Poster Presentation 8th Modern Solid Phase Peptide Synthesis & Its Applications Symposium 2022

Evaluation of efficient non-reducing enzymatic and chemical ligation strategies for complex disulfide-rich peptides (#142)

Hue Tran 1 , Poanna Tran 1 , Jennifer R Deuis 1 , KuoK Yap 1 , David J Craik 1 , Irina Vetter 1 2 , Christina I Schroeder 3
  1. Institute for Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia
  2. School of Pharmacy, The University of Queensland, Brisbane, QLD, Australia
  3. National Cancer Institute, National Institutes of Health, Frederick, MD, USA

Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are useful tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides comprise two individual cystine knots, each of which comprises 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. 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 through the use of alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. In order to further pursue these double-knotted peptides as pharmacological tools or leads for therapeutically relevant ion channels, we wanted 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 A, butelase 1, OaAEP1s, and peptiligase) and a mild chemical approach (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins, while maintaining the native disulfide connectivity of each pre-folded peptide. Two known NaV1.7 inhibitors–PaurTx3, a gate modifier spider-derived toxin, and KIIIA, a small pore blocker cone snail-derived peptide–whose bivalents were previously shown to increase affinity and inhibitory potency on hNaV1.7 were used. All correctly folded peptides were successfully ligated, without disulfide-bond shuffling or reduction, in varying yields using all methods, with sortase A5º being the most efficient. In addition, electrophysiology studies demonstrated that 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 and the importance of folding the individual peptides prior to ligation.