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 alpha nemertides. Following the prototypic alpha-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 alpha-2 to alpha-7 (2-7) were produced to characterize their effect on voltage-gated sodium channels (Blatella germanica BgNa(V)1 and mammalian Na(V)s1.1-1.8). 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(V)s. 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(V)1 channel. Structure-activity relationship analysis revealed key residues for Na-V-subtype selectivity. Combined with low EC50 values (e.g., Na(V)1.1: 7.9 nM (alpha-6); Na(V)1.3: 9.4 nM (alpha-5); Na(V)1.4: 14.6 nM (alpha-4)) this underscores the potential utility of alpha-nemertides for rational optimization to improve selectivity
