Assessing GPCR activation using protein complementation: a novel technique for HTS
Assessing GPCR activation using protein complementation: a novel technique for HTS
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DOI：
10.1042/bst0350746
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发表时间：
2007-08-01
影响因子：
3.9
通讯作者：
Eglen, R. M.
中科院分区：
文献类型：
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作者：
Eglen, R. M.
GPCR（G蛋白偶联受体）是药物发现的关键靶点。尽管大多数HTS（高通量筛选）测定常规用于鉴定功能性激动或拮抗，但它们作为筛选GPCR功能的其他新颖方面的调节剂的方法是次优的。事实上，现在很明显，GPCR是高度复杂的蛋白质，其与III（受体活性修饰伴侣）、β-抑制蛋白、G蛋白相互作用，以及在潜在的同源或异源聚体复合物中起作用。因此，现在需要新的HTS技术，这将有助于在其细胞蛋白质-蛋白质相互作用方面询问GPCR。一种方法是单体蛋白片段的寡聚化辅助互补和片段重组的检测。值得注意的是，酶的使用在这方面具有优势，因为互补导致催化活性蛋白质。以这种方式产生的测定信号导致高灵敏度的测定，从而使得能够在需要极低流体体积的HTS系统中开发方案。使用产生发光信号的互补蛋白质还提供了明显不受人为干扰的测定。
GPCRs (G-protein-coupled receptors) are critical targets in drug discovery. Although most HTS (high-throughput screening) assays are routinely used to identify functional agonism or antagonism, they are suboptimal as methods to screen for modulators of other, novel, aspects of GPCR function. Indeed, it is now evident that GPCRs are highly complex proteins that interact with Ill (receptor-activity-modifying partners), ss-arrestins, G-proteins, as well as functioning in potential homo- or hetero-meric complexes. Consequently, novel HTS technologies are now required that would facilitate interrogation of GPCRs in terms of their cellular protein-protein interactions. One approach is oligomerization-assisted complementation of monomeric protein fragments and detection of fragment reassembly. Notably, the use of enzymes has advantages in this regard, since complementation results in catalytically competent protein. The assay signal generated in this fashion results in assays of high sensitivity, thereby enabling protocols to be developed in HTS systems that require extremely low fluid volumes. The use of complementing proteins that generate a luminescent signal also provides assays that are markedly free from artefactual interferences.