Comprehensive mechanistic analysis of hits from high-throughput and docking screens against β-lactamase
Comprehensive mechanistic analysis of hits from high-throughput and docking screens against β-lactamase
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DOI：
10.1021/jm701500e
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发表时间：
2008-04-24
影响因子：
7.3
通讯作者：
Shoichet, Brian K.
中科院分区：
文献类型：
--
作者：
Babaoglu, Kerim;Simeonov, Anton;Shoichet, Brian K.
高通量筛选（HTS）广泛应用于药物发现。特别是对于无偏见的库的筛选，假阳性可以主导“命中列表”;它们的起源有很多争议。在这里，我们使用定量HTS（qHTS）从70，563个针对β-内酰胺酶的无偏倚分子的筛选中确定每个活性命中的机制。在1274种初始抑制剂中，95%对洗涤剂敏感，并被归类为聚集剂。在剩下的70种药物中，有25种是强效的共价作用β-内酰胺类药物。质谱、反筛和晶体学鉴定了12种混杂共价抑制剂。剩下的33个要么是聚合剂，要么是不可复制的。没有发现特异的可逆抑制剂，我们转向分子对接，从同一个库中优先选择分子，以进行更高浓度的测试。在16个测试中，2个是适度抑制剂。随后的X射线结构与对接预测相对应。类似物合成将亲和力提高到8 μ M。这些结果表明，这可能是有机分子的物理行为，而不是他们的反应，占大多数筛选文物。基于结构的方法可以在无偏文库筛选中优先考虑弱但新的化学型。
High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate "hit lists"; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against P-lactamase using quantitative HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent- sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting beta-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 mu M. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.