A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.
A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.
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DOI：
10.3390/bioengineering9100511
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发表时间：
2022-09-27
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产滋贺毒素E.大肠杆菌（STEC）是血性腹泻的常见原因。STEC感染的病理学来源于两种外毒素-滋贺毒素1（Stx 1）和滋贺毒素2（Stx 2）-其由STEC在肠道中分泌，从肠道中它们被全身吸收，引起严重的肾损伤，导致溶血性尿毒症综合征（HUS）。目前，HUS没有有效的治疗方法，仅推荐支持性治疗。我们报告了一组设计的锚蛋白重复序列蛋白（DARPin）的工程设计，对Stx 2a，与HUS相关的主要亚型具有有效的中和活性。通过定向进化和合理设计的组合产生的最佳二聚DARPin，SD 5，在体外以0.61 nM的半最大有效浓度（EC 50）中和Stx 2a。SD 5的两种单体DARPin组分表现出互补的功能-SHT靶向Stx 2a的酶促A亚基并抑制毒素的催化活性，而DARPin#3结合B亚基，基于冷冻-EM研究，并诱导B亚基中的新构象变化，其扭曲其五重对称性并可能干扰毒素附着到靶细胞。将SD 5融合到白蛋白结合DARPin，并且所得的三聚体DARPin DA 1-SD 5在毒素攻击模型中有效地保护小鼠，表明该DARPin作为STEC感染的治疗剂的高潜力。最后，DARPin#3诱导的前所未有的毒素构象变化代表了中和Stx 2毒性的新作用模式，并揭示了未来药物开发的新靶点。
Shiga toxin-producing E. coli (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins—Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)—that are secreted by STEC in the gut, from where they are systemically absorbed, causing severe kidney damage leading to hemolytic uremic syndrome (HUS). Currently, there is no effective treatment for HUS, and only supportive care is recommended. We report the engineering of a panel of designed ankyrin repeat proteins (DARPin) with potent neutralization activity against Stx2a, the major subtype associated with HUS. The best dimeric DARPin, SD5, created via a combination of directed evolution and rational design, neutralizes Stx2a with a half maximal effective concentration (EC50) of 0.61 nM in vitro. The two monomeric DARPin constituents of SD5 exhibit complementary functions—SHT targets the enzymatic A subunit of Stx2a and inhibits the toxin’s catalytic activity, while DARPin #3 binds the B subunit, based on the cryo-EM study, and induces a novel conformational change in the B subunit that distorts its five-fold symmetry and presumably interferes with toxin attachment to target cells. SD5 was fused to an albumin-binding DARPin, and the resulting trimeric DARPin DA1-SD5 efficiently protects mice in a toxin challenge model, pointing to a high potential of this DARPin as a therapeutic for STEC infection. Finally, the unprecedented toxin conformational change induced by DARPin #3 represents a novel mode of action for neutralizing Stx2 toxicity and reveals new targets for future drug development.
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影响因子：
5.6
作者：
Binz, HK;Stumpp, MT;Plückthun, A
通讯作者：
Plückthun, A
影响因子：
9.4
作者：
Boerlin, P;McEwen, SA;Gyles, CL
通讯作者：
Gyles, CL
DOI：
10.1007/978-0-387-79838-7_1
发表时间：
2009-01-01
期刊：
HOT TOPICS IN INFECTION AND IMMUNITY IN CHILDREN V
影响因子：
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作者：
Ahn, Christina K.;Holt, Nicholas J.;Tarr, Phillip I.
通讯作者：
Tarr, Phillip I.
DOI：
10.1107/s2059798319011471
发表时间：
2019-10-01
影响因子：
2.2
作者：
Liebschner, Dorothee;Afonine, Pavel V.;Adams, Paul D.
通讯作者：
Adams, Paul D.
影响因子：
11.8
作者：
Kimmitt, PT;Harwood, CR;Barer, MR
通讯作者：
Barer, MR