南海产α-芋螺毒素环化及其药理学性质研究

α-Conotoxins are selective antagonists of nicotinic acetylcholine receptors (nAChRs) which are ligand-gated ion channels that play an important role in synaptic transmission throughout the central nervous system. Previous researches have showed the nAChRs are implicated in the pathophysiology of a number of disease states including epilepsy, Parkinson’s disease, pain and addiction et al. Accordingly, α-conotoxins have proved to be valuable tools for identifying the composition and roles of nAChR subtypes and some of them have become either novel pharmaceutics or promising drug leads. We previously reported the discovery of several new α-conotoxins cloned from Conus of the South China Sea. These conopeptides inhibited acetylcholine-evoked currents mediated by various nAChR subtypes expressed in Xenopus oocytes and can be developed to tool drugs or drug leads. However, one potential drawback of α-conotoxins as drugs is that they may suffer the generic problems of peptides in vivo, such as poor absorption, susceptibility to proteolysis, and short biological half-lives. One promising strategy for improving the stability of α-conotoxins is backbone cyclization and cyclic peptides have been used in a variety of drug design applications of the pharmaceutical industry. The method can remove the N and C termini, and provide protection against exo-proteases, and an increase in rigidity of the structure that inhibited cleavage by other proteases. In this program, new chemical methods are going to be carried out to cyclize four new α-conotoxins from the South China Sea, including synthetic backbone cyclization of conotoxins, forming correct the disulfide-bonding pattern .Then these conotoxins analogies’ stability, electrophysiology activities, tridimensional structures will be elucidated by technology of biochemistry methods, patch clamping and NMR spectroscopy et al. Thus, the research activities of this program would provide clinically oriented cyclized α-conotoxins developing novel therapeutic and diagnostic applications.

α-芋螺毒素是烟碱型乙酰胆碱受体的选择性拮抗剂，因为乙酰胆碱受体与癫痫、帕金森症、疼痛、成瘾等多种疾病的发病、诊断和治疗密切相关，所以此类毒素在与乙酰胆碱受体相关的疾病研究和药物开发上具有重要价值。本实验室前期从栖息南海的芋螺中获得了多种能够作用于乙酰胆碱受体不同亚型且具有潜在药用开发价值的α-芋螺毒素，但芋螺毒素作为一类多肽，同样存在体内半衰期短，易被蛋白酶水解，生物利用度低等缺点。多肽骨架环化已经被用于多肽药物的设计中，且已证明可以提高多肽稳定性。因此，α-芋螺毒素的环化能克服其稳定性差这一缺点。本项目拟利用化学合成方法，对本实验室发现的4种新型α-芋螺毒素的结构进行N-C端环化，同时形成正确的二硫键和空间结构，再利用电生理方法、生化方法、核磁共振等方法对改造后芋螺毒素的活性、稳定性及其结构进行系统研究，以期获得更具应用药用价值的环化芋螺毒素，提高其成药性，为后续新药研发奠定基础。

芋螺毒素是由软体动物芋螺分泌出来的一类活性多肽，能够作用于多种离子通道受体，具有开发为药物的潜力。但芋螺毒素作为一类多肽，存在易被蛋白酶水解，导致生物利用度低等缺点。本项目利用化学环化等多种修饰方法，合成α-芋螺毒素的修饰突变体类似物，以提高芋螺毒素的活性、代谢稳定性和选择性。项目主要结果总结如下：1、利用多肽固相合成法，环化合成了α-芋螺毒素TxIB、TxID和LvIA等一系列突变体，对其药理活性、结构和稳定性进行测试，发现了cTxIB-7、c[S9A]TxID-6 和cLvIA-7等突变体，在活性保持的同时，稳定性有一定程度提高；2、对芋螺毒素TxID所含的易氧化甲硫氨酸进行替换，实验结果表明当用异亮氨酸替换甲硫氨酸，可以最大程度保持多肽活性，同时保障TxID不被氧化失活；3、对TxID、RgIA和GeXIVA等芋螺毒素进行不同策略的改造修饰，获得了选择性或稳定性改善的突变体，提高了这些芋螺毒素的药物开发潜力。4、对芋螺毒素改造后的突变体作用机制进行研究，阐明这些芋螺毒素与受体间相互作用关键氨基酸。.本项目建立了α-芋螺毒素的环化改造方法，研究了改造后芋螺毒素突变体活性及构效关系，并对其稳定性进行了测试，提升了这些芋螺毒素药用开发价值。.目前为止，发表标注本基金的论文共计12篇，其中SCI论文11篇，1篇SCI论文正在审稿修改中，作为副主编撰写专著1本，申请专利美国专利1项，培养博士和硕士研究生共4名。