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 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 EC 50 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 EC 50 values (e.g., Na V 1.1: 7.9 nM (α-6); Na V 1.3: 9.4 nM (α-5); Na V 1.4: 14.6 nM (α-4)) this underscores the potential utility of α-nemertides for rational optimization to improve selectivity.

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C–G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I–IV, II–III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC 50 0.64 μM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.