Institute for Molecular Bioscience , The University of Queensland , Brisbane , Queensland 4072 , Australia.
Bioconjug Chem. 2018 Oct 17;29(10):3309-3319. doi: 10.1021/acs.bioconjchem.8b00505. Epub 2018 Sep 12.
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 (Na1.7), 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 Na1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.
从蜘蛛毒液中分离出的门控修饰毒素是富含二硫键的肽，通常由稳定的抑制剂半胱氨酸结（ICK）组成。这些 knottin 肽被作为治疗一系列与跨膜蛋白相关疾病的治疗靶点。最近，在蜘蛛毒液中发现并通过重组表达产生的双 knottin 肽为研究跨膜通道的药理学提供了新的思路。在这里，我们使用化学酶连接来产生双 knottin 肽，以研究二价调节对电压门控钠离子通道亚型 1.7（Na1.7）的影响，Na1.7 与疼痛信号有关。单价 knottin 肽经过氧化折叠，然后使用 sortase A 进行生物化学偶联，形成双 knottin 肽。使用 NMR 确认了肽的结构完整性，荧光活性测定提供的证据表明，共孵育的单价和二价 knottin 可以协同调节 Na1.7。我们预计双 knottin 肽将为增强我们对治疗相关电压门控离子通道的理解和设计策略提供新的工具。
