Beijing Institute of Biotechnology, Beijing 100071, China.
Institute of Chemistry and Chemical Engineering, University of South China, Henyang 421000, China.
Mar Drugs. 2021 Dec 14;19(12):705. doi: 10.3390/md19120705.
α-Conotoxins GI and MI belong to the 3/5 subfamily of α-conotoxins and potently inhibit muscular nicotinic acetylcholine receptors (nAChRs). To date, no 3/4- or 3/6-subfamily α-conotoxins have been reported to inhibit muscular nAChRs. In the present study, a series of new 3/4-, 3/6-, and 3/7-subfamily GI and MI variants were synthesized and functionally characterized by modifications of loop2. The results show that the 3/4-subfamily GI variant GI[∆8G]-II and the 3/6-subfamily variants GI[+13A], GI[+13R], and GI[+13K] displayed potent inhibition of muscular nAChRs expressed in oocytes, with an IC of 45.4-73.4 nM, similar to or slightly lower than that of wild-type GI (42.0 nM). The toxicity of these GI variants in mice appeared to be about a half to a quarter of that of wild-type GI. At the same time, the 3/7-subfamily GI variants showed significantly lower in vitro potency and toxicity. On the other hand, similar to the 3/6-subfamily GI variants, the 3/6-subfamily MI variants MI[+14R] and MI[+14K] were also active after the addition of a basic amino acid, Arg or Lys, in loop2, but the activity was not maintained for the 3/4-subfamily MI variant MI[∆9G]. Interestingly, the disulfide bond connectivity "C1-C4, C2-C3" in the 3/4-subfamily variant GI[∆8G]-II was significantly more potent than the "C1-C3, C2-C4" connectivity found in wild-type GI and MI, suggesting that disulfide bond connectivity is easily affected in the rigid 3/4-subfamily α-conotoxins and that the disulfide bonds significantly impact the variants' function. This work is the first to demonstrate that 3/4- and 3/6-subfamily α-conotoxins potently inhibit muscular nAChRs, expanding our knowledge of α-conotoxins and providing new motifs for their further modifications.
α-芋螺毒素 GI 和 MI 属于α-芋螺毒素的 3/5 亚家族，能强有力地抑制肌肉型烟碱型乙酰胆碱受体（nAChRs）。迄今为止，尚未有报道称 3/4- 或 3/6-亚家族的 α-芋螺毒素能够抑制肌肉型 nAChRs。在本研究中，通过修饰环 2，合成了一系列新的 3/4-、3/6-和 3/7-亚家族 GI 和 MI 变体，并对其功能进行了表征。结果表明，3/4-亚家族 GI 变体 GI[∆8G]-II 和 3/6-亚家族变体 GI[+13A]、GI[+13R]和 GI[+13K]对在卵母细胞中表达的肌肉型 nAChRs 表现出强有力的抑制作用，IC 为 45.4-73.4 nM，与野生型 GI（42.0 nM）相似或略低。这些 GI 变体在小鼠中的毒性似乎约为野生型 GI 的一半至四分之一。与此同时，3/7-亚家族 GI 变体显示出明显较低的体外效力和毒性。另一方面，与 3/6-亚家族 GI 变体类似，在环 2 中添加碱性氨基酸精氨酸或赖氨酸后，3/6-亚家族 MI 变体 MI[+14R]和 MI[+14K]也具有活性，但 3/4-亚家族 MI 变体 MI[∆9G]的活性无法维持。有趣的是，3/4-亚家族变体 GI[∆8G]-II 中的二硫键连接“C1-C4，C2-C3”比野生型 GI 和 MI 中的“C1-C3，C2-C4”连接更有效，这表明在刚性 3/4-亚家族 α-芋螺毒素中二硫键连接容易受到影响，并且二硫键显著影响变体的功能。这项工作首次证明 3/4- 和 3/6-亚家族 α-芋螺毒素能强有力地抑制肌肉型 nAChRs，扩展了我们对 α-芋螺毒素的认识，并为进一步修饰它们提供了新的模式。
