Promiscuous aggregate-based inhibitors promote enzyme unfolding.
Promiscuous aggregate-based inhibitors promote enzyme unfolding.
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DOI：
10.1021/jm801605r
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发表时间：
2009-04-09
影响因子：
7.3
通讯作者：
Shoichet BK
中科院分区：
文献类型：
--
作者：
Coan KE;Maltby DA;Burlingame AL;Shoichet BK
早期药物发现中假阳性的主要来源之一是有机小分子聚集体的形成，其在水溶液中以微摩尔浓度非特异性地抑制酶。这一普遍问题的分子基础仍然模糊不清。为了在分子水平上研究抑制机制，我们使用氢氘交换质谱法确定了当酶与聚集体结合时溶剂可及性的变化。对于AmpC β-内酰胺酶，与小分子rottlerin聚集体的结合增加了所有10个可重复检测的肽的氘交换，其覆盖了β-内酰胺酶序列的41%。这表明聚合物结合后质子可及性总体增加，与变性一致。然后，我们研究了酶-聚集体复合物是否比未受抑制的酶更容易发生蛋白水解。对于5种聚集体，当β-内酰胺酶被聚集体抑制时，胰蛋白酶对β-内酰胺酶的降解显著增加，而未被抑制的酶通常对消化稳定。结合起来，这些结果表明，基于聚集体的抑制剂的作用机制通过结合到聚集体颗粒时的部分蛋白质解折叠进行。
One of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates, which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution. The molecular basis for this widespread problem remains hazy. To investigate the mechanism of inhibition at a molecular level, we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogen−deuterium exchange mass spectrometry. For AmpC β-lactamase, binding to aggregates of the small molecule rottlerin increased the deuterium exchange of all 10 reproducibly detectable peptides, which covered 41% of the sequence of β-lactamase. This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation. We then investigated whether enzyme−aggregate complexes were more susceptible to proteolysis than uninhibited enzyme. For five aggregators, trypsin degradation of β-lactamase increased substantially when β-lactamase was inhibited by aggregates, whereas uninhibited enzyme was generally stable to digestion. Combined, these results suggest that the mechanism of action of aggregate-based inhibitors proceeds via partial protein unfolding when bound to an aggregate particle.
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影响因子：
7.3
作者：
McGovern, SL;Helfand, BT;Shoichet, BK
通讯作者：
Shoichet, BK
影响因子：
--
作者：
Coan, Kristin E. D.;Shoichet, Brian K.
通讯作者：
Shoichet, Brian K.
影响因子：
15
作者：
Coan, Kristin E. D.;Shoichet, Brian K.
通讯作者：
Shoichet, Brian K.
影响因子：
14.8
作者：
Feng, Brian Y.;Toyama, Brandon H.;Shoichet, Brian K.
通讯作者：
Shoichet, Brian K.
影响因子：
7.4
作者：
Mandell, JG;Falick, AM;Komives, EA
通讯作者：
Komives, EA