芋螺毒素(conotoxin，CTx)是由软体动物芋螺分泌出来的一类活性多肽，主要用来捕食猎物和防御天敌。每种芋螺能够产生超过1000多种不同类型的芋螺毒素多肽，它们能够高效特异的作用于多种膜蛋白，其中多数种类能够抑制电压或配体门控离子通道。芋螺毒素可以根据其结构和功能特征进行分类，如半胱氨酸模式和药理学活性。芋螺毒素家族中，作用于烟碱型乙酰胆碱受体(nAChRs)的芋螺毒素种类最多，尤其是α-芋螺毒素。现有研究表明，nAChRs与多种神经系统疾病相关，包括疼痛，癫痫，帕金森病和成瘾等。因此，芋螺毒素不但可以开发为研究nAChRs的工具探针，而且还能作为先导药物分子直接作为药物进行研究。但是芋螺毒素作为一类多肽，存在易被蛋白酶水解，体内半衰期短，生物利用度低等缺点。本研究中，我们利用不同方法对4种作用nAChRs的芋螺毒素进行化学修饰改造，包括利用D-型氨基酸突变改造 α-CTx RgIA 和 αO-CTx GeXIVA、骨架环化 α-CTx LvIA、以及 α-CTx TxID序列中Met替换改造研究，以期获得活性或稳定性更好的芋螺毒素突变体，提高这4种芋螺毒素的药用价值。 α-CTx RgIA是α9α10 nAChR的竞争性拮抗剂，它对鼠源α9α10 nAChR的活性非常强，但对人源α9α10 nAChR的活性却很弱，且RgIA序列中含有四个精氨酸，容易被多种蛋白酶降解，为了改善RgIA的这些缺点，我们采用D-型氨基酸扫描方法合成RgIA突变体，研究氨基酸的立体异构改变对其活性的影响，希望能够获得更稳定的RgIA突变体，增强RgIA的生物利用度。本研究一共合成了 15个RgIA突变体，活性筛选实验结果表明其中14种突变体对鼠源α9α10 nAChR的活性显著降低，仅RgIA[13r]保留了与本体一致的生物活性。另外两个突变体RgIA [7，9，11，13r]和RgIA [7，9，11-r#]序列中的所有L-Args都被D-Args取代，其对鼠源α9α10 nAChR的活性丧失，但保持了对人源α9α10 nAChR的活性。稳定性实验显示这三种突变体在人血清中稳定性显著提高，而且RgIA[7，9，11，13-r]和RgIA[7，9，11-r#]在模拟肠液(SIF)中的稳定性也大幅度提升。 αO-CTx GeXIVA (GeXIVA)是本实验室从Conus generalis中克隆而来的新型芋螺毒素，同样是α9α10 nAChR的高选择性竞争性拮抗剂，能够治疗神经病理性疼痛。与结构紧凑的α-芋螺毒素相比，GeXIVA含有8个精氨酸，且结构较为松散，很容易被各种蛋白酶降解。为了提高GeXIVA的稳定性，我们合成了 7个GeXIVA的D-型氨基酸替代突变体，主要替换了 GeXIVA的C-和N-末端侧翼氨基酸和内部的L-Arg。电生理实验结果表明所有D-型氨基酸替代突变体都保持或提升了原有的活性，其中GeXIVA[15-18r]对α9α10nAChR的活性提升了约2.5倍。稳定性实验表明，所有突变体在人血清中的稳定性都有提升，其中GeXIVA [all-r]和GeXIVA [1t，28v，all-r]两种突变体稳定性增加最为明显，在人血清中的半衰期从30分钟提升至8-12小时，且GeXIVA [1t，28v，all-r]甚至可以在模拟肠液中存在90分钟。本研究与RgIA的改造共同揭示了恰当的D-型氨基酸取代既能够保留原始肽的生物活性，也可以增强芋螺毒素多肽的稳定性，从而提升芋螺毒素的药用价值。 α-CTx LvIA从Conus lividus中克隆获得，对α3β2 nAChR的活性非常强，且能够区分α3β2及非常接近的α6/α3β2β3 nAChR。本研究中，我们通过使用首尾化学连接法(HCL)合成了 3个具有不同氨基酸数量接头的LvIA环肽突变体，希望能够进一步提升它的稳定性。实验结果表明具有6个氨基酸接头的环化突变体能够最大程度保留其对α3β2nAChR的活性，同时在人血清中的稳定性也得到了提升，该研究结果表明环化是改善芋螺毒素稳定性的有效策略之一。 a-CTx TxID是本实验室从Conus textile中发现获得，属于4/6-型亚家族芋螺毒素，能够特异性抑制a3p4 nAChRo但TxID中的第11位甲硫氨酸(Met)在氧化环境下容易形成甲硫氨酸亚砜(MetO)结构，从而导致其活性显著降低。本研究中，我们选择了 8种不同的氨基酸替代甲硫氨酸，总共合成了 8个替换突变体。电生理实验结果显示大多数TxID的替换突变体对a3p4 nAChR活性降低超过10倍，其中有5个突变体对a3p4 nAChR完全没有抑制作用，唯有[M11I] TxID对a3(34 nAChR的活性保持最好，其半阻断剂量为69nM，与本体相比活性降低了 3.8倍。实验表明，采用较为疏水的异亮氨酸(Ile)替换Met是减少Met氧化对TxID影响的最佳策略，本研究也有助于设计改造序列中含有Met的芋螺毒素多肽。芋螺毒素含有的氨基酸数目较少，适合采用化学方法合成，这也让化学修饰改造芋螺毒素成为可能。上述研究表明，通过D-型氨基酸取代，环化和氨基酸突变等方法改造芋螺毒素，可以在保持芋螺毒素活性的同时提高其稳定性，从而提升芋螺毒素开发为药物的潜能。 关键词：α-芋螺毒素；D-型氨基酸取代；烟碱型乙酰胆碱受体；多肽合成；电生理；环化

 Conotoxins (CTxs) are a diverse group of evolved venom peptides used for prey capture or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage-or ligand-gated ion channels. The conopeptides are categorized into various superfamilies and classified by their structural and functional features, such as cysteine pattern and pharmacology. The largest family of conopeptides are competitive antagonists of nicotinic acetylcholine receptors (nAChRs), especially for α-CTxs. Previous researches have shown the nAChRs are implicated in the pathophysiology of a number of disease states including pain, epilepsy, Parkinson's disease, and addiction et al. Accordingly, CTxs inhibiting nAChRs have been proved to be valuable tools for identifying the composition and roles of nAChR subtypes and some of them have become either promising drug leads or novel pharmaceutics. However, one potential drawback of CTxs 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. In this study, three strategies for improving the stability or activity of CTxs has been successfully applied in four conotoxins. α-CTx RglA and αO-CTx GeXIVA were redesigned using D-amino acid substitution. α-CTx LvIA was cyclized and Met-11 of α-CTx TxID was mutated to other similar residues. α-CTx RglA is a selective and potent competitive antagonist of rat α9α10 nAChR, but it is much less potent on towards human versus rat α9α10 nAChR. Furthermore, RglA is more susceptible to proteolytic degradation due to containing four arginine residues. Therefore, we used the D-amino acid scan method to identify critical stereocenters of RglA and discover more stable analogues enhancing its bioavailability. The activity of each variant was investigated against rat and human α9α10 