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Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two Na V 1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNa V 1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.

μ-Conotoxins are components of cone snail venom, wellknown for their analgesic activity through potent inhibition of voltage-gated sodium channel (NaV) subtypes, including NaV1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a 'native' CysICysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at NaV1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIIICysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human NaV channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits NaV subtypes with a rank order of potency of NaV1.4 > 1.2 > 1.7 and Isomer 2 in the order of NaV1.4≈1.2 > 1.7, while the native isomer inhibited NaV1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNaV1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant NaV subtypes. [ABSTRACT FROM AUTHOR]