Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.
Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.
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DOI：
10.1177/1087057113510740
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发表时间：
2014-02
影响因子：
--
通讯作者：
Thomas DD
中科院分区：
文献类型：
--
作者：
Gruber SJ;Cornea RL;Li J;Peterson KC;Schaaf TM;Gillispie GD;Dahl R;Zsebo KM;Robia SL;Thomas DD
我们已经使用了“2色”SERCA（肌/内质网钙ATP酶）生物传感器和独特的高通量荧光寿命板读数器（FLT-PR）开发一种高精度的活细胞检测，旨在筛选干扰SERCA结构的小分子。其中红色荧光蛋白（RFP）融合到N末端并且绿色荧光蛋白（GFP）融合到内环的SERCA构建体在以单层或悬浮液生长的HEK细胞系中稳定表达。在FLT-PR中测量从GFP到RFP的荧光共振能量转移（FRET），其在不牺牲通量的情况下将精度提高30倍于基于强度的读板器。FRET对已知的SERCA调节剂高度敏感。我们筛选了一个小的化学库，并确定了10种化合物，显着影响2色SERCA FLT。这些化合物中的三种可重复地降低FRET并以剂量依赖性方式抑制SERCA。该检测方法可用于大规模HTS活动，并适用于许多其他靶标。
We have used a “2-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate-reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate-readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified ten compounds that significantly affected 2-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns, and is adaptable to many other targets.
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影响因子：
--
作者：
Cornea RL;Gruber SJ;Lockamy EL;Muretta JM;Jin D;Chen J;Dahl R;Bartfai T;Zsebo KM;Gillispie GD;Thomas DD
通讯作者：
Thomas DD
影响因子：
1.6
作者：
Muretta, Joseph M.;Kyrychenko, Alexander;Thomas, David D.
通讯作者：
Thomas, David D.
DOI：
10.1111/j.1749-6632.1998.tb08305.x
发表时间：
1998-01-01
期刊：
CARDIAC SARCOPLASMIC RETICULUM FUNCTION AND REGULATION OF CONTRACTILITY
影响因子：
--
作者：
Johnson, RG
通讯作者：
Johnson, RG
影响因子：
7.3
作者：
Tazzeo, T.;Worek, F.;Janssen, L. J.
通讯作者：
Janssen, L. J.
影响因子：
14.8
作者：
Yuan, Jing;Johnson, Ronald L.;Su, Xin-zhuan
通讯作者：
Su, Xin-zhuan