Molecular and Cellular PharmacologyDevelopment of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay
Introduction
The melanocortin circuitry of the CNS is a critical component of the adipostat (Cone, 2005). Activation of these circuits inhibits food intake and stimulates energy expenditure and thus the melanocortin MC4 receptor has been a target of the major pharmaceutical companies for the development of drugs for the treatment of common obesity (Wikberg and Mutulis, 2008). However, the first clinical trials of potent melanocortin MC4 receptor agonists failed due to pressor activity (Greenfield et al., 2009). Severe early onset obesity due to defective melanocortin signaling is linked, in up to 5% of cases, with non-synonymous coding mutations causing haploinsufficiency of the melanocortin MC4 receptor (Farooqi and O'Rahilly, 2006). It would not be unusual to expect that 10–30% of early onset childhood obesity may thus result from defective melanocortin signaling, assuming melanocortin MC4 receptor promoter mutations and mutations in additional genes in the pathway may ultimately be discovered. In the general population, these mutations are present at a frequency of around 0.6% (Calton et al., 2009, Hinney et al., 2006). The majority of these mutations disrupt trafficking of receptors to the cell surface, rather than affinity for ligand (Govaerts et al., 2005). In contrast to common obesity, treatment of severe obesity due to melanocortin receptor haploinsufficiency may involve returning melanocortin MC4 receptor signaling levels to normal, without causing unwanted pressor activity, suggesting a possible application for allosteric modulators of the melanocortin MC4 receptor.
Alternative approaches in other receptor systems based on development of allosteric ligands provided promising results relative to orthosteric agonist agents (Conn et al., 2009, Kenakin, 2007, May et al., 2007). Due to their mechanism of action, allosteric molecules should display agonism in a more physiological temporo-spatial pattern and may have an increased selectivity amongst melanocortin receptor subtypes. Given the rather unique pharmacological profile of melanocortin MC4 receptor involving the physiological expression of both agonists (proopiomelanocortin products) and inverse agonists (agouti-related protein, AgRP) (Cone, 2005), one might speculate that a variety of compounds targeting allosteric(s) site(s) on melanocortin MC4 receptor might be identified.
So far, most cAMP assays in use are static, and based on the accumulation of cAMP in the presence of a PDE blocker to enhance sensitivity. These static assays preclude the study of any complex time-dependent pattern of response. Live cell real-time cAMP imaging techniques based on downstream cAMP targets such as PKA (Zhang et al., 2001), EPAC (DiPilato et al., 2004) or cyclic nucleotide-gated channels (Rich et al., 2001) are just emerging (Willoughby and Cooper, 2008). Based on these indirect cAMP readouts, to our knowledge, only a single high-throughput screen was documented using a PDE blocker (Titus et al., 2008). So far, no highly sensitive real-time high-throughput screening was documented, therefore precluding the identification of allosteric modulators by HTS. We therefore developed an assay of human melanocortin MC4 receptor function based on real time cAMP detection, and validated this assay for high-throughput screening using a pilot screen designed to detect allosteric modulators.
Section snippets
Creation of the Human MC4R-GLO Cell Line
Human HEK293 cells were cotransfected with a plasmid encoding the human melanocortin MC4 receptor cDNA (pCDNA3.1 vector) and with a plasmid encoding an engineered cAMP sensitive luciferase (pGLO sensor™-20F cAMP plasmid, Promega) by the Lipofectamin method (Invitrogen). These cells were grown in 90% minimum essential medium (MEM), 10% fetal bovine serum (FBS), geneticin (700 μg/mL) and hygromycin B (200 μg/mL). Resistant clones were isolated, expanded and selected for their ability to respond to
A Cell Line Expressing a cAMP-dependent Luminescent Reporter Monitors Human Melanocortin MC4 Receptor Signaling in Real Time
To monitor the cAMP response of the human melanocortin MC4 receptor in living cells, we generated clones of HEK293 cells stably coexpressing the human melanocortin MC4 receptor and a modified luciferase that carries the cAMP binding B domain from the RIIβ subunit of cAMP dependent protein kinase (pGloSensor™, Promega). cAMP binding is required for the activity of this modified luciferase enzyme. Double transfectants were selected for further experiments based on their response to forskolin, as
Discussion
We document here a cell line expressing a modified cAMP-sensitive luciferase allowing real time detection of cAMP concentrations resulting from human melanocortin-4 receptor signaling. In this cell line, the known melanocortin agonists and the physiological inverse agonist (AgRP) exert their expected activities. Furthermore, the sensitivity of this technique further allows one to monitor real time activity of endogenous PDEs. The sensitivity of this assay also allowed us to examine the inverse
Acknowledgments
We thank Dr. Larry Marnett (Vanderbilt) for providing celecoxib and rofecoxib, and Dr. Marco Conti (UCSF) for recombinant human PDE4D3. This study was supported by National Institutes of Health [Grant DK70332 to RDC and JP], and the Bristol-Myers Squibb Foundation (Freedom to Discover Unrestricted Metabolic Diseases Research Grant). JP is a recipient of the Sabbatical Leave Programme from the European Society for Paediatric Endocrinology, supported by E. Lilly Co.
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