Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay

Abstract

The melanocortin MC₄ receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC₄ receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC₄ receptor, we created HEK293 cell lines coexpressing the human melanocortin MC₄ receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z′ = 0.50). A pilot screen run on the Microsource Spectrum compound library (n = 2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β₂AR agonists – the β₂AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.

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.