nAChRs, which were expressed in Xenopus oocytes. Experimental assays showed that 14 out of 15 analogues had a substantial reduction in potency towards rat α9α10 nAChR. Noticeably, RgIA[13r] retained full biological activity compared with RglA. Meanwhile, two other analogues RglA[7,9,11,13-r] and RgIA[7,9,11-r#], of which L-Args were substituted with D-Args, had significantly increased potency at the towards human α9α10 nAChR, although these analogues showed decreased their activities at the against rat α9α10 nAChR. Additionally, these three analogues exhibited high resistance against enzymatic degradation in human serum and simulated intestinal fluid (SIF). αO-CTx GeXIVA was cloned from Conus generalis in our lab which is an antagonist of α9α10 nAChRs and has therapeutic potential for the treatment of neuropathic pain. In contrast to other well-structured α-conotoxins, αO-GeXIVA has eight arginines and a flexible structure which lead to being degraded by proteases easily. In order to improve the stability of GeXIVA, we synthesized eight GeXIVA analogues which replaced the C- and N-terminal flanking residues or Arg residues with their D-amino acid at counterparts. Our data showed that all D-amino acid substitutions exhibited similar potency at α9α10 nAChR and other subtypes. Significantly, GeXIVA[15-l8r] analogs exhibited about 2.5-fold more potency for the α9α10 nAChR compared with the original peptide. Furthermore, GeXIVA[all-r] and GeXIVA[1t, 28v, all-r] exhibited high resistance against enzymatic degradation in human serum, and the half-life in human serum is extended from 30 min to 8-12 hours. Especially for GeXIVA[1t, 28v, all-r], it even existed in SIF for 90 min. Together, these findings suggest that suitable D-amino acid substitution in GeXIVA retain original activity and enhance stability which may improve potential therapeutic utility. α-CTx LvIA was cloned from Conus lividus, which was found the high potency on α3β2 nAChR. In addition, it has the ability to discriminate between similar neuronal α3β2 and α6/α3β2β3 nAChRs. Here, we improve the stability of LvIA by backbone cyclization using linkers head-to-tail chemical ligation method. Cyclization with a six-residue linker preserve the activity of α3β2 nAChR and improves stability in human serum. The results provide insights to further improve the therapeutic properties of LvIA and confirm that cyclization is an applicable strategy to improve the stability of peptides. α-CTx TxID was discovered from Conus textile by gene cloning in our lab, which has 4/6 inter-cysteine loop spacing and selectively inhibits α3β4 nAChR. However, TxID is susceptible to modification due to it containing a methionine (Met) residue that easily forms methionine sulfoxide (MetO) in an oxidative environment. In this study, we investigated how Met-11 and its derivatives affect the activity of TxID. The results showed most TxID analogues had substantially decreased activities on α3β4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on α3β4 nAChR. However, one mutant, [M11I]TxID, displayed potent inhibition at α3β4 nAChR with an IC₅₀ of 69 nM, which only exhibited 3.8-fold less compared with TxID. The results indicate replacement of Met with a hydrophobic moderate-sized Ile in TxID is an alternative strategy to reduce the impact of Met oxidation, which may help to redesign CTxs containing methionine residue. In summary, the relatively small size of conotoxins makes them very amenable to chemical synthesis. This opens the possibility of improving stability through synthetic chemical modifications. Our findings suggest that the substitution of D-amino acids, cyclization and amino acid mutations affect the structure and function of peptides and may facilitate the development of more stable analogues to increase conotoxins' therapeutic potential. Key words: Conotoxins; D-amino acid substitution; Nicotinic acetylcholine receptors; peptide synthesis; Electrophysiology; Cyclization