A specific mechanism of nonspecific inhibition
A specific mechanism of nonspecific inhibition
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DOI：
10.1021/jm030266r
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发表时间：
2003-09-25
影响因子：
7.3
通讯作者：
Shoichet, BK
中科院分区：
文献类型：
--
作者：
McGovern, SL;Helfand, BT;Shoichet, BK
混杂的小分子困扰着筛选库和黑名单。先前的研究发现，几种非特异性化合物形成亚微米聚集体，并提出这种聚集体对许多不同的酶有抑制作用。目前尚不清楚聚集体如何抑制它们的目标。为了解决这个问题，生物物理、动力学和显微镜方法被用来研究混杂的、聚集形成抑制剂与模型蛋白的相互作用。通过离心和凝胶电泳，发现聚集体和蛋白质直接相互作用。这与随后共聚焦荧光显微镜观察到的绿色荧光蛋白聚集一致。与野生型酶相比，β -内酰胺酶突变体的热力学稳定性增加或降低，同样受到聚集形成化合物的抑制，这表明通过展开变性不是相互作用的主要机制。相反，通过电子显微镜的可视化显示，酶与抑制剂聚集体的表面结合。这种关联可以通过添加Triton X-100来逆转或阻止。这些观察结果表明，混杂化合物形成的聚集体可逆地隔离酶，导致明显的抑制。他们还提出了一种简单的方法来识别或逆转基于聚集体的抑制剂的作用，这种抑制剂似乎很普遍。
Promiscuous small molecules plague screening libraries and hit lists. Previous work has found that several nonspecific compounds form submicrometer aggregates, and it has been suggested that this aggregate species is responsible for the inhibition of many different enzymes. It is not understood how aggregates inhibit their targets. To address this question, biophysical, kinetic, and microscopy methods were used to study the interaction of promiscuous, aggregate-forming inhibitors with model proteins. By use of centrifugation and gel electrophoresis, aggregates and protein were found to directly interact. This is consistent with a subsequent observation from confocal fluorescence microscopy that aggregates concentrate green fluorescent protein. beta-Lactamase mutants with increased or decreased thermodynamic stability relative to wild-type enzyme were equally inhibited by an aggregate-forming compound, suggesting that denaturation by unfolding was not the primary mechanism of interaction. Instead, visualization by electron microscopy revealed that enzyme associates with the surface of inhibitor aggregates. This association could be reversed or prevented by the addition of Triton X-100. These observations suggest that the aggregates formed by promiscuous compounds reversibly sequester enzyme, resulting in apparent inhibition. They also suggest a simple method to identify or reverse the action of aggregate-based inhibitors, which appear to be widespread.