Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides

Abstract

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 NaV1.7 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 hNaV1.7 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.

Site-Specific Fluorescent Labeling of the Cysteine-Rich Toxin, DkTx, for TRPV1 Ion Channel Imaging and Membrane Binding Studies

“…47,48 Toward this end, we were encouraged by the Schroeder research group's work on ligating disulfide-rich toxins by using sortase A. 49,50 To perform sortase A-mediated bioconjugation on DkTx, we recombinantly produced wild-type sortase A in E. coli (Figure 5a), a DkTx variant containing the G 3 SLPETG 2 H 6 sequence on its C-terminus, and a triglycine (G 3 )-conjugated fluoresceinamine dye (3) (Figure 5b). HPLC analysis of the sortase A-catalyzed bioconjugation reaction of the DkTx variant and compound 3 yielded the desired labeled toxin at 32.3 min, along with peaks for sortase A and the unreacted dye at 25.5 and 17.5 min, respectively (Figure 5c).…”

“…Next, we attempted to label the C -terminus of DkTx by employing the sortase A enzyme that catalyzes the formation of a peptide linkage between the threonine residue of the LPXTG sequence (X being any amino acid) present on the C -terminus of a protein with a probe containing N -terminal poly-Gly. , Toward this end, we were encouraged by the Schroeder research group’s work on ligating disulfide-rich toxins by using sortase A. , …”

Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors

“…A number of synthetic strategies were utilized to join the NTD and CTD using different linkers and ligation chemistries. 17 As each CTD of VxXXB has four disulfide bonds and the NTD is formed by two interchain disulfide bonds, we considered that the minimum requirement for the synthesis of VxXXB was for each domain to be oxidized prior to ligation to ensure correct disulfide bond connectivities are achieved. A requirement of this approach is to avoid reducing conditions that would potentially allow disulfide bond shuffling during the ligation reaction.…”

“…Recent work characterizing enzymatic ligation approaches to chemical ligation by KAHA, revealed the latter efficiently ligated complex disulfide-rich peptides without disulfide bond rearrangement. 17 Remarkably, KAHA ligated peptides had high serum stability, possibly owing to the unnatural amino acid h Ser resisting the recognition of protease enzyme. 17 Both the full length and truncated NTD variants had no significant effect on potency of the NC variant compared to CTD(19–50).…”

“…17 Remarkably, KAHA ligated peptides had high serum stability, possibly owing to the unnatural amino acid h Ser resisting the recognition of protease enzyme. 17 Both the full length and truncated NTD variants had no significant effect on potency of the NC variant compared to CTD(19–50). Although the insertion of –L h S– between the NTD and CTD may affect potency, this finding supports a secondary role of NTD in VxXXB potency.…”