A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates
A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates
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DOI：
10.1016/j.ab.2009.03.049
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发表时间：
2009-07-15
影响因子：
2.9
通讯作者：
Sisido, Masahiko
中科院分区：
文献类型：
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作者：
Nakata, Hidetaka;Ohtsuki, Takashi;Sisido, Masahiko
我们开发了新的底物，用于用荧光相关光谱(FCS)评价蛋白酶活性。底物用N端附近的荧光团以位置特异的方式标记，并包括一个C末端，30 kDa，高度可溶的蛋白质(伸长因子ts[Ef-ts])。C-末端蛋白提高了底物多肽的溶解度，增加了分子量，使FCS能够灵敏地检测。利用标记的底物，FCS检测caspase-3和基质金属蛋白酶-9(MMP9)的活性。为了证明这种基于FCS的检测方法在高通量筛选中的适用性，我们筛选了各种化合物作为基质金属蛋白酶-9抑制剂。筛选结果证实了一种化合物的抑制活性，也揭示了另一种潜在的基质金属蛋白酶-9抑制剂。因此，这种位置特异性标记蛋白底物和FCS的结合可以作为一种有用的工具来评估各种蛋白酶的活性和筛选蛋白酶抑制剂。(C)2009 Elsevier Inc.保留所有权利。
We developed novel Substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide Solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the Suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein Substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening. (C) 2009 Elsevier Inc. All rights reserved.