Background

A natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A).

Methods

In this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC.

Results

Studies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site.

Conclusion

The NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT.

General significance

Insights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.

中文翻译：

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与药物设计相关的肉毒杆菌神经毒素抑制剂结合动力学和动力学

背景

天然产物类似物 3-(4-nitrophenyl)-7 H - furo[3,2 - g ]chromen-7-one，是一种硝基苯基补骨脂素 (NPP)，被发现是 A 型肉毒杆菌神经毒素的有效抑制剂(BoNT/A)。

方法

在这项工作中，我们进行了酶抑制动力学，并采用等温量热法 (ITC) 和荧光光谱法等生化技术以及分子建模来检查 NPP 抑制剂与 BoNT/A LC 的动力学和结合机制。

结果

NPP 与 BoNT/A 轻链 (BoNT/A LC) 的抑制机制和结合动力学研究表明，NPP 是锌内肽酶活性的混合型抑制剂，这意味着至少部分抑制剂-酶结合位点可能不同来自底物-酶结合位点。通过使用生化技术，我们证明了 NPP 与 BoNT/A LC 形成稳定的复合物。这些观察结果通过分子动力学 (MD) 模拟得到证实，这表明 NPP 与活性位点附近的位点结合。

结论

NPP 结合会干扰 BoNT/A LC 与 SNAP-25 的结合，从而抑制其切割。基于这些结果，我们提出了一种改进的策略来设计一种分子，以提高对 BoNT 神经毒性作用的抑制效率。

一般意义

使用生化和计算方法深入了解 NPP 与 BoNT/A LC 的相互作用，将有助于未来开发有效的对策和更好的对抗肉毒杆菌中毒的药理学策略。