High-throughput FRET assay yields allosteric SERCA activators.
High-throughput FRET assay yields allosteric SERCA activators.
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DOI：
10.1177/1087057112456878
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发表时间：
2013-01
影响因子：
--
通讯作者：
Thomas DD
中科院分区：
文献类型：
--
作者：
Cornea RL;Gruber SJ;Lockamy EL;Muretta JM;Jin D;Chen J;Dahl R;Bartfai T;Zsebo KM;Gillispie GD;Thomas DD
使用荧光共振能量转移（FRET），我们在重构膜系统中进行了高通量筛选（HTS），寻找通过其内源性调节剂受磷蛋白（PLB）逆转肌质/内质网Ca-ATP酶（SERCA）抑制的化合物。长期以来，人们一直在寻找这样的化合物来纠正心力衰竭中的异常Ca 2+调节。供体-SERCA在具有或不具有受体-PLB的磷脂膜中重构，并且在稳态荧光酶标仪中测量FRET。一式两份地测试20，000个化合物文库。降低FRET超过三个标准偏差的化合物被认为是命中。在43个主要命中（0.2%）中，经过更彻底的测试发现31个（72%）是假阳性。在Ca-ATP酶活性测定中测试了其余12个命中，其中6个显著激活SERCA，高达60%，并且几个还增强了心肌细胞的收缩性。这些化合物直接激活心脏和其他组织的SERCA。这些结果验证了我们的FRET方法，并为药物化学和临床前测试奠定了基础。我们担心由于稳态荧光的低精度而导致的高假阳性率。使用新型荧光寿命酶标仪的初步研究显示出高20倍的精度。该仪器可以大大提高未来HT的质量。
Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.
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DOI：
10.1152/ajpcell.2000.279.3.c724
发表时间：
2000-09-01
影响因子：
5.5
作者：
Fruen, BR;Bardy, JM;Louis, CF
通讯作者：
Louis, CF
影响因子：
7.3
作者：
Gobbini, Mauro;Armaroli, Silvia;Cerri, Alberto
通讯作者：
Cerri, Alberto
影响因子：
5.6
作者：
Gustavsson, Martin;Traaseth, Nathaniel J.;Veglia, Gianluigi
通讯作者：
Veglia, Gianluigi
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：
6
作者：
Jaski, Brian E.;Jessup, Mariell L.;Mancini, Donna M.;Cappola, Thomas P.;Pauly, Daniel F.;Greenberg, Barry;Borow, Kenneth;Dittrich, Howard;Zsebo, Krisztina M.;Hajjar, Roger J.
通讯作者：
Hajjar, Roger J.