Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.
Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.
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DOI：
10.1021/acs.bioconjchem.1c00452
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发表时间：
2021-11-17
影响因子：
4.7
通讯作者：
Schroeder, Christina, I
中科院分区：
文献类型：
--
作者：
Tran, Hue N. T.;Tran, Poanna;Deuis, Jennifer R.;McMahon, Kirsten L.;Yap, Kuok;Craik, David J.;Vetter, Irina;Schroeder, Christina, I
在毒液中鉴定的双结肽和靶向离子通道的合成二价肽构建体是用于研究离子通道药理学和生理学的新兴工具。这些高度复杂且富含二硫键的肽含有两个单独的胱氨酸结，每个胱氨酸结包含六个半胱氨酸和三个二硫键。到目前为止，天然的双结肽，如Hi 1a和DkTx，仅从毒液中分离或重组产生，而工程化的双结肽已成功地通过酶促连接使用分选酶A在两个打结毒素之间的连接位点形成无缝酰胺键，并通过炔/叠氮化物点击化学，通过三唑键连接两个肽结。为了进一步追求这些双结肽作为治疗相关离子通道的药理学工具或探针，我们试图确定一种稳健的方法，该方法产生高产率产物，该产物适合于快速生产和容易的突变研究。在这项研究中，我们评估了酶促（分选酶A5°、butelase 1、野生型OaAEP 1、C247 A-OaAEP 1和肽酶）和温和化学方法（α-酮酸-羟胺，KAHA）用于形成连接毒素的天然酰胺键同时保持每个预折叠肽的天然二硫键连接的连接效率。我们使用了两种NaV1.7抑制剂：PaurTx 3，一种蜘蛛衍生的门控修饰肽，和KIIIA，一种小锥螺衍生的孔阻断肽，这两种肽先前已被证明在连接在一起时增加对hNaV1.7的亲和力和抑制效力。正确折叠的肽被成功地连接在不同的产量，没有二硫键改组或还原，分选酶A5°是最有效的，导致60%的连接转化率在15分钟内。此外，电生理学研究表明，这两个肽，接头的氨基酸组成不影响双结肽的活性。本研究证明了酶在高效连接富含复杂二硫键的肽中的强大应用，为轻松生产双结肽铺平了道路。
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.
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影响因子：
14.8
作者：
通讯作者：
--
影响因子：
4.8
作者：
Deuis, Jennifer R.;Dekan, Zoltan;Vetter, Irina
通讯作者：
Vetter, Irina
影响因子：
56.9
作者：
DAWSON, PE;MUIR, TW;KENT, SBH
通讯作者：
KENT, SBH
DOI：
10.1007/978-1-4939-9546-2_6
发表时间：
2019-01-01
期刊：
ENZYME-MEDIATED LIGATION METHODS
影响因子：
--
作者：
Hemu, Xinya;Zhang, Xiaohong;Tam, James P.
通讯作者：
Tam, James P.
影响因子：
7.3
作者：
Gunasekera, Sunithi;Foley, Fiona M.;Daly, Norelle L.
通讯作者：
Daly, Norelle L.