Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV 1.7

Bioconjugate Chemistry
Akello J AgwaChristina I Schroeder

Abstract

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.

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Citations

Sep 14, 2019·Journal of Peptide Science : an Official Publication of the European Peptide Society·Sutharsana YathursanVijayalekshmi Sarojini
Dec 19, 2019·Frontiers in Chemistry·Timo NuijensWalter Cabri
Jun 17, 2020·Biochemical Society Transactions·Clara L Frazier, Amy M Weeks
Mar 4, 2021·Bioconjugate Chemistry·Mengxin QianJun Feng

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