A highly specific cell-based high-throughput screening assay for ligands of cyclic adenosine monophosphate receptor protein in gram-negative bacteria.
A highly specific cell-based high-throughput screening assay for ligands of cyclic adenosine monophosphate receptor protein in gram-negative bacteria.
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一种高度特异性的基于细胞的高通量筛选方法，用于检测革兰氏阴性细菌中环磷酸腺苷受体蛋白的配体。
DOI：
10.1089/adt.2013.514
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发表时间：
2013
影响因子：
1.8
通讯作者：
Benitez,JorgeA
中科院分区：
文献类型：
--
作者：
Wang,Hongxia;Silva,AnisiaJ;Rasmussen,Lynn;White,ELucile;Benitez,JorgeA
群体感应是细菌细胞间的一种通讯过程，涉及被称为自诱导剂的化学信号分子的产生、释放和随后的检测。在霍乱弧菌中，多个输入信号激活群体感应调节因子HapR的表达，其作用是抑制毒力因子的表达。我们已经证明，CRP，环磷酸腺苷（cAMP）受体蛋白，通过激活霍乱自诱导剂1的生物合成来增强群体感应，霍乱自诱导剂1是促进HapR激活的主要信号分子。因此，原感c反应蛋白激动剂可以抑制毒力，从而开发出治疗重症霍乱的新药。在这项研究中，我们发现群体感应调节的luxcdabeoperon的表达可以作为CRP活性的可靠读数。此外，我们描述并验证了一种高度特异性的基于细胞的发光高通量筛选方法，用于检测CRP配体。在9425种化合物的试点筛选中，命中率为0.02%，其中一种是cAMP本身。阳性对照化合物的Z值为0.76，变异系数为8%。据我们所知，这是革兰氏阴性菌高度保守的CRP蛋白配体的第一个基于细胞的检测。利用这种方法筛选大型化学文库，可以确定治疗霍乱的先导化合物，以及探测CRP分子中配体-受体相互作用的小分子。
Quorum sensing is a cell–cell communication process in bacteria that involves the production, release, and subsequent detection of chemical signal molecules called autoinducers. InVibrio cholerae, multiple input signals activate the expression of the quorum sensing regulator HapR, which acts to repress the expression of virulence factors. We have shown that CRP, the cyclic adenosine monophosphate (cAMP) receptor protein, enhances quorum sensing by activating the biosynthesis of cholera autoinducer 1, the major signaling molecule that contributes to the activation of HapR. Thus, proquorum sensing CRP agonists could inhibit virulence and lead to new drugs to treat severe cholera. In this study, we show that expression of the quorum sensing-regulatedluxCDABEoperon can be used as a robust readout for CRP activity. Further, we describe and validate a highly specific cell-based luminescence high-throughput screening assay for proquorum sensing CRP ligands. A pilot screen of 9,425 compounds yielded a hit rate of 0.02%, one hit being cAMP itself. The Z′ value for this assay was 0.76 and its coefficient of variance 8% for the positive control compound. To our knowledge, this is the first cell-based assay for ligands of the highly conserved CRP protein of Gram-negative bacteria. The use of this assay to screen large chemical libraries could identify lead compounds to treat cholera, as well as small molecules to probe ligand–receptor interactions in the CRP molecule.
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DOI：
10.1073/pnas.94.1.265
发表时间：
1997-01-07
影响因子：
11.1
作者：
Skorupski, K;Taylor, RK
通讯作者：
Taylor, RK
影响因子：
4.8
作者：
Krueger, S;Gorshkova, I;Schwarz, FP
通讯作者：
Schwarz, FP
影响因子：
3.9
作者：
Chattopadhyay, R;Parrack, P
通讯作者：
Parrack, P
cAMP 类似物对大肠杆菌体内 lac 转录刺激的研究。
DOI：
10.1111/j.1432-1033.1984.tb07887.x
发表时间：
1984
期刊：
European journal of biochemistry
影响因子：
--
作者：
Hans‐Gerd Scholübbers;P. H. V. Knippenberg;J. Baraniak;Wojciech J. Stec;Michael Morr;Bernd Jastorff
通讯作者：
Bernd Jastorff
影响因子：
2.8
作者：
Liang, Weili;Pascual-Montano, Alberto;Benitez, Jorge A.
通讯作者：
Benitez, Jorge A.