Peptide toxins find use in medicine, biotechnology, and agriculture. They are exploited as pharmaceutical tools, particularly for the investigation of ion channels. Here, we report the synthesis and activity of a novel family of peptide toxins: the cystine-knotted α nemertides. Following the prototypic α-1 and -2 (1 and 2), six more nemertides were discovered by mining of available nemertean transcriptomes. Here, we describe their synthesis using solid phase peptide chemistry and their oxidative folding by using an improved protocol. Nemertides α-2 to α-7 (2–7) were produced to characterize their effect on voltage-gated sodium channels (Blatella germanica BgNa_V1 and mammalian Na_Vs1.1–1.8). In addition, ion channel activities were matched to in vivo tests using an Artemia microwell assay. Although nemertides demonstrate high sequence similarity, they display variability in activity on the tested Na_Vs. The nemertides are all highly toxic to Artemia, with EC₅₀ values in the sub-low micromolar range, and all manifest preference for the insect BgNa_V1 channel. Structure–activity relationship analysis revealed key residues for Na_V-subtype selectivity. Combined with low EC₅₀ values (e.g., Na_V1.1: 7.9 nM (α-6); Na_V1.3: 9.4 nM (α-5); Na_V1.4: 14.6 nM (α-4)) this underscores the potential utility of α-nemertides for rational optimization to improve selectivity.

中文翻译：

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Nemertide α 肽毒素家族的功能表征

肽毒素可用于医学、生物技术和农业。它们被用作制药工具，特别是用于研究离子通道。在这里，我们报告了一个新的肽毒素家族的合成和活性：胱氨酸结的 α nemertides。在原型 α-1 和 -2（1和2）之后，通过挖掘可用的纽虫转录组发现了另外六种纽虫。在这里，我们使用改进的协议描述了它们使用固相肽化学的合成及其氧化折叠。生产 Nemertides α-2 到 α-7 ( 2 – 7 ) 以表征它们对电压门控钠通道（德国小蠊BgNa _V 1 和哺乳动物 Na_V s1.1–1.8）。此外，离子通道活性与使用卤虫微孔分析的体内测试相匹配。尽管 nemertides 表现出高度的序列相似性，但它们在测试的 Na _V s上显示出活性的可变性。nemertides 对卤虫都具有高毒性，EC ₅₀值在亚低微摩尔范围内，并且都表现出对昆虫 BgNa _V 1 通道的偏好。构效关系分析揭示了 Na _V亚型选择性的关键残基。结合低 EC ₅₀值（例如，Na _V 1.1：7.9 nM (α-6)；Na _V 1.3：9.4 nM (α-5)；Na _V1.4: 14.6 nM (α-4)) 这强调了 α-nemertides 在合理优化以提高选择性方面的潜在效用。