Epitope targeting of tertiary protein structure enables target-guided synthesis of a potent in-cell inhibitor of botulinum neurotoxin.
Epitope targeting of tertiary protein structure enables target-guided synthesis of a potent in-cell inhibitor of botulinum neurotoxin.
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DOI：
10.1002/anie.201502451
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发表时间：
2015-06-08
期刊：
影响因子：
--
通讯作者：
Heath JR
中科院分区：
文献类型：
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作者：
Farrow B;Wong M;Malette J;Lai B;Deyle KM;Das S;Nag A;Agnew HD;Heath JR
肉毒神经毒素（BoNT）血清型A是已知的最致命的毒素，并且具有封闭结构，该封闭结构防止其活性位点在其进入胞质溶胶之前被直接抑制。我们将联合收割机原位点击靶向引导合成与合成表位靶向结合起来，利用BoNT蛋白的三级结构作为组装竞争性抑制剂的景观。模拟肽大环的底物用作BoNT的直接抑制剂。利用靶向表位的原位点击筛选来鉴定第二肽大环配体，所述第二肽大环配体结合在折叠BoNT结构中与活性位点相邻的表位。第二个原位点击屏幕识别两个大环之间的分子桥。所得二价抑制剂在体外对BoNT/A催化链表现出165 pM的抑制常数。该抑制剂由完整的全毒素携带到细胞中，并在人神经元模型中证明了对BoNT中毒的保护和拯救。
Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure which prevents direct inhibition of its active site before it enters the cytosol. We combine in situ click target-guided synthesis with synthetic epitope-targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope targeted in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active site adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an inhibition constant of 165 pM in vitro against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model.