基于α4β2 nAChR阻断作用的南海产新型α-芋螺毒素的发现、合成及作用机制研究

α4β2 nAChR has been proven to be closely related to nicotine addiction through reward pathway, and α4β2 nAChR antagonists have been a new trend on discovery of new anti-tobacco drug. α-conotoxins are an array of bioactive peptides derived from Conus snails’ venom and target nAChRs with high potency and good selectivity. However, low potency and poor selectivity of the existing α-conotoxins which are available as α4β2 nAChR antagonists, may cause potential side effects and inevitably limit their development. In order to acquire new potent and selective α4β2 nAChR antagonists, here we will isolate α-conotoxins from South China Sea-derived venom fractions which have shown inhibitory activity against α4β2 nAChR. Then the electrophysiology activities of the isolated conotoxins on α4β2 nAChR and other subtypes will be tested. The bioactive conotoxins will be subjected to structure elucidation, including sequencing, posttranslational modification identification and disulfide mapping. According to the structure elucidation, these bioactive conotoxins will be synthesized to obtain enough amount for the NMR test for their 3D structure analysis. The molecular mechanism of interactions between bioactive conotoxins and α4β2 nAChR will also be investigated using computational methods, such as docking and molecular dynamic simulations. This project predicts to obtain 1-2 promising new leads inhibiting α4β2 nAChR with high potency, good selectivity and explicit mechanism. The result will provide a basis for developing new specific antagonist for α4β2 nAChR.

α4β2 nAChR与尼古丁成瘾的奖赏机制密切相关，α4β2 nAChR阻断剂的开发已成为新型戒烟药物研发的新方向。α-芋螺毒素是来源于芋螺毒液的一类选择性作用于nAChRs的多肽毒素，具有选择性好、作用强等特点，但目前已发现的阻断α4β2 nAChR的α-芋螺毒素活性弱、选择性差，限制了它们的开发应用。本项目拟从具有α4β2 nAChR阻断活性的南海产芋螺粗毒流分中分离纯化获得芋螺毒素单体，利用电生理技术测定其对α4β2及其他亚型 nAChRs的阻断活性，鉴定活性芋螺毒素的氨基酸序列、翻译后修饰及二硫键连接方式，然后人工合成获得足量芋螺毒素进行NMR三维结构解析，最后利用计算机分子模拟及对接技术，阐明活性芋螺毒素与α4β2 nAChR相互作用机制。本项目预期发现1-2个活性显著、选择性优良、作用机制明确的阻断α4β2 nAChR的先导分子，为新型α4β2 nAChR阻断剂的开发奠定基础。

α4β2 nAChR与尼古丁成瘾的奖赏机制密切相关，α4β2 nAChR阻断剂的开发已成为新型戒烟药物研发的新方向。但目前已发现的阻断α4β2 nAChR的α-芋螺毒素活性弱、选择性差，限制了它们的开发应用。本项目从大理石芋螺、织锦芋螺、玉女芋螺等毒液组分中分离得到了69个芋螺毒素单体。通过高分辨串联质谱技术（MALDI-TOF-MS/MS）和edman降解法鉴定了它们的氨基酸序列和翻译后修饰（PTM），其中22个为新芋螺毒素单体。通过分步还原及分步烷基化反应，结合MALDI-TOF-MS/MS分析，鉴定了芋螺毒素的二硫键连接，发现了半胱氨酸模式为III的芋螺毒素的两种新颖的二硫键连接方式（I-V,II-VI,III-IV和I-III,II-VI,IV-V），为该类芋螺毒素的结构研究提供了参考。以Fmoc多肽合成法及一步氧化折叠，人工合成了31个芋螺毒素单体。通过对氧化折叠条件的优化，含三对二硫键的芋螺毒素的折叠产率得到显著提高，为该类芋螺毒素的高效人工合成提供了方法学指导。通过双电极电压膜片钳技术，检测发现二硫键连接为I-V, II-VI, III-IV和I-VI, II-IV, III-V的Tx3h折叠肽对α4β2 nAChR有较好的选择性，为其后续作为α4β2 nAChR阻断剂进行结构改造奠定了基础。此外，我们还检测了芋螺毒素单体对钠离子通道受体（Nav1.4和Nav1.8）的抑制作用以及对卵巢癌细胞ID8的细胞毒作用，发现芋螺毒素Mr3e.1, Mr3.8, Tx3a.1和TCP对Nav1.4和Nav1.8的阻断率为21.51~24.32%，芋螺毒素Tx3a.1, Mr3e.1和Vi14b对ID8细胞生长抑制的IC50值分别为24.29 µM，54.97 µM和111.6 µM，为芋螺毒素后续的抗卵巢癌研究奠定了基础。