇痛活性中心。研究发现中华眼镜蛇毒神经毒蛋白cobrotoxin（即"科博肽"）单独口服亦有效镇痛，但起效慢，其中可能因素是需消化酶将其部分水解成小肽片段后再起效，这一可能性一直被忽视。本项目组前期研究表明，眼镜蛇毒三指蛋白混合多肽经胃蛋白酶水解成小肽混合物后，具有灌胃10min内速效镇痛效果。现已分离纯化出一个胃蛋白酶水解后镇痛小肽，经质谱分析及数据库比对，推断为neurotoxin c的 Loop II上10氨基酸片段，合成肽证实与水解肽HPLC、镇痛活性等基本一致。本项目将以此小肽为配基，分离纯化并鉴定其镇痛受体，探讨镇痛通路及分子机制；优化序列，筛选镇痛活性更高多肽，结合本项目组在该蛇毒三指蛋白中做的大量前期工作，推进三指蛋白镇痛机制、家族进化与分化、功能演化研究。

长效不成瘾镇痛药物一直是药物研发及科学研究的热点之一。一些毒素蛋白，包括眼镜蛇神经毒蛋白(neurotoxin)、芋螺毒素(conotoxin)等均表现出了长效不成瘾镇痛特征，但目前只能注射给药、有强烈致死毒性等缺点，而且镇痛机制还有大量未知。通过本项目的研究，设计了一个十肽(KDHRGTRIER)，用热板法、醋酸扭体法、福尔马林注射法验证其确有良好的口服和腹腔注射镇痛活性；其LD50为8.4 g/kg，95%可信限为7.3-9.7 g/kg，表明小肽的毒性很低或无毒性，也表明蛇毒镇痛活性与毒性无关，是两个独立的活性中心。基于此10肽，我们开展了序列优化工作，设计合成出了3个13、15、16氨基酸残基小肽，并进行了活性比较，确认16氨基酸环肽具有更好的镇痛活性。活体动物实验和切片染色实验，发现了荧光标记10AA小肽在背根神经节部位富集的实验现象，从而首次证实了外周镇痛的假说。通过荧光标记镇痛小肽及活体动物成像观察，还发现了镇痛小肽在疼痛通路上的富集，提示有主动镇痛的现象，这也是首次发现。结合宏基因组比较分析，我们认为镇痛小肽很可能还通过作用于炎症因子或其调节因子从而达到主动镇痛和长效镇痛的功能。我们的研究还发现，镇痛小肽的镇痛功能还可能存在性别差异。.本项目的研究总体达到了最初的各项研究目标，证实了外周镇痛的假说。新发现镇痛小肽有富集于疼痛通路、炎症部位和伤口的现象，具有主动镇痛特性，有别于小分子镇痛化合物；新发现镇痛小肽的长效镇痛机制很可能与炎症因子或其调节因子有关；还发现镇痛小肽在镇痛活性及其机制方面可能存在性别差异。.虽然目前镇痛实验数据表明我们设计合成的小肽镇痛活性的效价与现有小分子镇痛药物相比还有些差距，但镇痛小肽的其长效镇痛、不成瘾也是其有别于小分子镇痛药物的显著优势，还能口服，安全便捷；加上在镇痛机制方面有一些新发现，值得继续深入研究。