Case Study: HTS of GPR-54 with IP-One Assay

Using HTRF IP-One and IP-One ELISA for agonist and antagonist screening (by Havard Neuroscience center).

Marcy Glicksman


Harvard Neuroscience Center, Brigham and Women's Hospital, Cambridge, MA, USA

Assays and Cellular Targets 2006

GPR54 and its ligand kisspeptin have been shown to inhibit tumor invasion and metastasis and to regulate gonadotropin releasing hormone physiology and reproduction. The discovery of drugs that modify GPR54 activation may hence be clinically useful, agonists for infertility or delayed puberty and antagonists for hormone-dependent cancers. Our high throughput screening (HTS) campaigns aims to identify small molecule agonists that activate GPR54 and antagonists that block GPR54 activation by kisspeptin. We have developed and characterized a stably transfected cell line overexpressing human GPR54 for use in HTS. A fluorescence resonance energy transfer (FRET)-based assay measuring intracellular inositol monophosphate production (IP-One HTRF, Cisbio) was optimized for HTS in 384-well format and a 120,000 small molecule library was screened for agonists. A single agonist hit was verified on the primary IP-One HTRF assay and other secondary assays including ERK phosphorylation, a radioactive IP3 assay and the IP-One ELISA (Cisbio) in a 96-well plate format. An antagonist assay was developed using a pentapeptide as ligand. The performance of the IP-One assay format for both screens will be discussed and verification with other secondary assays will be demonstrated.

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