title: Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.
abstract: The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) 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 MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) 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: β(2)AR agonists - the β(2)AR 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.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Jacques
authors.initials: J
authors.lastname: Pantel
authors.forename: Savannah Y
authors.initials: SY
authors.lastname: Williams
authors.forename: Dehui
authors.initials: D
authors.lastname: Mi
authors.forename: Julien
authors.initials: J
authors.lastname: Sebag
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
authors.forename: C David
authors.initials: CD
authors.lastname: Weaver
authors.forename: Roger D
authors.initials: RD
authors.lastname: Cone
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D011564:Furocoumarins / Q000737:chemistry / Q000494:pharmacology; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D010865:Pilot Projects; D044105:Receptor, Melanocortin, Type 4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects*; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D000096926:Benzenesulfonamides
chemical_list: D000242:Cyclic AMP; D011564:Furocoumarins; D010726:Phosphodiesterase Inhibitors; D044105:Receptor, Melanocortin, Type 4; D013449:Sulfonamides; C031534:imperatorin; C471996:deracoxib; D000096926:Benzenesulfonamides