A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution.
A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution.
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DOI：
10.1021/jm8014525
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发表时间：
2009-03-12
影响因子：
7.3
通讯作者：
Inglese J
中科院分区：
文献类型：
--
作者：
Auld DS;Zhang YQ;Southall NT;Rai G;Landsman M;MacLure J;Langevin D;Thomas CJ;Austin CP;Inglese J
我们测量了来自萤火虫 Photuris pennsylvanica 的 ATP 依赖性荧光素酶的“成药性”，该荧光素酶使用定向进化（Ultra-Glo™，Promega）进行了优化。使用定量高通量筛选 (qHTS) 来确定 198,899 个样品针对 Ultra-Glo 荧光素酶 (Kinase-Glo™) 制剂的 IC50。我们发现，只有 0.1% 的激酶-Glo 抑制剂表现出 IC50 < 10 μM，而之前的 qHTS 针对萤火虫荧光素酶 (lucPpy) 的 IC50 为 0.9%。此外，lucPpy qHTS 中鉴定的最大亲和力为 50 nM，而对于 Kinase-Glo，该值增加至 600 nM。 Ultra-Glo 荧光素酶使具有延伸到荧光素结合袋之外的相互作用的化合物大部分丢失。因此，与 lucPpy 相比，Ultra-Glo 荧光素酶用作 ATP 传感器时会表现出更少的化合物干扰。这项研究展示了结构-活性关系（>100K 化合物）的大规模定量分析在解决靶标成药性等重要问题方面的力量。
We measured the “druggability” of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo™, Promega). Quantitative high throughput screening (qHTS) was used to determine IC50’s of 198,899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo™). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC50 < 10 μM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.
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影响因子：
4.1
作者：
Lembert, N
通讯作者：
Lembert, N
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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作者：
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通讯作者：
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