Category: Assay Development and Screening
Although GPCRs represent the most predominant therapeutic targets, the fundamental biology and drug discovery potential of a large portion of these receptors remains to be explored. ‘Orphan’ receptors (oGPCRs), whose endogenous ligand(s) remain to be identified, represent putative novel therapeutic targets in a variety of diseases, and consequently studies aiming to functionally characterize these receptors and to elucidate their physiological and therapeutic relevance are critical. One such receptor awaiting pharmacological characterization is GPR151; an orphan GPCR whose distribution is specifically and highly enriched in the brain habenula complex. This structure is ideally located, both anatomically and functionally, to regulate emotional, motivational, and cognitive behaviors. Given that GPR151 is highly conserved from lower vertebrates to mammals, including humans, is enriched in a specific brain region, and belongs to the highly druggable Family A of GPCRs, this receptor has tremendous therapeutic targeting potential for the treatment of common and devastating psychiatric disorders such as anxiety & depression, schizophrenia, and drug dependence. Hence, the identification of GPR151 molecular probes is highly warranted.
Conventional cell-based assays for known GPCRs rely on the coupling of the ligand-bound receptor to heterotrimeric G proteins, as, historically, it has been assumed that all signaling events mediated by ‘GPCRs’ occur due to G protein activation. However, the lack of knowledge regarding GPR151 endogenous ligands, lack of synthetic ligands, and a poor understanding of how it interacts with other signaling pathways hinder efforts to enable HTS campaigns and validate its potential. New assay methods have become available that are not based on G protein activation, but instead exploit the molecular mechanism underlying the attenuation of G protein signaling (desensitization) mediated byarrestin. Despite the lack of GPR151 ligands, we have demonstrated that GPR151 has the potential to recruit arrestin in an ‘agonist-dependent’ manner and therefore most likely utilizes the classical mechanism of agonist-driven arrestin-mediated desensitization, such that, an assay that monitors ligand-drivenarrestin recruitment to GPR151, is relevant and can be enabled in an HTS-compatible format. Moreover, we validated this assay via pilot screening of the Sigma LOPAC library and miniaturized to 1536-well format in order to screen the Scripps Drug Discovery Library (SDDL); a large, diverse >650K small molecule collection empowered to deliver novel, tractable, and target-selective chemical scaffolds as hits. Herein, we describe the development and implementation of a series of novel, cell-based functional assays that encompass primary screening for hit identification, confirmation screening to triage artifacts, and counterscreens to assess selectivity versus structurally related GPCRs. These assays have facilitated the identification of potent, GPR151-selective ligands that can be used as molecular probes to interrogate GPR151 receptor function in regulating the habenula complex, its role in normal physiological processes, and its function in disease states.
Ainhoa Nieto Gutierrez– Senior Research Associate, The Scripps Research Institute, Jupiter, FL
Senior Research Associate
The Scripps Research Institute
I have a BS in chemistry and a MS in Pharmaceutical and Chemical R & D, followed by a year of experience in the pharmaceutical industry. In 2009 I returned to academia to pursue my Ph.D. in applied pharmacology focused on interrogating drug-related adverse effects mediated by the 5HT2B receptor. In addition, I had the opportunity to participate in several collaborations with industry and public EU research consortia, wherein I expanded my skill set to include in vitro and in vivo pharmacological characterization of compounds relevant to various stages of small molecule drug discovery.
Currently, as a member of the McDonald Lab at TSRI, my major research interests lie in identifying novel, tractable, CNS and anti-diabetes targets, developing selective molecular probes of these targets, and in collaboration with lead identification, medicinal chemistry, and DMPK, optimizing these probes to generate in vivo lead molecules for testing in rodent pre-clinical models.