High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase
High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase
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DOI：
10.1016/j.bmc.2007.01.004
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发表时间：
2007-03-01
影响因子：
3.5
通讯作者：
Matts, Robert L.
中科院分区：
文献类型：
--
作者：
Galam, Lakshmi;Hadden, M. Kyle;Matts, Robert L.
以前，我们已经证明热变性萤火虫荧光素酶的复性依赖于兔网织红细胞裂解物中Hsp 90的活性。在这里，我们表明，该试验可以确定抑制剂，阻碍伴侣蛋白的活性热休克蛋白90通过直接结合到其N-末端或C-末端核苷酸结合位点或通过干扰的伴侣蛋白的能力，以开关构象。该测定法进行了调整和优化，用于高通量筛选。筛选了超过20，000种化合物，以证明大规模使用该测定法的可行性。该测定法是可重复的（平均Z因子= 0.62），并确定了120种化合物，抑制荧光素酶复性大于70%，浓度为12.5 μ g/mL。测定了20种不同结构化合物抑制荧光素酶重折叠的IC 50值，并在基于细胞的测定中测定了Hsp 90抑制。几种化合物的IC 50值< 10 μ M，代表了许多新的先导结构，具有进一步开发和优化作为有效的Hsp 90抑制剂的潜力。(c)2007爱思唯尔有限公司版权所有。
Previously, we have demonstrated that the renaturation of heat denatured firefly luciferase is dependent Upon the activity of Hsp90 in rabbit reticulocyte lysate. Here, we demonstrate that this assay may identify inhibitors that obstruct the chaperone activity of Hsp90 either by direct binding to its N-terminal or C-terminal nucleotide binding sites or by interference with the ability of the chaperone to switch conformations. The assay was adapted and optimized for high-throughput screening. Greater than 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (av Z-factor = 0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 mu g/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several Compounds had IC50 values < 10 mu M and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors. (c) 2007 Elsevier Ltd. All rights reserved.