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 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= 2,000) 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 an HTS for allosteric modulators for a Gs protein coupled receptor.

SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

“…Despite a clear increase of cAMP production in response to PACAP 38 or forskolin, no significant increase could be detected in response to GnRHa, 5 consistent with data previously reported by others using these cells (46). We further re-explored GnRHR signaling to cAMP pathway in ␣T3-1 cells by taking advantage of a recent highly sensitive technology based on the use of a cAMP biosensor to monitor cAMP mobilization in living cells (47). This strategy allowed us to demonstrate for the first time that GnRHa increases cAMP production in ␣T3-1 cells.…”

Biased Agonism as a Novel Strategy To Harness the Proresolving Properties of Melanocortin Receptors without Eliciting Melanogenic Effects

“…In this article, we characterize the orally active small molecule AP1189 as a biased agonist with the additional advantage of targeting both tissue-protective MC 1 and MC 3 receptors. AP1189 activation of MC receptors did not induce cAMP accumulation, the canonical pathway ascribed to MC agonists and used for drug screening programs (e.g., see Pantel et al [14]). AP1189 interaction with MC 1 and MC 3 activated a second pathway centered on ERK1/2 phosphorylation, as well as intracellular Ca 2+ mobilization.…”

“…Allosteric modulation consists in the ability of a molecule to enhance (positive modulation) or reduce (negative modulation) the effect of the endogenous ligand by binding to a distinct site of the receptor protein, termed allosteric site (13). A higher degree of selectivity is expected as allosteric regions are less conserved among the five MCRs, and indeed, allosteric modulators at MC 4 are currently under development for the treatment of obesity (14).…”

A Screen of Approved Drugs Identifies the Androgen Receptor Antagonist Flutamide and Its Pharmacologically Active Metabolite 2-Hydroxy-Flutamide as Heterotropic Activators of Cytochrome P450 3A In Vitro and In Vivo

“…The SPECTRUM library of compounds [MicroSource Discovery Systems Inc., Gaylordsville, CT; http://www.msdiscovery.com (Pantel et al, 2011;Kumar et al, 2014)] includes a wide range of clinically approved and structurally diverse compounds, including over 60% marketed drugs. In an attempt to understand the prevalence of CYP heterotropic activation among the top classes of administered drugs, we conducted a semiautomated functional screen of the SPECTRUM chemical library via a cocktail probe substrate assay design in HLM (Fig.1).…”