环肽是环肽,是设计候选药物和化学工具的有希望的支架。然而,尽管有数百个环潮报告,药物设计研究通常集中在精选的几个原型例子上。这里,我们探讨了祖先序列重建是否可以用于生成新的环肽以进行进一步优化。我们证明了重建的“伪祖先”序列,命名为Ancy-m(代表莫比乌斯亚家族的祖先环潮)和Ancy-b(代表手镯亚家族),有明确的结构,就像它们现有的成员一样,包括环状胱氨酸结的核心结构特征。此主题支持重新设计用于农业化学和治疗应用的环肽的努力。我们进一步表明,重建序列对接近沸腾的温度具有抵抗力,以微摩尔亲和力结合磷脂酰乙醇胺脂质双层,并在微摩尔范围内的抑制浓度下抑制昆虫细胞的生长。有趣的是,Ancy-bcyclotide的氧化折叠产率高于其比较的cyclotidecyO2,后者属于众所周知难以折叠的手镯cyclotide亚家族。总的来说,这项研究提供了尚未自然发现的新的环肽序列,这可能是理解环肽进化和进一步优化药物先导的有价值的起点。
Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed \'pseudo-ancestral\' sequences, named Ancy-m (for the ancestral cyclotide of the Möbius sub-family) and Ancy-b (for the bracelet sub-family), have well-defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re-engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl-ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy-b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.